Supraspinal and spinal effects of [Phe1psi(CH2-NH)Gly2]-nociceptin(1-13)-NH2 on nociception in the rat

A new derivative of the neuropeptide nociceptin (NC) has recently been developed. This molecule, the pseudopeptide [Phe1psi(CH2-NH)Gly2]-nociceptin(1-13)-NH2 was found to antagonize NC inhibitory effects in peripheral smooth muscle preparations in vitro. However, contrasting results have appeared as regards its pharmacodynamic profile in the CNS. Here, we investigated the pseudopeptide effects, in vivo, on nociceptive responses in the rat. [Phe1psi(CH2-NH)Gly2]-nociceptin(1-13)-NH2 was administered intracerebroventricularly (i.c.v.) or intrathecally (i.t.) (alone or in combination with NC), and tail-flick latencies (TFL) to radiant heat were assessed. I.c.v. [Phe1psi(CH2-NH)Gly2]-nociceptin(1-13)-NH2 (1-10 nmol/rat) caused a short-lasting decrease (5 min) of TFL and did not antagonize the threshold lowering effect of i.c.v. NC (1 nmol/rat). At the spinal level, the i.t. administration (0.2-10 nmol/rat) of [Phe1psi(CH2-NH)Gly2]-nociceptin(1-13)-NH2 produced a dose-dependent and long-lasting antinociceptive effect that was not modified by the administration of a high dose (30 nmol/rat i.t.) of the opioid antagonist naloxone. The i.t. co-administration of the pseudopeptide (10 nmol/rat) did not block the antinociceptive effect of i.t. NC (10 nmol/rat). These data indicate that the pseudopeptide behaves as an NC agonist at supraspinal and spinal levels in the rat tail-flick test of nociception. These different profiles in the periphery and the CNS could suggest differences between central and peripheral NC receptor/s and provide a basis for further development of antagonist molecules suitable for their characterization.     

Nociceptin/orphanin FQ and neurotransmitter release in the central nervous system

In this article, the effect of nociceptin (orphanin FQ) on transmitter release in the central nervous system in vitro and in vivo is reviewed. Nociceptin inhibits the electrically or K(+)-evoked noradrenaline, dopamine, serotonin, and glutamate release in brain slices from guinea-pig, rat, and mouse. This effect is usually naloxone-resistant but antagonized by OP(4) receptor antagonists like [Phe(1)psi(CH(2)-NH)Gly(2)]-nociceptin(1-13)NH(2). In the rat in vivo, nociceptin diminishes acetylcholine release in the striatum, reduces dopamine release, and prevents the stimulatory effect of morphine on this transmitter in the nucleus accumbens and also elevates extracellular glutamate and gamma-aminobutyric acid levels in mesencephalic dopaminergic areas. The effect of nociceptin on the mesencephalic dopaminergic system might explain its actions on motor behavior.     

Species differences in the efficacy of compounds at the nociceptin receptor (ORL1)

Recent studies have identified compounds with reduced efficacy relative to nociceptin/orphanin FQ at the opioid-like receptor ORL1. Utilizing stimulation of [(35)S]GTPgammaS binding as in vitro assays, it was determined that both [Phe(1)psi(CH(2)-NH)Gly(2)]N/OFQ(1-13)NH(2) and the hexapeptide Ac-RYYRIK-NH(2) act as partial agonists in CHO cells transfected with either human or mouse ORL1. Maximal activity for both [Phe(1)psi(CH(2)-NH)Gly(2)]N/OFQ(1-13)NH(2) and Ac-RYYRIK-NH(2) was significantly greater in cells transfected with the human receptor (90% and 73% in a high expressing clone, 76% and 68% in low expressing clone) rather than the mouse receptor (37.5 and 33%), regardless of receptor number in individual clones. In vitro studies in cells transfected with exaggerated receptor numbers can lead to unreliable estimates of agonist and antagonist activity, however, these studies suggest that animal experiments on the activity of novel compounds may not always be better predictors of the ultimate activity in humans.     

