Christine Gouardères

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.

Autoradiographic localization of mu, delta and kappa opioid receptor binding sites in rat and guinea pig spinal cord

The autoradiographic distribution of mu, delta and kappa opioid binding sites was evaluated in various segments of the rat and guinea pig spinal cord. Mu opioid receptor binding sites are highly concentrated in the superficial layers of the dorsal horn (laminae II and III) in both species, without any marked gradient along the cord. Delta binding sites are somewhat concentrated in the superficial layers of the dorsal horn. However, delta binding sites are also present and evenly distributed in other areas of the gray matter. The highest density of delta sites is found in the cervical segment with only low levels in the lumbo-sacral region of the rat and guinea pig spinal cord. Kappa opioid binding sites are highly concentrated in the superficial layers of the dorsal horn of the spinal cord. Lower levels are seen in the rest of the gray matter with some enrichment in lamina X. Moreover, the lumbo-sacral portion of the spinal cord is enriched in kappa sites as compared to the cervical and thoracic segments. These data demonstrate the differential laminar distribution of mu, delta and kappa opioid binding sites in rat and guinea pig spinal cord.

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 β‐funaltrexamine (β‐FNA, 2 nmol) or naltrindole (2.2 nmol). The doses of the μ‐antagonists (CTOP and β‐FNA) or the δ‐antagonist (naltrindole) used in 1DMe and 3D experiments blocked the antinociceptive effects of μ‐or δ‐receptor‐selective agonists. 4 When administered in combination with antinociceptive doses of the μ‐receptor agonist, morphine (13.2 nmol) or the δ‐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 μ‐and δ‐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.

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

Abstract By using an optimized [ 35 S]GTPγS 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 GTPγS 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 Gα i3 and Gα s are the main transducers of NPFF 1 receptors while NPFF 2 are probably coupled with Gα i2 , Gα i3 , Gα o and Gα s proteins. Our data indicate that NPPF 1 and NPFF 2 receptors are differently coupled to G proteins in CHO cells.

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.

Agonist and antagonist activities on human NPFF2 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 NPFF2 receptors. NPY Y1 antagonist BIBP3226 and mixed Y1 antagonist/Y4 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 Y2, Y4 or Y5 receptors. Furthermore, BIBP3226 which is unable to inhibit the forskolin‐stimulated cyclic AMP production mediated by NPFF2 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.

Opioid binding sites in different levels of rat spinal cord

Opioid receptor binding sites were analyzed in various segments of rat spinal cord. Mu and delta types were labelled with [3H]-DHM or [3H]-DAGO and [3H]-DADLE or [3H]-DSLET respectively. Kappa 1 (kappa) and kappa 2 (benzomorphan) binding sites were individually detected by the overall labeling of opioid binding sites with [3H]-etorphine followed by the elimination of binding to particular sites by the use of appropriate selective unlabelled ligands. Whereas lumbo sacral region contained mainly the kappa 2 site, thoracic membranes had a proportion of approximately 20% mu, 20% kappa 1 and 60% kappa 2 and cervical region contained much less kappa 2 sites (25% mu, 20% delta, 28% kappa 1 and 32% kappa 2).

Benzomorphan binding sites in rat lumbo-sacral spinal cord

The rat lumbo-sacral spinal cord contains a homogeneous population of opiate binding sites labelled with high affinity (KD = 0.21 +/- 0.04 nM) by [3H]etorphine and lower affinity (KD = 2.2 +/- 0.4 nM) by [3H]ethylketocyclazocine. Benzomorphan drugs are potent competitors for these binding sites while morphine and enkephalin display a low affinity. These binding sites have binding properties which are distinct from the mu-, delta-, and also kappa-sites but are very similar to those of the benzomorphan sites characterized in rat brain.

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.

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.