Functional differences between NPFF1 and NPFF2 receptor coupling: high intrinsic activities of RFamide-related peptides on stimulation of [35S]GTPgammaS binding.

By using an optimized [(35)S]GTPgammaS binding assay, the functional activities (potency and efficacy) of peptides belonging to three members of the RFamide family; Neuropeptide FF (NPFF), prolactin-releasing peptide (PrRP) and 26RFamide, were investigated on NPFF(1) and NPFF(2) receptors stably expressed in Chinese Hamster Ovary (CHO) cells. Despite their large differences in affinity and selectivity, all analogues tested behaved as agonists toward NPFF(1) and NPFF(2) receptors. High NaCl concentration in the assay strongly increased the efficacy toward NPFF(2) receptors and augmented differences among agonists. In low sodium conditions, whereas the potencies of agonists correlated with their affinities for NPFF(1) receptors, NPFF(2) receptors exhibited an extraordinary activity since all compounds tested displayed EC(50) values of GTPgammaS binding lower than their K(I) values. Comparisons of functional values between NPFF(1) and NPFF(2) receptors revealed unexpected potent selective NPFF(2) agonists especially for the PLRFamide and the VGRFamide sequences. By using blocker peptides, we also show that Galpha(i3) and Galpha(s) are the main transducers of NPFF(1) receptors while NPFF(2) are probably coupled with Galpha(i2), Galpha(i3), Galpha(o) and Galpha(s) proteins. Our data indicate that NPPF(1) and NPFF(2) receptors are differently coupled to G proteins in CHO cells.

Facilitation of spinal morphine analgesia in normal and morphine tolerant animals by neuropeptide SF and related peptides.

Neuropeptide FF and related synthetic amidated peptides have been shown to elicit sustained anti-nociceptive responses and potently augment spinal anti-nociceptive actions of spinal morphine in tests of thermal and mechanical nociception. Recent studies have described the occurrence of another octapeptide, neuropeptide SF (NPSF) in the spinal cord and the cerebrospinal fluid and demonstrated its affinity for the NPFF receptors. This study examined the effects of NPSF and two putative precursor peptides, EFW-NPSF and NPAF, on the spinal actions of morphine in normal and opioid tolerant rats using the tailflick and pawpressure tests. In normal rats, NPSF demonstrated weak intrinsic activity but sub-effective doses of the peptide significantly increased the magnitude and duration of spinal morphine anti-nociception in both tests. A low-dose of NPSF also augmented the spinal actions of a delta receptor agonist, deltorphin. The morphine-potentiating effect of NPSF was shared by EFW-NPSF and the octadecapeptide NPAF. In animal rendered tolerant by continuous intrathecal infusion of morphine for 6 days, low dose NPSF itself elicited a significant anti-nociceptive response and potently increased morphine-induced response in both tests. In animals made tolerant by repeated injections of intrathecal morphine, administration of NPSF, EFW-NPSF, and NPAF with morphine reversed the loss of the anti-nociceptive effect and restored the agonist potency. The results demonstrate that in normal animals NPSF and related peptides exert strong potentiating effect on morphine anti-nociception at the spinal level and in tolerant animals these agents can reverse the loss of morphine potency.

Rodent strain differences in the NPFF1 and NPFF2 receptor distribution and density in the central nervous system.

The present study evaluates the putative differences between NPFF1 and NPFF2 receptor distribution and density throughout the central nervous system between rat and mouse strains by using in vitro quantitative autoradiography. The binding of [125I]YVP ([125I]YVPNLPQRF-NH2) and [125I]EYF ([125I]EYWSLAAPQRF-NH2), used to label NPFF1 and NPFF2 receptors, respectively, was compared between Sprague-Dawley and Wistar rats and between Swiss and C57BL/6-SV129 mice. In contrast to Wistar, Sprague-Dawley brains contained NPFF1 binding sites in the cortical and spinal cord areas, the accumbens nucleus, the anterodorsal thalamic nucleus, the parafascicular thalamic nucleus, the inferior colliculus and the nucleus of the solitary tract. The distribution of NPFF2 binding sites was also different between the two strains of rats. As compared to Swiss, C57BL/6-SV129 mice showed higher basal NPFF2 receptor levels in cortical areas, telencephalon and some other regions. In contrast, they showed lower amounts in thalamic structures, except the reuniens nucleus, and in mesencephalic and rhombencephalic regions. In the cervical spinal cord the levels of NPFF2 receptors were similar. The NPFF1 binding levels were nearly the same in telencephalic structures while distinct in the forebrain. Differences in amount of NPFF receptor subtypes among these strains of rats or mice could lead to differences in NPFF control of opioid nociception.