Structure-activity relationships of nociceptin and related peptides: comparison with dynorphin A

Nociceptin and its receptor (OP(4)) share sequence homologies with the opioid peptide ligand dynorphin A and its receptor OP(2). Cationic residues in the C-terminal sequence of both peptides seem to be required for selective receptor occupation, but the number and the distribution of these basic residues are different and quite critical. Both receptors are presumably activated by the peptides N-terminal sequence (Xaa-Gly Gly-Phe, where Xaa = Phe or Tyr); however, although OP(4) requires Phe(4) as a determinant pharmacophore, OP(2) requires Tyr(1) as do the other opioid receptors. An extensive structure-activity analysis of the N-terminal tetrapeptide has led to conclude that the presence of aromatic residues in position one and four, preferably Phe, as well as the distance between Phe(1) and Phe(4) are extremely critical for occupation and activation of OP(4) in contrast with other opioid receptors (e.g. OP(1), OP(3), OP(2)). Modification of distance between the side chains of Phe(1) and Phe(4) (as obtained with Nphe(1) substitution in both NC and NC(1-13)-NH(2)) and/or conformational orientation of Phe(1) (as in Phe(1)psi(CH(2)-NH)-Gly(2)) has brought to discovery of pure antagonist ([Nphe(1)]-NC(1-13)-NH(2)) and a partial agonist ([Phe(1) psi(CH(2)-NH)-Gly(2)]-NC(1-13)-NH(2)), which have allowed us to characterize and classify the OP(4) receptor in several species. Thus, although antagonist activities at the OP(4) receptor are obtained by chemical modification of Phe(1)-Gly(2) peptide bond or by a shift of Phe(1) side chain of NC peptides, antagonism at the OP(2) receptor requires the diallylation of the N-terminal amino function, for instance, of dynorphin A. These considerations support the interpretation that the two systems nociceptin/OP(4) and dynorphin A/OP(2) are distinct pharmacological entities that differs in both their active sites (Tyr(1) for Dyn A and Phe(4) for NC) and the number and position of cationic residues in the C-terminal portions of the molecules. The chemical features of novel OP(4) receptor ligands either pseudopeptides obtained by combinatorial library screening or molecules of nonpeptide structure are reported and discussed in comparison with NC and NC related peptides.     

Nociceptin receptor activation inhibits tachykinergic non adrenergic non cholinergic contraction of guinea pig isolated bronchus

We studied the action of nociceptin (NC) on the atropine-resistant contractions of the guinea pig isolated bronchus evoked by the electrical field stimulation (EFS), an effect that is mediated by the activation of excitatory non adrenergic-non cholinergic (eNANC) nerves and the subsequent release of tachykinins. The functional site by which NC acts in this preparation was investigated using few different NC receptor agonists and the newly discovered NC receptor antagonist, [Phe1psi(CH2-NH)Gly2]NC(1-13)NH2 ([F/G]NC(1-13)NH2). NC inhibited in a concentration dependent manner (pEC50 7.14; Em - 87 +/- 3% of control values) EFS induced contractions. NC effect was mimicked by the NC analogues, NCNH2 and NC(1-13)NH2, but not by NC(1-9)NH2. NC (1 microM) did not affect the contractile effects of exogenously applied neurokinin A (1 microM). [F/G]NC(1-13)NH2 (10 microM) completely prevented the inhibition induced by NC (1 microM), whereas naloxone (1 microM) was found inactive. Both naloxone and ([F/G]NC(1-13)NH2 were per se inactive on basal resting tone as well as on the electrically induced contractions. The present findings show that NC inhibits the atropine-resistant EFS-induced contraction in the guinea pig bronchus by inhibiting eNANC nerves, and suggest the presence of NC receptors, distinct from opioid receptors, on the nerves of the guinea pig bronchus.     

