N/OFQ Receptor Research Articles

Stimulation of δ opioid receptor and blockade of nociceptin/orphanin FQ receptor synergistically attenuate parkinsonism.

δ opioid peptide (DOP) receptors are considered a therapeutic target in Parkinson's disease, although the use of DOP agonists may be limited by side effects, including convulsions. To circumvent this issue, we evaluated whether blockade of nociceptin/orphanin FQ (N/OFQ) tone potentiated the antiparkinsonian effects of DOP agonists, thus allowing for reduction of their dosage. Systemic administration of the N/OFQ receptor (NOP) antagonist J-113397 [(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H benzimidazol-2-one] and the DOP receptor agonist SNC-80 [(+)-4-[(αR)-α-(2S,5R)-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxy-benzyl]-N-N-diethylbenzamide] revealed synergistic attenuation of motor deficits in 6-hydroxydopamine hemilesioned rats and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mice. In this model, repeated administration of the combination produced reproducible antiparkinsonian effects and was not associated with rescued striatal dopamine terminals. Microdialysis studies revealed that either systemic administration or local intranigral perfusion of J-113397 and SNC-80 led to the enhancement of nigral GABA, reduction of nigral Glu, and reduction of thalamic GABA levels, consistent with the view that NOP receptor blockade and DOP receptor stimulation caused synergistic overinhibition of nigro-thalamic GABA neurons. Whole-cell recording of GABA neurons in nigral slices confirmed that NOP receptor blockade enhanced the DOP receptor-induced effect on IPSCs via presynaptic mechanisms. Finally, SNC-80 more potently stimulated stepping activity in mice lacking the NOP receptor than wild-type controls, confirming the in vivo occurrence of an NOP-DOP receptor interaction. We conclude that endogenous N/OFQ functionally opposes DOP transmission in substantia nigra reticulata and that NOP receptor antagonists might be used in combination with DOP receptor agonists to reduce their dosage while maintaining their full therapeutic efficacy.

Molecular Mechanisms Mediating Nociceptin/Orphanin FQ Receptor Signaling, Desensitization and Internalization

In 1994, the isolation of an opioid receptor-related clone soon led to the isolation and characterization of a novel neuropeptide, termed nociceptin or orphanin FQ (N/OFQ). This heptadecapeptide binds to the N/OFQ receptor (NOP) with high affinity, but does not interact directly with classical opioid receptors. The regional distribution of N/OFQ and of its receptor suggest any possible involvement of this neurotransmission system in motor and balance control, reinforcement and reward, nociception, stress response, sexual behavior, aggression and autonomic control of physiological processes as well as of immune functions. The actions of N/OFQ may also be uniquely dependent on contextual factors, both genetic and environmental. As for most of the G protein coupled receptors, NOP C-terminal sequences are believed to interact with proteins that are mandatory for anchoring receptor at the plasma membrane, internalization, recycling, or degradation after ligand binding. Increasing details of how NOP receptors are activated and removed from the plasma membrane have been elucidated in vitro, and more importantly in a physiological context. Details of how these receptors travel and recycle following internalization have also shed light on the importance of such mechanisms for any potential therapeutic use of NOP ligands. A picture of the pathways and proteins involved in these processes is beginning to emerge. This review will address molecular events contributing to NOP receptor signaling and trafficking.

Role of nociceptin/orphanin FQ and NOP receptors in the response to acute and repeated restraint stress in rats.