Quantitative autoradiographic distribution of NPFF1 neuropeptide FF receptor in the rat brain and comparison with NPFF2 receptor by using [125I]YVP and [(125I]EYF as selective radioligands.

The selectivity of two new radioligands, [(125)I]YVP ([(125)I]YVPNLPQRF-NH(2)) and [(125)I]EYF ([(125)I]EYWSLAAPQRF-NH(2)), for neuropeptide FF (NPFF) receptor subtypes was determined using HEK293 cells expressing hNPFF(1) and CHO cells expressing hNPFF(2) receptors. Saturation binding and displacement experiments showed that [(125)I]YVP and [(125)I]EYF bound selectively with a very high affinity, K(D)=0.18 nM and 0.06 nM, to NPFF(1) and NPFF(2) receptors respectively. By using in vitro autoradiography with these radioligands and frog pancreatic polypeptide (PP) as selective unlabelled competitor of NPFF(2) binding sites, NPFF(1) and NPFF(2) receptor distribution was analyzed throughout the rat CNS. The highest densities of [(125)I]EYF binding sites were seen in the most external layers of the dorsal horn of the spinal cord, the parafascicular thalamic nucleus, laterodorsal thalamic nucleus and presubiculum of hippocampus. All specific binding of this radioligand was inhibited by 200 nM frog PP. The density of 0.1 nM [(125)I]YVP binding was much smaller in all brain areas and frog PP-insensitive binding sites (NPFF(1) receptor subtype) were detected in septal, thalamic and hypothalamic areas but were absent in the spinal cord. The restricted distribution of NPFF(1) receptors in the CNS supports its specific role in a limited number of neuronal functions. In contrast to the rat spinal cord where the NPFF(1) system is absent, there is no strict separation between NPFF(1) and NPFF(2) system at the supraspinal level.

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.

[(125)I]EYF: a new high affinity radioligand to neuropeptide FF receptors.

[(125)I]EYF ([(125)I]EYWSLAAPQRFamide), a new radioiodinated probe derived from a peptide present in the rat Neuropeptide FF precursor (EFWSLAAPQRFamide, EFW-NPSF) was synthesized and its binding characteristics investigated on sections of the rat spinal cord and on membranes of mouse olfactory bulb. In both tissues, [(125)I]EYF binding was saturable and revealed a very high affinity interaction with a single class of binding sites in rat and mouse (K(D) = 0.041 and 0.019 nM, respectively). Competition studies showed that [(125)I]EYF bound to one class of binding sites exhibiting a high affinity for all the different peptides the precursor could generate (NPA-NPFF, SPA-NPFF, NPFF, EFW-NPSF, QFW-NPSF) with the exception of NPSF which displayed a low affinity. Autoradiographic studies demonstrated that [(125)I]EYF binding sites were fully inhibited by a synthetic Neuropeptide FF agonist (1DMe) in all areas of the rat brain. The density of [(125)I]EYF binding sites was high in the intralaminar thalamic nuclei, the parafascicular thalamic nucleus and in the superficial layers of the dorsal horn. Non specific binding reached 5-10% of the total binding in all brain areas. Similarly, in mouse brain experiments, the non-specific binding was never superior to 10%. These findings demonstrate that putative neuropeptides generated by the Neuropeptide FF precursor and containing the NPFF or NPSF sequences should bind to the same receptor. Furthermore, these data indicate that [(125)I]EYF is a useful radiolabeled probe to investigate the NPFF receptors; its major advantages being its high affinity and the very low non-specific binding it induces.

Agonist and antagonist activities on human NPFF(2) receptors of the NPY ligands GR231118 and BIBP3226.

Neuropeptide FF (NPFF) is a part of a neurotransmitter system acting as a modulator of endogenous opioid functions. At this time, no non-peptide or peptide NPFF-antagonists have been discovered. Here, we demonstrate that Neuropeptide Y (NPY) ligands, in fact possess significant ability to interact with the human NPFF(2) receptors. NPY Y(1) antagonist BIBP3226 and mixed Y(1) antagonist/Y(4) agonist GR231118 are able to displace with low affinity, 50 -- 100 nM, the specific binding on NPFF receptors expressed in CHO cells as well as in rat dorsal spinal cord, an affinity however superior to those determined against Y(2), Y(4) or Y(5) receptors. Furthermore, BIBP3226 which is unable to inhibit the forskolin-stimulated cyclic AMP production mediated by NPFF(2) receptors, antagonizes the effect of NPFF, revealing the first antagonist of NPFF receptors. These properties of NPY ligands on Neuropeptide FF receptors must be considered when evaluating pharmacological activities of these drugs.