Effects of [Nphe1]nociceptin(1-13)NH2, [Phe1(CH2-NH)Gly2]nociceptin(1- 13)NH2, and nocistatin on nociceptin inhibited constrictions of guinea pig isolated bronchus

Electric field stimulation (EFS) causes excitatory non adrenergic-non cholinergic (eNANC) and cholinergic constrictions in the guinea pig isolated bronchus, the activation of eNANC and cholinergic nerves respectively. We investigated the effects of [Nphe1]nociceptin(1-13)NH2 ([Nphe1]NC(1-13)NH2), [Phe1(CH2-NH)Gly2]nociceptin(1- 13)NH2 ([F/G] NC(1-13)NH2), and nocistatin (NST) on nociceptin (NC) inhibited constrictions in isolated bronchus of guinea pig. The results show that NC (1 micromol/L) inhibited EFS-induced eNANC and cholinergic constrictions compared with the control, in which nociceptin was not applied. After pretreatment with [Nphe1]NC(1-13)NH2, [F/G]NC(1-13)NH2, or NST, the inhibitions of NC were antagonized by [Nphe1]NC(1-13)NH2 and [F/G]NC(1-13)NH2 but not NST. However, [Nphe1]NC(1-13)NH2, [F/G]NC(1-13)NH2, and NST did not affect the inhibitions induced by morphine. Furthermore, [Nphe1]NC(1-13)NH2, [F/G]NC(1-13)NH2 and NST did not cause any appreciable effects on EFS-induced eNANC and cholinergic constrictions in guinea pig bronchi. The results demonstrate that [Nphe1]NC(1-13)NH2 and [F/G]NC(1- 13)NH2 but not NST act as selective antagonists of the NC receptor and the effects of NC on EFS-induced constrictions of guinea pig isolated bronchus.     

Synthesis and biological activities of linear and cyclic enkephalin analogues containing a psi (E,CH = CH) or psi (CH2CH2) isosteric replacement.

The peptide CO-NH function was replaced by a trans carbon-carbon double bond or by a CH2-CH2 isostere in enkephalin analogues of DADLE, DCDCE-NH2 or DPDPE. In DADLE the 2-3 and the 3-4 peptide bond was modified, whereas in the cyclic analogues the Gly3-Phe4 bond was replaced by the isosteres Gly psi (E,CH = CH)Phe [5-amino-2-(phenylmethyl)-3(E)-pentenoic acid] or Gly psi (CH2CH2)Phe [5-amino-2-(phenylmethyl)pentanoic acid]. In general, the modification results in a drop in potency which is the largest for the flexible CH2-CH2 replacement. The Gly3 psi (E,CH = CH)Phe4 DCDCE-NH2 analogue retains considerable potency. These results confirm the importance of the peptide function at the 2-3 and 3-4 position in enkephalin analogues for biological potency.     

Pharmacological characterization of the nociceptin receptor, ORL1

A novel opioid receptor-like orphan receptor (ORL1) was cloned and identified to be homologous to classical opioid receptors but insensitive to traditional opioids. A heptadecapeptide, termed orphanin FQ or nociceptin (OFQ/N), was identified as its endogenous ligand. OFQ/N shares overlapping distribution sites in pain-processing areas and common cellular mechanisms with opioids but exerts diverse effects on nociceptive responses. Of the two reported ORL1 antagonists, [Phe(1)psi(CH(2)-NH)- Gly(2)] nociceptin-(1-13)-NH(2) (Phepsi) and naloxone benzoylhydrazone (NBZ), antagonisms were validated in the activation of inward rectifying K channels induced by OFQ/N, using the patch clamp technique in ventrolateral periaqueductal gray slices. Results showed that Phepsi acted as a partial agonist and NBZ was a weak nonselective antagonist of ORL1. It is comparable with most but not all of the findings from other tissues. Comparing all the reports supports the above inference for these two antagonists. The possible causes for the discrepancy were discussed. A brief review on the putative ORL1 antagonists, acetyl-RYYRIK-NH2, some sigma-ligands and the functional antagonist, nocistatin, is also included. It indicates that a potent and selective ORL1 antagonist is expecting to elucidate the physiological role of OFQ/N.     

