N/OFQ Peptide Receptor Receptor Research Articles

Nociceptin/Orphanin FQ receptor expression in primary human umbilical vein endothelial cells is not regulated by exposure to breast cancer cell media or angiogenic stimuli.

Opioid receptors are naloxone-sensitive (MOP [mu: μ], DOP [delta: δ], and KOP [kappa: κ]) and naloxone-insensitive Nociceptin/Orphanin FQ (N/OFQ) peptide receptor (NOP). Clinically, most opioid analgesics target MOP. Angiogenesis is the formation of new blood vessels and involves endothelial cell activation, proliferation, and migration. The effect of opioids on this process is controversial with no data for NOP receptor ligands. We used patient-derived human umbilical vein endothelial cells (HUVECs) treated with media from the Michigan Cancer Foundation-7 (MCF-7) breast cancer cells or vascular endothelial growth factor (VEGF; 10 ng ml-1) and fibroblast growth factor (FGF; 10 ng ml-1) as angiogenic stimuli. We have measured (i) NOP/MOP messenger RNA, (ii) receptor protein using N/OFQATTO594 and DermorphinATTO488 as fluorescent probes for NOP and MOP, and (iii) NOP/MOP function in a wound healing assay (crude measure of migration that occurs during angiogenesis). HUVEC lines from 32 patients were used. Using all 32 lines, mRNA for NOP but not MOP was detected. This was unaffected by media from MCF-7 cells or VEGF/FGF. There was no binding of either N/OFQATTO594(NOP) or DermorphinATTO488(MOP) in the absence or presence of angiogenic stimuli (six lines tested). In the absence of MOP mRNA, this was expected. Whilst MCF-7 conditioned medium (not VEGF/FGF) reduced wound healing per se (14 lines tested), there was no effect of N/OFQ (NOP ligand) or morphine (MOP ligand). Media from MCF-7 breast cancer cells or VEGF/FGF as angiogenic stimuli did not influence NOP translation into receptor protein. MOP was absent. In the absence of constitutive or inducible MOP/NOP, there was no effect on wound healing as a measure of angiogenesis.

Loss of Nociceptin/Orphanin FQ peptide receptor protects against traumatic brain injury (TBI)‐induced deficits in male and female rats

Introduction : Traumatic brain injury (TBI) is a leading cause of death and disability in the United States and often chronically affects mental and physical health and overall quality of life. We previously reported that acute treatment of blast TBI with a Nociceptin/OrphaninFQ (N/OFQ) peptide receptor (NOP) antagonist improved vestibulomotor deficits in male rats, corresponding to TBI-induced increases in NOP and its endogenous neuropeptide N/OFQ. The goal of this study is to determine whether the absence of NOP protects against neurological and behavioral sequelae of TBI in male and female KO rats compared to WT. Methods Male and female Wistar NOP receptor knockout (NOP KO) and wildtype (WT) rats received either a sham craniotomy, mild TBI (mTBI) or moderate TBI (ModTBI) controlled cortical impact (CCI) to the left cerebral hemisphere. Injury severity and recovery were assessed using modified neurological severity score (mNSS) on days 1 and 8 post-TBI. Vestibulomotor function was determined using the rotarod. Nociceptive sensitivity to mechanical and thermal stimuli were determined by measuring hind paw withdrawal threshold (PWT) to plastic filaments and by measuring tail flick latency (TFL) from radiant heat, respectively, prior to injury (baseline) and on days 2, 4, and 7 post-TBI. The presence of anxiety-like symptoms was assessed using the elevated plus maze (EPM) on day 7 post-TBI. Data were analyzed by two-way ANOVA with or without repeated measures (as appropriate). Results Rats of both genotypes and sexes showed complete neurological recovery (mNSS<1) following mTBI, and partial recovery following ModTBI by day 8. Mild and ModTBI produced significant vestibular deficits in rat rotarod performance on days 1-8 post-TBI in WT male (n=7-8). In WT females (n=8), ModTBI produced vestibular deficits on days 1-7, whereas mTBI only produced vestibular deficits on days 1-4. Interestingly, neither male (n=5-6) nor female (n=3) mTBI NOP KO rats exhibited vestibulomotor deficits. ModTBI caused significant vestibular deficits only on day 1 post-TBI in male and female NOP KO rats. Rotarod performance in WT females was significantly worse than KO females on days 2-4 post TBI following moderate injury (*p<0.05), and on days 4-7 following mild injury (*p<0.05). WT males (n=7-8) and females (n=8) exhibited thermal hyperalgesia and mechanical allodynia throughout the study, with little difference between mild and moderate TBI groups. KO males (n=3) developed no thermal hyperalgesia after either mild or ModTBI. Only ModTBI rats developed mechanical allodynia, and it lasted throughout the study. KO females (n=3) showed both thermal and mechanical hypersensitivity on day 2 and only mechanical hypersensitivity on day 4. No differences in anxiety-like behaviors were found between any of the groups. Conclusion The N/OFQ-NOP system contributes to vestibular deficits and hyperalgesia associated with TBI; the absence of NOP protects against TBI-induced vestibulomotor deficits and nociceptive hypersensitivity.