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.

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.

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-NH2) at the spinal level in the rat. This hypothesis was directly assessed by investigating the effects of a NPFF analogue, 1DMe ([D-Tyr1,(NMe)Phe3]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 microM-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 micro-opioid receptor antagonist CTOP (1 microM) did not significantly affect the spinal MELM overflow due to 0.1 mM 1DMe. In contrast, both naltrindole and nor-binaltorphimine, at concentrations (10 microM) that allow the selective blockade of alpha- and kappa-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.

Nociceptin receptors in the rat spinal cord during morphine tolerance.

The anatomical localization of nociceptin receptors was examined by in vitro quantitative autoradiography techniques in rat spinal cord sections by using [(125)I-Tyr(14)]nociceptin. [(125)I-Tyr(14)]nociceptin appeared to interact with a single class of binding sites (K(D)=0.1 nM) present in the grey matter in all laminae of the spinal cord from cervical to sacral levels. Pre-incubation of sections in the presence of 150 mM NaCl, did not modify the radioligand affinity but significantly augmented the number of accessible binding sites and increased specific binding of [(125)I-Tyr(14)]nociceptin differentially on each laminae. In particular, the superficial layers of the dorsal horn exhibited the highest density of sites after pre-wash. Continuous intrathecal infusion of morphine produced a tolerance accompanied by a significant increase in nociceptin site density in the superficial layers. Thus, nociceptin binding sites may have different properties dependent upon the layer and may be up-regulated during the process of opioid-induced tolerance.

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.

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-NH2, NPFF) exhibiting both supraspinal anti-opioid and spinal analgesic activities were studied for their abilities to interact with specific mu, delta, and kappa 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 Ki values ranged from 5 to 400 microM. 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.

Autoradiographic characterization of rat spinal neuropeptide FF receptors by using [125I][D.Tyr1, (NMe)Phe3]NPFF.

The binding properties of neuropeptide FF (NPFF) receptors were investigated in different laminae of the rat spinal cord by using quantitative autoradiography and [125I][D.Tyr1, (NMe)Phe3]NPFF as radioligand. In the superficial layers, the specific binding of [125I][D.Tyr1, (NMe)Phe3]NPFF was time-dependent, reversible, and saturable (KD = 0.1 nM). Preincubation of spinal sections increased the maximal number of [125I][D.Tyr1, (NMe)Phe3]NPFF binding sites. Bestatin, an inhibitor of aminopeptidases, increased significantly the apparent affinity of NPFF. Optimal binding of [125I][D.Tyr1, (NMe)Phe3]NPFF was observed in the presence of 120 mM NaCl in all laminae of the spinal cord. No significant differences were noted in the salt dependence in laminae I-II, IV-V, and X, and the pharmacological profile of [125I][D.Tyr1, (NMe)Phe3]NPFF binding was similar in each laminae. These results do not support the existence of NPFF receptors subtypes differentially localized in different area of the spinal cord. Our data reveal the effects of tissue treatments on binding characteristics of NPFF receptors and indicate that [125I][D.Tyr1, (NMe)Phe3]NPFF is a useful radioactive probe for the characterization of NPFF receptors in discrete brain areas.

Presence of neuropeptide FF receptors on primary afferent fibres of the rat spinal cord.

A radioiodinated analogue of neuropeptide FF, [125I][D. Tyr1,(NMe) Phe3]neuropeptide FF, was used as a selective probe to label neuropeptide FF receptors in the rat spinal cord. Following neonatal capsaicin treatment, dorsal rhizotomy or sciatic nerve section, the distribution and possible alterations of spinal cord specific [125I][D.Tyr1,(NMe)Phe3]neuropeptide FF binding sites were evaluated using in vitro quantitative receptor autoradiography. In normal rats, the highest densities of sites were observed in the superficial layers of the dorsal horn (laminae I-II) whereas moderate to low amounts of labelling were seen in the deeper (III-VI) laminae, around the central canal, and in the ventral horn. Capsaicin-treated rats showed a bilateral decrease (47%) in [125I][D.Tyr1,(NMe)Phe3]neuropeptide FF binding in all spinal areas. Unilateral sciatic nerve section and unilateral dorsal rhizotomy induced significant depletions (15-27%) in [125I][D.Tyr1,(NMe)Phe3]neuropeptide FF labelling in the ipsilateral dorsal horn. These results suggest that a proportion of neuropeptide FF receptors is located on primary afferent terminals of the dorsal horn and could thus play a role in the modulation of nociceptive transmission.