Central nociceptin/orphanin FQ (N/OFQ)-expressing neurones are abundantly expressed in the hypothalamus and limbic system and are implicated in the regulation of activity of the hypothalamic-pituitary-adrenal axis (HPA) and stress responses. We investigated the role of the endogenous N/OFQ receptor (NOP) system using the nonpeptidic NOP antagonist, JTC-801 [N-(4-amino-2-methylquinolin-6-yl)-2-(4-ethylphenoxy-methyl)benzamide monohydrochloride], during the HPA axis response to acute physical/psychological stress (60 min of restraint). Although i.v. JTC-801 (0.05 mg/kg in 100 μl) had no significant effect on restraint-induced plasma corticosterone release at 30 or 60 min post-injection, i.v. JTC-801 (0.05 mg/kg in 100 μl) in quiescent rats significantly increased basal plasma corticosterone at the 30-min time-point compared to i.v. vehicle (1% dimethysulphoxide in sterile saline). Central injection of JTC-801 i.c.v. was associated with increased Fos expression in the parvocellular paraventricular nucleus 90 min after infusion compared to vehicle control. These findings contrast to the effects of i.c.v. UFP-101, a NOP antagonist that we have previously shown to have no effect on HPA activity in quiescent rats. To determine whether restraint stress was associated with compensatory changes in N/OFQ precursor (ppN/OFQ) or NOP receptor mRNAs, in a separate study, we undertook reverse transcriptase-polymerase chain reaction and in situ hybridisation analysis of ppN/OFQ and NOP transcripts in the brains of male Sprague–Dawley rats. In support of an endogenous role for central N/OFQ in psychological stress, we found that acute restraint significantly decreased preproN/OFQ transcript expression in the hippocampus 2 h after stress compared to unstressed controls. PpN/OFQ mRNA was also reduced in the mediodorsal forebrain 4 h after stress. NOP mRNA was reduced in the hypothalamus 2 h after restraint and at 4 h in mediodorsal forebrain and hippocampus. In situ hybridisation analysis showed that acute restraint significantly decreased ppNN/OFQ in the central amygdala, with significantly increased expression in bed nucleus and reticular thalamus associated with repeated restraint. There was a strong trend for reduced NOP mRNA in the bed nucleus of acute and repeated restraint groups, although there were no other significant changes seen. Although the exact mechanisms require elucidation, the findings obtained in the present study provide evidence indicating that the endogenous N/OFQ system is involved in both acute and chronic restraint stress responses. In summary, our findings confirm the significant role of endogenous NOP receptors and tonic N/OFQ function in the response to the psychological stress of restraint.

Nociceptin/orphanin FQ inhibition with SB612111 ameliorates dextran sodium sulfate-induced colitis

Inflammatory bowel diseases, primarily Crohn's disease and ulcerative colitis, are chronic inflammatory disorders of the gastrointestinal tract with unknown etiology. The majority of current therapeutic agents focus on controlling proinflammatory molecules. The neuropeptide nociceptin/orphanin FQ (N/OFQ) has been described as a potential immunomodulator for inflammatory bowel diseases. In this study, we asked whether the small molecule N/OFQ antagonist (−)-cis-1-methyl-7-[[4-(2,6-dichlorophenyl)piperidin-1-yl]methyl]-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ol (SB612111) would inhibit the development of dextran sodium sulfate-induced colitis in C57BL/6 mice. Inhibition of the N/OFQ receptor (NOP) by SB612111 significantly ameliorated the clinical disease course in these animals, as indicated by reduced fecal bleeding, improved recovery from diarrhea and weight loss, and a reduction in histopathological alterations. In addition, the inflammatory response in the colon was diminished, as demonstrated by reduced cytokine protein and messenger RNA expression for CXCL1/keratinocyte-derived chemokine, interferon-γ, interleukin-1β, interleukin-6, and tumor necrosis factor-α, some of which are known targets for the treatment of this devastating disease. Our results strongly support a role for the receptor–ligand pair NOP-N/OFQ in the pathogenesis of colitis. We conclude that inhibition of NOP receptors with small molecule inhibitors may constitute a novel, urgently needed approach for the treatment of inflammatory bowel diseases.