Nociceptin receptor activation produces nitric oxide-mediated systemic hypotension

The purpose of the present study was to investigate the effects of L-N5-(1-iminoethyl)ornithine hydrochloride (L-NIO), an inhibitor of nitric oxide (NO) formation, and [Phe1-psi(CH2NH)-Gly2]Nociceptin(1-13)-NH2 (Phe-NOC), a nociceptin receptor antagonist, on the systemic vasodepressor response to nociceptin in the anesthetized rat. The systemic vasodepressor response to bolus intravenous (i.v.) injections of nociceptin was significantly reduced by L-NIO and Phe-NOC. The present data suggest activation of nociceptin receptors dilates the systemic vascular bed through a NO-dependent pathway. These data also demonstrate Phe-NOC is an efficacious and selective nociceptin receptor antagonist in vivo.     

Involvement of sympathetic efferents but not capsaicin-sensitive afferents in nociceptin-mediated dual control of rat synovial blood flow

This study set out to examine the vasomotor effects of the opioid-like peptide nociceptin on knee joint capsular blood flow in urethane-anaesthetized rats. Topical application of nociceptin (10(-15)-10(-8) mol) caused a progressive fall in joint perfusion that was significantly inhibited by the specific nociceptin receptor antagonist [Phe(1)-(CH(2)-NH)-Gly(2)] Nociceptin(1-13)-NH(2) as well as the nonspecific opioid antagonist naloxone. To test whether this constrictor response was sympathetically mediated, we administered nociceptin in animals treated with guanethidine to produce sympathetic blockade or in the presence of the alpha-adrenoceptor antagonist phentolamine. Both guanethidine treatment and phentolamine coadministration attenuated the constrictor response to nociceptin. Inhibition of nociceptin-mediated vasoconstriction revealed a supplementary hyperemic response that persisted in animals whose knee joints were treated with 1% capsaicin to destroy the articular unmyelinated nerve supply. These results show that, in the rat knee, peripheral administration of nociceptin primarily causes a sympathetically mediated vasoconstriction. In addition, high-dose nociceptin produces a vasodilatatory response that is likely due to the direct action of nociceptin on vascular smooth muscle and not by a neurogenic mechanism.     

Nphe1]nociceptin(1-13)-NH2 antagonizes nociceptin-induced hypotension, bradycardia, and hindquarters vasodilation in the anesthetized rat

The purpose of this study was to investigate the effects of [Nphe1]nociceptin(1-13)-NH2 on nociceptin-induced decreases in mean arterial pressure (MAP), heart rate (HR), and hindquarters vascular bed resistance (HVBR) of the anesthetized rat. The results showed that i.c.v. or i.v. [Nphe1]nociceptin(1-13)-NH2 (1.5-12 nmol/kg and 5-120 nmol/kg, respectively) could antagonize the depressor effects of i.c.v. or i.v. nociceptin (3 and 30 nmol/kg, respectively) on MAP and HR. Furthermore, [Nphe1]nociceptin(1-13)-NH2 (5-120 nmol/kg) could reverse nociceptin (30 nmol/kg)-induced decrease of HVBR. However, [Nphe1]nociceptin(1-13)-NH2 had no significant effects on similar effects induced by morphine. Our results suggest that [Nphe1]nociceptin(1-13)-NH2 acts as a selective antagonist of the nociceptin receptor in the cardiovascular system of the rat.     

Nociceptin/orphanin FQ increases ANP secretion in neonatal cardiac myocytes

Nociceptin (N/OFQ) is a novel heptadecapeptide with an amino acid sequence similar to that of endogenous opioid peptide dynorphin A. Dynorphin have been reported to increase the secretion of atrial natriuretic peptide (ANP) via selective activation of kappa-opioid receptor in cultured atrial cardiocytes. The present study was designed to investigate the direct effect of N/OFQ on the ANP secretion in cultured neonatal rat cardiac myocytes via N/OFQ receptor (NOP) activation. The secretion of ANP from cultured neonatal cardiac myocytes was increased in terms of incubation time. N/OFQ, at a dose of 0.3, 1, 3, and 10 microM, caused increases in ANP secretion in a dose-dependent manner. The N/OFQ-induced ANP secretion was completely antagonized by antagonists of NOP, 1 microM each of [Phe1 (CH2-NH) Gly2] nociceptin (1-13)-NH2 ([FG]N/OFQ(1-13)NH2) or naloxone benzoylhydrazone. In contrast, naloxone (1 microM), the non-selective opioid receptor antagonist, did not alter ANP response to N/OFQ. N/OFQ at 3 microM inhibited basal and forskolin-stimulated cAMP production, which was partially antagonized with the pretreatment of [FG]N/OFQ(1-13)NH2. An increase in ANP secretion by N/OFQ was also partially blocked by the pretreatment of forskolin. Homologous competition studies in neonatal cardiomyocyte membranes revealed the presence of two distinct sites. The high affinity site (10.9 +/- 1.6 nM) was far less abundant than the low affinity site. Therefore, these results suggest that N/OFQ causes an increase in ANP secretion in cultured neonatal cardiac myocytes by decreasing cAMP through its binding sites.     