Characterization of the Cardiovascular and Renal Effects of Synthetic Nociceptin/Orphanin FQ Receptor Partial Agonists

Following discovery of the endogenous opioid‐like peptide, nociceptin/orphanin FQ (N/OFQ) and the N/OFQ peptide receptor (NOP), numerous peptide and non‐peptide NOP receptor ligands have been developed for potential treatment of disease states including Parkinson's, depression, anxiety, and chronic pain. In addition, our laboratory has demonstrated that NOP receptor peptide ligands produce changes in cardiovascular and renal function. However, we have yet to determine whether non‐peptide NOP receptor ligands have similar cardiovascular and renal effects. Therefore, the aim of the present study was to characterize the cardiovascular and renal profile of the non‐peptide NOP receptor compounds AT‐039, AT‐127, AT‐090, and AT‐403 (Astraea Therapeutics).MethodsMale Sprague‐Dawley rats were surgically implanted with a venous, arterial, and bladder catheter and continuously infused intravenously (i.v.) with isosaline. After stabilization, conscious rats received an i.v. bolus injection of AT‐039, 127, 090, or 403 (30, 100, or 300 nmol/kg) while mean arterial pressure (MAP), heart rate (HR), and urine flow rate (V) were measured for 90 minutes. Urine samples were analyzed for urinary sodium excretion (UNaV) and urinary potassium excretion (UKV).ResultsFollowing i.v. bolus administration all AT compounds produced a dose‐dependent diuresis with a decrease in MAP, HR, MAP, UNaV, and UKV. Each ligand also produced sedation with the exception of AT‐039. Further studies with AT‐039 demonstrated that i.v. infusion of this ligand produced dose‐dependent effects on water and electrolyte excretion and blood pressure, but did not alter HR or produce sedation.ConclusionsThese findings indicate that i.v. injection of all non‐peptide NOP receptor AT compounds produced a significant diuretic and hypotensive effects. However, sedation and bradycardia were absent with AT‐039 i.v. infusion treatment. Further studies are needed to explore the mechanisms by which these compounds produce their cardiorenal responses.Support or Funding InformationFunding for this work was supported by Astraea Therapeutics (NZ), R01DA027811 (NZ), R01014026 (NZ), and NIH P30GM106392 (DRK).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

NOP Ligands for the Treatment of Anxiety and Mood Disorders.

Many studies point toward the nociceptin/orphanin FQ (N/OFQ) and the N/OFQ peptide receptor (NOP) as targets for the development of innovative drugs for treating anxiety- and mood-related disorders. Evidence supports the view that the activation of NOP receptors with agonists elicits anxiolytic-like effects, while its blockade with NOP antagonists promotes antidepressant-like actions in rodents. Genetic studies showed that NOP receptor knockout mice display an antidepressant-like phenotype, and NOP antagonists are inactive in these animals. In contrast, the genetic blockade of NOP receptor signaling generally displays an increase of anxiety states in the elevated plus-maze test. In this chapter we summarized the most relevant findings of NOP receptor ligands in the modulation of anxiety and mood disorders, and the putative mechanisms of action are discussed.

Structure activity studies of nociceptin/orphanin FQ(1–13)-NH2 derivatives modified in position 5

Nociceptin/orphanin FQ (N/OFQ) is a heptadecapeptide acting as the endogenous ligand of the N/OFQ peptide receptor (NOP). N/OFQ(1–13)-NH2 is the shortest N/OFQ sequence maintaining the same potency and efficacy as the natural peptide. Thus N/OFQ(1–13)-NH2 was used as chemical template for investigating the structure activity relationship of threonine in position 5. 28 [X5]N/OFQ(1–13)-NH2 derivatives, in which Thr was substituted with natural and unnatural residues, were synthesized and characterized pharmacologically for their effects at the human NOP receptor. Two different functional assays were used: agonist stimulated [35S]GTPγS binding in cell membranes and calcium mobilization in whole cells co-expressing chimeric G proteins. All [X5]N/OFQ(1–13)-NH2 derivatives behaved as full NOP agonists showing large differences in their potency. There was an excellent correlation between the results obtained in the two assays. The results of this study suggest that: position 5 does not play a pivotal role in receptor activation; the secondary alcoholic function of Thr is not important for receptor binding; side chain size, lipo/hydrophilic balance as well as hydrogen bond capability are also not crucial for receptor binding; an aliphatic amino function positively charged with at least 3 carbon atom distance from the peptide backbone has a huge disrupting effect on receptor binding. In conclusion this study demonstrates that a simple ethyl side chain as in compound 23 is sufficient in N/OFQ position 5 for maintaining bioactivity.