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.

Opioid and substance P receptor adaptations in the rat spinal cord following sub-chronic intrathecal treatment with morphine and naloxone.

The effect of continuous intrathecal infusion with morphine (5 mu/h) or naloxone (2 micrograms/h) was investigated with regard to analgesia and the apparent density of mu- and delta-opioid and neurokinin-I/substance P receptors in the rat spinal cord. Morphine infusion increased tail-flick and paw-pressure responses until day 4 after the mini-osmotic pump implant. A decline in antinociception, reflecting tolerance to morphine, was then apparent in both tests. Quantitative in vitro receptor autoradiography of [125I]FK-33824, [125I][D.Ala2]deltorphin-I and [125I] Bolton-Hunter substance P binding sites, as ligands of mu, delta and neurokinin-I/substance P receptors, respectively, was performed on lumbosacral spinal cord sections of seven-days tolerant animals. Treatments with morphine and naloxone induced a similar increase (37%) in the number of delta binding sites in the superficial laminae of the dorsal horn. In contrast, the density of mu-opioid receptors was only affected by naloxone (50% increase). Neurokinin-I/substance P binding parameters were not altered by these treatments. Thus, it appears that delta-opioid binding sites may be of special relevance with regard to the development of tolerance to opiates in the spinal cord.

Antinociceptive effects of intrathecally administered F8Famide and FMRFamide in the rat.

The effects of intrathecal injections of F8Famide (Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2, 0.05-17.5 nmol) and FMRF-amide (Phe-Met-Arg-Phe-NH2, 0.002-25 nmol), known as anti-opioid agents, were investigated by using noxious thermal (tail flick) and mechanical (paw pressure) tests in the rat. Both peptides produced significant long-lasting (24-48 h) analgesia in both tests without causing detectable motor dysfunction. Pretreatment with systemic naloxone (5.5 mumol/kg i.p.) attenuated the initial antinociceptive effects (first hour) induced by both peptides (8.8 nmol) in the tail flick test and only by FMRFamide in the paw pressure test. A subeffective dose of F8Famide (0.05 nmol) enhanced both the intensity and the duration of spinal morphine (6.6 nmol) analgesia in both tests. In contrast, a subanalgesic dose of FMRFamide (0.002 nmol) decreased the intensity and enhanced the duration of the effect of morphine. These results show that, besides acting as antinociceptive agents in the spinal cord, F8Famide and FMRFamide could differentially modulate spinal opioid functions.

Quantitative autoradiographic mapping of delta-opioid receptors in the rat central nervous system using [125I][D.Ala2]deltorphin-I.

The distribution of delta-opioid binding sites was studied by quantitative autoradiography in rat brain and spinal cord using the highly selective ligand [125I][D.Ala2]deltorphin-I. The binding properties of [125I][D.Ala2]deltorphin-I were investigated by microdensitometry of autoradiographic films with the aid of a computer-assisted image-analysis system. [125I][D.Ala2]deltorphin-I appeared to interact with a single class of sites in all brain areas (KD = 0.9 nM). In 23 regions tested, whatever the delta site concentration, DTLET, a delta agonist, appears to be 2 orders of magnitude more effective than DAGO, a mu agonist, in inhibiting specific [125I][D.Ala2]deltorphin-I binding. The distribution of [125I][D.Ala2]deltorphin-I sites is globally consistent with that of other delta ligands and does not support the existence of a delta-receptor subtype recognized by [D.Ala2]deltorphin-I. [125I][D.Ala2]deltorphin-I binding sites were highly confined, exhibiting selective localization in the neocortex and a diffuse pattern in the striatum, accumbens nucleus, claustrum, layer of bulb, amygdaloid nucleus, pontine nuclei, and inferior colliculus. In several areas a rostro-caudal gradient of site concentration was indicated. [D.Ala2]deltorphin-I binding sites were also present in the substantia gelatinosa at all levels of the spinal cord and, unexpectedly, in deeper laminae and the ventral horn. These results demonstrate the ability of [125I][D.Ala2]deltorphin-I to characterize low concentrations of binding sites and to reveal new localizations of delta receptors.