The nociceptin/orphanin FQ receptor ligand acetyl-RYYRIK-amide exhibits antagonistic and agonistic properties

The hexapeptide acetyl-RYYRIK-amide (Ac-RYYRIK-NH(2)) has recently been reported to act as partial agonist of the nociceptin/orphanin FQ (noc/OFQ) receptor expressed in CHO cells. In addition, this peptide acts as a competitive antagonist of noc/OFQ-stimulated GTPgamma(35)S binding in rat brain membranes as well as of the noc/OFQ-evoked chronotropic effect in rat cardiomyocytes. In contrast to this antagonism, in the present study, Ac-RYYRIK-NH(2) was found to behave as an agonist at noc/OFQ receptors, affecting spontaneous locomotor activity. When administered intracerebroventricularly (i.c.v.), noc/OFQ and Ac-RYYRIK-NH(2) inhibited spontaneous locomotor activity in mice with ID(50) of 1.1 and 0.07 nmol, respectively. Co-administration of both peptides lead to additive effects. The higher potency of Ac-RYYRIK-NH(2) could not be clearly explained by differential metabolism, because in vivo microdialysis in rat striatum and in vitro metabolic inactivation by rat and mouse brain membranes revealed extensive inactivation of both peptides. Similar to Ac-RYYRIK-NH(2), [Phe(1)psi(CH(2)-NH)Gly(2)]noc/OFQ(1-13)-NH(2) ([F/G]NC(1-13)NH(2)) inhibited the noc/OFQ-stimulated GTPgamma(35)S binding in rat brain membranes (Schild constant 3.83 nM) and mouse brain sections, although several reports have shown that this peptide exhibits agonist activity of noc/OFQ in the CNS. Changes in the optimum conditions of the in vitro assay for GTP binding increased low partial agonism of Ac-RYYRIK-NH(2) in GTP binding response. To explain the discrepancy between the in vitro antagonism of G protein coupling of the noc/OFQ receptor and in vivo agonism of Ac-RYYRIK-NH(2) and of [F/G]NC(1-13)NH(2), it is suggested that low partial agonism of receptor/G protein coupling in native systems may be sufficient to evoke full biologic responses. The extent of partial agonism for GTP binding and of coupling reserve may vary in different systems, thus explaining why [F/G]NC(1-13)NH(2) and Ac-RYYRIK-NH(2) were reported to exhibit antagonist, partial agonist, or even full agonist properties, depending on the system studied.     

Synthesis and binding characteristics of [3H] H-Tyr-Ticpsi[CH2-NH] Cha-Phe-OH, a highly specific and stable delta-opioid antagonist.

Substitution of the Phe3 aromatic ring in H-Tyr-Ticpsi[CH2-NH]Phe-Phe-OH with cyclohexylalanine (Cha) has been reported to result in a compound, H-Tyr-Ticpsi[CH2-NH]Cha-Phe-OH (TICP[psi]), showing substantially increased delta-opioid antagonist potency and high delta selectivity. TICP[psi] was radiolabeled by catalytic tritiation of its precursor Tyr(3',5'-I2)1TICP[psi]. Binding characteristics of the new tritiated pseudopeptide were determined using the radioligand binding assay in rat brain membranes. On the basis of the results of saturation binding studies performed at 25 degrees C, an equilibrium dissociation constant (Kd) of 0.35 nM and a receptor density (Bmax) of 112 fmol/mg protein were calculated. This new tritiated ligand exhibits high affinity for delta-opioid receptors, whereas its binding to mu and kappa receptors is weak. A study of [H3]TICP[psi] binding displacement by various receptor-selective opioids showed the following rank order of potency: delta > kappa = mu. These receptor binding characteristics of the ligand, together with its high specific radioactivity (41.3 Ci/mmol) and stability, makes it a useful tool for labeling delta-opioid receptors, both in vitro and in vivo.     