P.2.018 Behavioural and cognitive alterations of the young megencephaly (BALB/cByJ-Kv1.1 mceph/mceph) mouse

Two novel ligands for the nociceptin/orphanin FQ (N/OFQ) receptor (NOP), [(pF)Phe4,Aib7, Aib11,Arg14,Lys15]N/OFQ-NH2 (peptide-1) and [Nphe1,(pF)Phe4,Aib7,Aib11,Arg14,Lys15]N/OFQ-NH2 (peptide-2), have been generated by combining different modifications of N/OFQ sequence. In the present study, we investigated the actions of two analogues and compared them with those of N/OFQ in four assays. Peptide-1 mimicked N/OFQ effects in mouse vas deferens and mouse colon and showed similar maximal effects but higher potency relative to N/OFQ. The effects of peptide-1 were sensitive to NOP receptor selective antagonist ([Nphe1]N/OFQ(1-13)-NH2) but not to naloxone in vitro. Peptide-1 (25 pmol, i.c.v.) mimicked the pronociceptive action of N/OFQ (2.5 nmol, i.c.v.) in mouse tail withdrawal assay, displaying higher potency and longer lasting effects. In anesthetized rats, peptide-1 (1 nmol/kg, i.v.) produced a marked decrease in mean arterial pressure, which was comparable to that evoked by i.v. N/OFQ (100 nmol/kg). Peptide-2 did not produce any effect per se but antagonized N/OFQ actions in mouse vas deferens and mouse colon assays. Peptide-2 is active in vivo where it prevented the pronociceptive effect induced by 2.5 nmol N/OFQ i.c.v. in the mouse tail withdrawal assay. Furthermore, peptide-2 at 5 nmol produced alone a robust and long lasting antinociceptive effect. Moreover, peptide-2 (10 and 40 nmol/kg i.v.) didn't produce any effect per se but antagonized hypotensive actions produced by i.v. administration of N/OFQ. Collectively, these findings demonstrate that [(pF)Phe4,Aib7,Aib11, Arg14,Lys15]N/OFQ-NH2 behaves as a highly potent NOP receptor agonist which produces long lasting effects in vivo and [Nphe1,(pF)Phe4,Aib7,Aib11,Arg14,Lys15]N/OFQ-NH2 acts as a pure and competitive antagonist of the NOP receptor.

Synthesis and biological activity of novel small peptides with aminophosphonates moiety as NOP receptor ligands

The aim of the present study was the synthesis and the biological screening of new analogs of Ac-RYYRWK-NH2, modified at the N-terminal with 1-[(methoxyphosphono)methylamino]cycloalkanecarboxylic acids. The four newly synthesized ligands for the nociceptin/orphanin FQ (N/OFQ) receptor (NOP) have been prepared by solid-phase peptide synthesis--Fmoc-strategy. These compounds were tested for agonistic activity in vitro on electrically stimulated smooth-muscle preparations isolated from vas deferens of Wistar rats. Our data showed that substitution of Arg at position 1 with aminophosphonates moiety decreased significantly the affinity of ligands to the NOP receptor. Furthermore, the enlargement of the cycle (with 5-8 carbon atoms) additionally diminished both the activity and the selectivity for NOP-receptor.

P.1.c.047 Hippocampal nociceptin receptors and memory: possible implications for "anti-alcohol" effects of nociceptin ligands

The opioid system comprises four receptor subtypes: μ (MOP), κ (KOP), δ (DOP), now called the ‘classical’ opioid receptors, and the ‘non-classical’ nociceptin/orphanin FQ peptide (N/OFQ) receptor (NOP). Selective endogenous peptides, cleaved from larger precursor proteins, have been identified for all subtypes. Both classical and non-classical opioid receptors couple to inhibitory, pertussis toxin-sensitive G-proteins. Opioid receptors activate the same major intracellular pathways, which include: closing of voltage-sensitive calcium channels; opening of potassium channels and subsequent cellular hyperpolarization; and inhibition of cyclic AMP production through inhibition of the enzyme adenylate cyclase. All current, clinically used opioids work through activation of the MOP receptor. In an experimental setting, co-administration of MOP and DOP agonists has been shown to have a synergistic analgesic action. Administration of DOP-receptor antagonists has also been shown to reduce tolerance, physical dependence and other side effects of MOP-receptor agonists, without detriment to their analgesic action. In animal models NOP agonists are analgesic when administered spinally and have a pronociceptive/anti-analgesic (or anti-opioid) effect supraspinally. NOP knockout mice show a partial loss of tolerance to morphine and there is an up-regulation of N/OFQ production in chronic morphine tolerant mice. Analgesic tolerance that develops from repeated exposure to morphine is markedly attenuated in NOP knockout mice. The development of ligands with mixed action at MOP, DOP and NOP receptors offer new opportunities for opioid pharmacology.

Structure–activity studies on the nociceptin/orphanin FQ receptor antagonist 1-benzyl- N-{3-[spiroisobenzofuran-1(3 H),4′-piperidin-1-yl]propyl} pyrrolidine-2-carboxamide

Twelve derivatives of the nociceptin/orphanin FQ (N/OFQ) receptor (NOP) antagonist 1-benzyl- N-{3-[spiroisobenzofuran-1(3 H),4′-piperidin-1-yl]propyl} pyrrolidine-2-carboxamide (Comp 24) were synthesized and tested in binding experiments performed on CHO hNOP cell membranes. Among them, a novel interesting NOP receptor antagonist (compound 35) was identified by blending chemical moieties taken from different NOP receptor ligands. In vitro in various assays, Compound 35 consistently behaved as a pure, highly potent (pA 2 in the range 8.0–9.9), competitive and NOP selective antagonist. However compound 35 was found inactive when challenged against N/OFQ in vivo in the mouse tail withdrawal assay. Thus, the usefulness of the novel NOP ligand compound 35 is limited to in vitro investigations.

Nociceptin receptor antagonist JTC-801 inhibits nitrous oxide-induced analgesia in mice

The mechanism of the analgesic effect of nitrous oxide (N(2)O) has not been completely clarified. Although we have reported that the analgesic effect of N(2)O was significantly decreased in nociceptin-orphanin FQ (N/OFQ) receptor (NOP)-deficient mice, the effect of nociceptin receptor antagonists on N(2)O-induced analgesia has not been reported. In this investigation, we examined the effect of the NOP antagonist JTC-801 on N(2)O-induced analgesia in 129Sv mice by the writhing test and tail flick test, and demonstrated that the analgesic effect of N(2)O was suppressed by the intraperitoneal administration of JTC-801.

The nociceptin/orphanin FQ receptor: a target with broad therapeutic potential

Identification of the enigmatic nociceptin/orphanin FQ peptide (N/OFQ) in 1995 represented the first successful use of reverse pharmacology and led to deorphanization of the N/OFQ receptor (NOP). Subsequently, the N/OFQ-NOP system has been implicated in a wide range of biological functions, including pain, drug abuse, cardiovascular control and immunity. Although this could be considered a hurdle for the development of pharmaceuticals selective for a specific disease indication, NOP represents a viable drug target. This article describes potential clinical indications and highlights the current status of the very limited number of clinical trials.

Dysregulation of Nociceptin/Orphanin FQ Activity in the Amygdala Is Linked to Excessive Alcohol Drinking in the Rat