Biological effects and receptor binding affinities of new pseudononapeptide bombesin/GRP receptor antagonists with N-terminal D-Trp or D-Tpi.

In an attempt to produce more powerful (effective) bombesin/GRP receptor antagonists, the D forms of Trp or Trp analog (Tpi) were introduced at position 6 in two pseudononapeptides, Leu13 psi (CH2NH)Leu14-bombesin(6-14) and Leu13 psi(CH2NH)Phe14-bombesin (6-14). These antagonists were tested for their ability to inhibit basal and gastrin releasing peptide (GRP) (14-27)-induced amylase release from rat pancreatic acini in a superfusion assay. They were also assessed for the inhibition of 125I-Tyr4-bombesin binding to Swiss 3T3 and small cell lung carcinoma cell line H-345 and the mitogenic response of Swiss 3T3 cells induced by GRP(14-27). The peptides, when given alone, did not stimulate amylase secretion, but were able to inhibit gastrin releasing peptide (14-27)-induced amylase release. All of the antagonists showed strong binding affinities for Swiss 3T3 and H-345 cells and suppressed the GRP(14-27)-induced increase of [3H]thymidine incorporation into DNA of Swiss 3T3 cells at nanomolar concentrations. Antagonist D-Tpi6,Leu13 psi (CH2NH)Leu14-bombesin (6-14)(RC-3095) was slightly more potent in these assays than D-Trp6,Leu13 psi (CH2NH)Leu14-bombesin (6-14)(RC-3125). Nevertheless, D-Trp6,Leu13 psi (CH2NH)Phe14-bombesin (6-14) showed the highest binding affinity for Swiss 3T3 and H345 cells and it was the most potent inhibitor of GRP(14-27)-induced amylase secretion. This antagonist RC-3420 was particularly effective in inhibiting the growth of Swiss 3T3 cells, exhibiting an IC50 value less than 1 nM.(ABSTRACT TRUNCATED AT 250 WORDS)     

NOC/oFQ PKC-dependent superoxide generation contributes to hypoxic-ischemic impairment of NMDA cerebrovasodilation

This study determined whether nociceptin/orphanin FQ (NOC/oFQ) generates superoxide anion (O(2)(-)) in a protein kinase C (PKC)-dependent manner and whether such production contributes to hypoxic-ischemic (H-I) impairment of N-methyl-D-aspartate (NMDA)-induced pial artery dilation in newborn pigs equipped with closed cranial windows. Superoxide dismutase (SOD)-inhibitable nitroblue tetrazolium (NBT) reduction was an index of O(2)(-) generation. Under non-H-I conditions, topical NOC/oFQ (10(-10) M, concentration present in cerebrospinal fluid after I or H-I) increased SOD-inhibitable NBT reduction from 1 +/- 1 to 20 +/- 3 pmol/mm(2). PKC inhibitors staurosporine and chelerythrine (10(-7) M) blunted NBT reduction (1 +/- 1 to 7 +/- 2 pmol/mm(2) for chelerythrine), whereas the NOC/oFQ receptor antagonist [F/G]NOC/oFQ (1-13)-NH(2) (10(-6) M) blocked NBT reduction. [F/G]NOC/oFQ(1-13)-NH(2) and staurosporine also blunted the NBT reduction observed after I or H-I. NMDA (10(-8), 10(-6) M)-induced pial artery dilation was reversed to vasoconstriction after H-I. The NOC/oFQ antagonist staurosporine and free radical scavengers partially prevented this impaired dilation (sham: 9 +/- 1 and 16 +/- 1; H-I: -5 and -10 +/- 1; H-I staurosporine pretreated: 3 +/- 1 and 6 +/- 1%). These data show that NOC/oFQ increased O(2)(-) production in a PKC-dependent manner and contributed to this production after insult and that NOC/oFQ contributed to impaired NMDA-induced pial artery dilation after H-I, suggesting, therefore, that PKC-dependent O(2)(-) generation by NOC/oFQ links NOC/oFQ release to impaired NMDA dilation after H-I.     