Alcoholism is a complex behavioral disorder in which interactions between stressful life events and heritable susceptibility factors contribute to the initiation and progression of disease. Neural substrates of these interactions remain largely unknown. Here, we examined the role of the nociceptin/orphanin FQ (N/OFQ) system, with an animal model in which genetic selection for high alcohol preference has led to co-segregation of elevated behavioral sensitivity to stress (Marchigian Sardinian alcohol-preferring [msP]). The msP and Wistar rats trained to self-administer alcohol received central injections of N/OFQ. In situ hybridization and receptor binding assays were also performed to evaluate N/OFQ receptor (NOP) function in naïve msP and Wistar rats. Intracerebroventricular (ICV) injection of N/OFQ significantly inhibited alcohol self-administration in msP but not in nonselected Wistar rats. The NOP receptor messenger RNA expression and binding was upregulated across most brain regions in msP compared with Wistar rats. However, in msP rats [(35)S]GTPgammaS binding revealed a selective impairment of NOP receptor signaling in the central amygdala (CeA). Ethanol self-administration in msP rats was suppressed after N/OFQ microinjection into the CeA but not into the bed nucleus of the stria terminalis or the basolateral amygdala. These findings indicate that dysregulation of N/OFQ-NOP receptor signaling in the CeA contributes to excessive alcohol intake in msP rats and that this phenotype can be rescued by local administration of pharmacological doses of exogenous N/OFQ. Data are interpreted on the basis of the anti-corticotropin releasing factor (CRF) actions of N/OFQ and the significance of the CRF system in promoting excessive alcohol drinking in msP rats.

Nociceptin/orphanin FQ receptor blockade attenuates MPTP-induced parkinsonism

Endogenous nociceptin/orphanin FQ (N/OFQ) inhibits the activity of dopamine neurons in the substantia nigra and affects motor behavior. In this study we investigated whether a N/OFQ receptor (NOP) antagonist, J-113397, can modify movement in naive mice and nonhuman primates and attenuate motor deficits in MPTP-treated parkinsonian animals. J-113397 facilitated motor activity in naïve mice at low doses (0.1–1 mg/kg) and inhibited it at higher ones (10 mg/kg). Likewise, in MPTP-treated mice, J-113397 reversed motor deficit at 0.01 mg/kg but worsened hypokinesia at higher doses (1 mg/kg). In naïve nonhuman primates, J-113397, ineffective up to 1 mg/kg, produced inconsistent motor improvements at 3 mg/kg. Conversely, in parkinsonian primates J-113397 (0.01 mg/kg) reversed parkinsonism, being most effective against hypokinesia. We conclude that endogenous N/OFQ modulates motor activity in mice and nonhuman primates and contributes to parkinsonian symptoms in MPTP-treated animals. NOP receptor antagonists may represent a novel approach to Parkinson's disease.

Human Peripheral Blood Mononuclear Cells Produce Pre-Pro-Nociceptin/Orphanin FQ mRNA

Peripheral blood mononuclear cells (PBMC) transcribe mRNA for the nonclassical opioid nociceptin/orphanin FQ (N/OFQ) receptor (NOP). We probed for the N/OFQ precursor, pre-pro-N/OFQ (ppN/OFQ). Using PBMC from 10 healthy volunteers we probed for ppN/OFQ using polymerase chain reaction (PCR) based experimental paradigms. In gel-based PCR, we detected amplicons consistent with ppN/OFQ mRNA in all samples. This was confirmed in quantitative real-time PCR with cycle thresholds (representing quantity of mRNA) of 30.91 +/- 0.18 (n = 10). These data indicate that PBMCs transcribe ppN/OFQ which, coupled with NOP expression, suggest NOP may be involved in the autoregulation of PBMCs.

Kainic Acid Down-regulates NOP Receptor Density and Gene Expression in Human Neuroblastoma SH-SY5Y Cells

Nociceptin (N/OFQ) is involved in neuronal excitability and in certain types of seizures. Kainate-induced seizures are associated with increased N/OFQ release in the rat thalamus and hippocampus, causing down-regulation of the N/OFQ receptor (NOP). In this study, we used the neuroblastoma SH-SY5Y cell line as a model to investigate the effects of kainate on NOP receptor density and gene expression. Exposure to kainate (10-50 microM) for 3 h did not affect NOP receptor density. In contrast, a NOP Bmax down-regulation was detected in cells exposed to 10 microM kainate for both 6 and 24 h. Moreover, our data show that kainate causes a decrease in NOP mRNA levels after 3, 6, and 24 h, an effect blocked by the AMPA/kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). These findings show that kainate is able to affect the NOP system, both at biosynthesis and receptor density levels in SH-SY5Y cells, and that the kainate ionotropic receptor can contribute to the regulation of the NOP receptor. These data are in agreement with data obtained in vivo and provide new evidence concerning the existence of a cross-talk between NOP and kainate receptors, leading to an interplay between glutamate and N/OFQ circuits.