Structural modelling and mutation analysis of a nociceptin receptor and its ligand complexes

We recently modelled and proposed four ligand-bound conformations for a G-protein-coupled receptor, namely, forms I, II, III and IV, based on the 3D structure and functional evidences for rhodopsin. In this study, the same strategy was applied to a human nociceptin receptor (NR), in order to predict ligand-bound receptor structures. Additionally, site-directed mutagenesis studies were carried out to evaluate these structures. A Thr138Ala mutant demonstrated the same affinity for [Phe(1)Psi(CH(2)-NH)Gly(2)]nociceptin(1-13)NH(2) as the wild-type receptor; however, the affinity of this mutant for nociceptin was 20-fold lower than that of the wild type. A Ser223Ala mutation showed the same characteristics as those of the wild type. On the other hand, a Gln280Ala mutation reduced the affinity to nociceptin by more than 60-folds. These results suggested that a change in the conformation of NR following agonist binding did not accompany the rigid-body rotation of the sixth transmembrane segment that was reported for an adrenergic receptor and a kappa-opioid receptor. NR is potently activated not only by nociceptin but also a synthetic peptide, i.e. Ac-RYYRIK-NH(2), although the amino acid sequences of both these ligands are completely different. The model explains why both the ligands activate NR and shows that their receptor-bound conformations have similar 3D structures.     

Novel nociceptin analogues

A series of new nociceptin analogues containing cysteine was tested for their nociceptive effects in tail-flick test on mice after icv injection. The cysteines were introduced in order to get irreversibly binding analogues based on the assumption that the cysteines in the ligand can interact with the cysteines from the receptor to form an S-S bridge. In vivo tests revealed that Cys1-nociceptin (1-13)-NH2 (Cys1-NC) is an antagonist, whereas Cys7-NC is an agonist. Gly1[Phe(p-NO2)]4-NC was less active indicating that the antagonist properties of Cys1-NC are associated with the presence of the sulfhydryl group of cysteine. The analogues D-Cys2 and Cys3 were also almost inactive.     

An active pseudopeptide analog of the leucokinin insect neuropeptide family.

A pseudopeptide analog of the active core of the leucokinin insect neuropeptide family was synthesized and found to retain myotropic activity. No reports of active pseudopeptide analogs of an insect or other invertebrate neuropeptide have previously appeared in the literature. The pseudopeptide (Phe psi [CH2-NH] Phe-Ser-Trp-Gly-NH2) contains a reduced-amide linkage between the two N-terminal Phe residues. Unlike its amide-bond containing counterpart, the activity of the pseudopeptide was not destroyed upon exposure to aminopeptidase M.     

Structure-activity relationship of [Nphe1]-NC-(1-13)-NH2, a pure and selective nociceptin/orphanin FQ receptor antagonist.

A series of analogs of the ORL1 receptor antagonist [Nphe1]-NC(1-13)-NH2 was prepared and tested for agonistic and antagonistic activities in the mouse vas deferens, a preparation that shows high sensitivity to nociceptin and related peptides. The purpose of this study was to determine the role of the aromatic residue at the N-terminal for antagonism and eventually identify compounds with improved potency. Results indicated that all 23 compounds are inactive as agonists, and the antagonistic potency of the initial template [Nphe1]-NC(1-13)-NH2 is high (pKB 6.43) compared with those of all other compounds except [(S)(betaMe)Nphe1]NC(1-13)-NH2 (pK(B) 6.48). The other 22 compounds can be divided into two groups: 10 show antagonistic potencies (pK(B)) ranging from 5.30 to 5.86, whereas the other 12 compounds are inactive. This study clearly shows that the aromatic ring of Nphe is very critical for the interaction with the ORL1 receptor and can not be enlarged or sterically modified without significant loss of antagonistic potency.