In vitro pharmacological characterisation of a novel cyclic nociceptin/orphanin FQ analogue c[Cys7,10]N/OFQ(1–13)NH2

Nociceptin/orphanin FQ (N/OFQ) is the endogenous 17 amino acid peptide ligand for the G(i)-protein-coupled N/OFQ receptor (NOP). In an attempt to improve the metabolic stability of N/OFQ, we have produced a truncated cyclic analogue with cysteine residues at positions 7 and 10, c[Cys(7,10)]N/OFQ(1-13)NH(2) (c[Cys(7,10)]). c[Cys(7,10)], the template N/OFQ(1-13)NH(2) and N/OFQ displaced the binding of [(3)H]N/OFQ to Chinese hamster ovary cells expressing recombinant human NOP (CHO(hNOP)) with pK ( i ) values of 9.98, 9.83 and 9.18, respectively. In addition, c[Cys(7,10)], N/OFQ(1-13)NH(2) and N/OFQ stimulated the binding of guanosine triphosphate gamma [(35)S] to CHO(hNOP) cells with pEC(50)/E (max) (stimulation factor) of 9.16/5.5, 9.11/4.9 and 8.35/5.5, respectively. c[Cys(7,10)], N/OFQ(1-13)NH(2) and N/OFQ inhibited forskolin-stimulated cyclic adenosine monophosphate (cAMP) formation with pEC(50) values of 10.08, 10.11 and 9.78, respectively. All ligands produced complete inhibition of cAMP formation. In both functional assays, c[Cys(7,10)] was a full agonist. In a series of metabolism experiments, incubation of 1 nM c[Cys(7,10)], N/OFQ(1-13)NH(2) and N/OFQ with a rat brain homogenate produced a time-dependent loss of peptide that was greatest for the native peptide N/OFQ. Amidation in N/OFQ(1-13)NH(2) produced some metabolic protection, but this was not significantly improved by further inclusion of c[Cys(7,10)]. In summary, c[Cys(7,10)] is a high-affinity, high-potency full agonist of the NOP receptor. However, we were unable to demonstrate clear metabolic protection.

Novel potent agonist [(pF)Phe 4,Aib 7,Aib 11,Arg 14,Lys 15]N/OFQ-NH 2 and antagonist [Nphe 1,(pF)Phe 4,Aib 7,Aib 11,Arg 14,Lys 15]N/OFQ-NH 2 of nociceptin/orphanin FQ receptor

Two novel ligands for the nociceptin/orphanin FQ (N/OFQ) receptor (NOP), [(pF)Phe 4,Aib 7, Aib 11,Arg 14,Lys 15]N/OFQ-NH 2 (peptide-1) and [Nphe 1,(pF)Phe 4,Aib 7,Aib 11,Arg 14,Lys 15]N/OFQ-NH 2 (peptide-2), have been generated by combining different modifications of N/OFQ sequence. In the present study, we investigated the actions of two analogues and compared them with those of N/OFQ in four assays. Peptide-1 mimicked N/OFQ effects in mouse vas deferens and mouse colon and showed similar maximal effects but higher potency relative to N/OFQ. The effects of peptide-1 were sensitive to NOP receptor selective antagonist ([Nphe 1]N/OFQ(1-13)-NH 2) but not to naloxone in vitro. Peptide-1 (25 pmol, i.c.v.) mimicked the pronociceptive action of N/OFQ (2.5 nmol, i.c.v.) in mouse tail withdrawal assay, displaying higher potency and longer lasting effects. In anesthetized rats, peptide-1 (1 nmol/kg, i.v.) produced a marked decrease in mean arterial pressure, which was comparable to that evoked by i.v. N/OFQ (100 nmol/kg). Peptide-2 did not produce any effect per se but antagonized N/OFQ actions in mouse vas deferens and mouse colon assays. Peptide-2 is active in vivo where it prevented the pronociceptive effect induced by 2.5 nmol N/OFQ i.c.v. in the mouse tail withdrawal assay. Furthermore, peptide-2 at 5 nmol produced alone a robust and long lasting antinociceptive effect. Moreover, peptide-2 (10 and 40 nmol/kg i.v.) didn't produce any effect per se but antagonized hypotensive actions produced by i.v. administration of N/OFQ. Collectively, these findings demonstrate that [(pF)Phe 4,Aib 7,Aib 11, Arg 14,Lys 15]N/OFQ-NH 2 behaves as a highly potent NOP receptor agonist which produces long lasting effects in vivo and [Nphe 1,(pF)Phe 4,Aib 7,Aib 11,Arg 14,Lys 15]N/OFQ-NH 2 acts as a pure and competitive antagonist of the NOP receptor.

The opioid peptide nociceptin/orphanin FQ mediates prostaglandin E2‐induced allodynia, tactile pain associated with nerve injury

Pain often outlasts its usefulness as warning and aid in wound healing, and becomes chronic and intractable after tissue damage and nerve injury. Many molecules have been implicated as mediators and modulators in persistent pain such as hyperalgesia and tactile pain (allodynia). We previously showed that prostaglandin (PG) E(2), PGF(2alpha) or the neuropeptide nociceptin, also called orphanin FQ (N/OFQ) administered intrathecally (i.t.) produced allodynia in conscious mice. In the present study, we examined the relationship of pain responses between PGs and N/OFQ using the N/OFQ receptor (NOP) antagonist, N-(4-amino-2-methylquinolin-6-yl)-2-(4-ethylphenoxy-methyl)benzamide monohydrochloride (JTC-801), and in mice lacking the N/OFQ prepropeptide (ppN/OFQ(-/-)) and the NOP receptor (NOP(-/-)). JTC-801 dose-dependently blocked the N/OFQ- and PGE(2)-induced allodynia, but not the PGF(2alpha)-induced one. Neither N/OFQ nor PGE(2) induced allodynia in NOP(-/-) mice. By contrast, the N/OFQ-induced allodynia was not affected by inhibition of PG production by a 60-min pretreatment with the non-steroidal anti-inflammatory drug, indomethacin. Among PGE receptor (EP) subtype-selective agonists, the EP4 agonist, AE1-329, markedly stimulated the release of N/OFQ from spinal slices and induced allodynia. AE1-329 also increased nitric oxide production in spinal slices using fluorescent nitric oxide detection, which was blocked by pretreatment with JTC-801. Conversely, PGE(2)-induced allodynia was not observed in ppN/OFQ(-/-) mice. N/OFQ immunoreactive puncta were colocalized with EP4. Taken together, these results demonstrate that PGE(2) induced allodynia by stimulation of N/OFQ release in the spinal cord via EP4 receptor subtypes.

Agonist activity of naloxone benzoylhydrazone at recombinant and native opioid receptors

1. In the present study, we examined the pharmacological activity of the putative kappa3-opioid receptor agonist naloxone benzoylhydrazone (NalBzoH) at recombinant human opioid receptors individually expressed in Chinese hamster ovary (CHO) cells and native opioid receptors present in rat striatum. 2. At the mu-opioid receptor (MOR), NalBzoH stimulated guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS) binding (pEC50=8.59) and inhibited cyclic AMP accumulation (pEC50=8.74) with maximal effects (Emax) corresponding to 55 and 65% of those obtained with the MOR agonist DAMGO, respectively. The MOR antagonist CTAP blocked the stimulatory effects of NalBzoH and DAMGO with similar potencies. 3. At the kappa-opioid receptor (KOR), NalBzoH stimulated [35S]GTPgammaS binding (pEC50=9.70) and inhibited cyclic AMP formation (pEC50=9.45) as effectively as the selective KOR agonist (-)-U-50,488. The NalBzoH effect was blocked by the KOR antagonist nor-binaltorphimine (nor-BNI) (pKi=10.30). 4. In CHO cells expressing the delta-opioid receptor (DOR), NalBzoH increased [35S]GTPgammaS binding (pEC50=8.49) and inhibited cyclic AMP formation (pEC50=8.61) almost as effectively as the DOR agonist DPDPE. Naltrindole (NTI), a selective DOR antagonist, completely blocked the response to NalBzoH (pKi of 10.40). 5. In CHO cells expressing the nociceptin/orphanin FQ (N/OFQ) receptor (NOP), NalBzoH failed to exert agonist effects and antagonized the agonist-induced receptor activation. 6. When compared to other opioid receptor ligands, NalBzoH showed an efficacy that was lower than that of morphine at MOR, but higher at KOR and DOR. 7. In rat striatum, NalBzoH enhanced [35S]GTPgammaS binding and inhibited adenylyl cyclase activity. These effects were antagonized by either CTAP, nor-BNI or NTI, each antagonist blocking a fraction of the NalBzoH response. 8. These data demonstrate that NalBzoH displays agonist activity at MOR, DOR and KOR expressed either in a heterologous cell system or in a native environment.

Tryptophan replacement in the nociceptin/orphanin FQ receptor ligand Ac‐RYYRWK‐NH2

In the present study we describe the in vitro pharmacological characterization of the nociceptin/orphanin FQ (N/OFQ) receptor (NOP) ligand Ac-RYYRWK-NH2 and the synthesis and biological evaluation of 13 Trp5 substituted Ac-RYYRWK-NH2 analogs. Results indicate that Ac-RYYRWK-NH2 behaves as a highly potent and selective partial agonist at the NOP receptors and that the whole indole moiety of the Trp5 side chain is not required, being a phenyl-ethyl side chain already sufficient for maintaining high potency.

Nociceptin/orphanin FQ inhibits electrically induced contractions of the human bronchus via NOP receptor activation

Nociceptin/orphanin FQ (N/OFQ) has been reported to inhibit neurogenic contractions in various tissues, including guinea pig airways. In the present study, we investigated the ability of N/OFQ to affect cholinergic contractions of human bronchi elicited by electrical field stimulation (EFS). Tissues were obtained from 23 patients undergoing surgery for lung cancer. EFS (20 Hz, 320 mA, 1.5 ms, 10 s) was applied five times every 20 min. Contractions induced by EFS were abolished by either TTX (1 μM) or atropine (1 μM) and concentration-dependently (10 nM–1 μM) inhibited by N/OFQ ( E max, 11.5 ± 1.8% inhibition). The inhibitory effects of N/OFQ were mimicked by the N/OFQ receptor (NOP) ligand [Arg 14, Lys 15]N/OFQ which displayed however, higher significant maximal effects (17.7 ± 2.9% inhibition, P < 0.05). The actions of N/OFQ and [Arg 14, Lys 15]N/OFQ were not affected by naloxone (1 μM) while prevented by the selective NOP receptor antagonist UFP-101 (10 μM). Moreover, the inhibitory effects of NOP agonists were no longer evident in tissues treated with tertiapin (10 μM), an inhibitor of inward-rectifier potassium channels. In conclusion, the present data demonstrate that N/OFQ inhibited acetylcholine (ACh) release in the human bronchi via NOP receptor activation. This effect may involve stimulation of potassium currents.