UFP-112 a Potent and Long-Lasting Agonist Selective for the Nociceptin/Orphanin FQ Receptor

Nociceptin/orphanin FQ (N/OFQ) controls several biological functions via selective activation of the N/OFQ peptide receptor (NOP). [(pF)Phe(4) Aib(7) Arg(14) Lys(15) ]N/OFQ-NH(2) (UFP-112) is an NOP receptor ligand designed using a combination of several chemical modifications in the same peptide sequence that increase NOP receptor affinity/potency and/or reduce susceptibility to enzymatic degradation. In the present review article, we summarize data from the literature and present original findings on the in vitro and in vivo pharmacological features of UFP-112. Moreover, important biological actions and possible therapeutic indications of NOP receptor agonists are discussed based on the results obtained with UFP-112 and compared with other peptide and nonpeptide NOP receptor ligands.

Pharmacological profile and antiparkinsonian properties of the novel nociceptin/orphanin FQ receptor antagonist 1-[1-cyclooctylmethyl-5-(1-hydroxy-1-methyl-ethyl)-1,2,3,6-tetrahydro-pyridin-4-yl]-3-ethyl-1,3-dihydro-benzoimidazol-2-one (GF-4)

In this study we provided a pharmacological characterization of the recently synthesized nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP) antagonist 1-[1-Cyclooctylmethyl-5-(1-hydroxy-1-methyl-ethyl)-1,2,3,6-tetrahydro-pyridin-4-yl]-3-ethyl-1,3-dihydro-benzoimidazol-2-one (GF-4) and investigated its antiparkinsonian properties. GF-4 inhibited N/OFQ binding to CHO hNOP cell membranes (p K i 7.46), and antagonized N/OFQ effects in a calcium mobilization assay and electrically stimulated isolated tissues (p K B 7.27–7.82), showing a ∼5-fold selectivity over classical opioid receptors. In vivo, GF-4 dually modulated stepping activity in wild-type mice, causing facilitation in the 0.01–10 mg/kg dose range and inhibition at 30 mg/kg. These effects were mediated by NOP receptors since GF-4 was ineffective in NOP receptor knock-out mice. Antiparkinsonian properties of GF-4 were investigated in 6-hydroxydopamine hemilesioned rats. GF-4 ameliorated akinesia, bradykinesia and overall gait ability in the 0.1–10 mg/kg dose range, but inhibited motor activity at 30 mg/kg. To investigate the circuitry underlying motor facilitating and inhibitory effects of GF-4, microdialysis coupled to behavioral testing (akinesia test) was performed. An anti-akinetic dose of GF-4 (1 mg/kg) reduced glutamate (GLU) and enhanced GABA release in SNr, while the pro-akinetic dose of GF-4 (30 mg/kg) evoked opposite effects. Moreover, the anti-akinetic dose of GF-4 reduced GABA and increased GLU release in ventro-medial thalamus, the pro-akinetic dose decreasing GABA without affecting GLU release in this area. We conclude that GF-4 is an effective NOP receptor antagonist able to attenuate parkinsonian-like symptoms in vivo via inhibition of the nigro-thalamic pathway.

Pharmacological characterization of the nociceptin/orphanin FQ receptor non peptide antagonist Compound 24

Compound 24, 1-benzyl-N-{3-[spiroisobenzofuran-1(3H),4′-piperidin-1-yl]propyl} pyrrolidine-2-carboxamide was recently identified as a nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP) ligand. In this study, the in vitro and in vivo pharmacological profiles of Compound 24 were investigated. In vitro studies were performed measuring receptor and [35S]GTPγS binding and calcium mobilization in cells expressing the recombinant NOP receptor as well as using N/OFQ sensitive tissues. In vivo studies were conducted using the tail withdrawal assay in mice. Compound 24 produced a concentration-dependent displacement of [3H]N/OFQ binding to CHOhNOP cell membranes showing high affinity (pKi 9.62) and selectivity (1000 fold) over classical opioid receptors. Compound 24 antagonized with high potency the following in vitro effects of N/OFQ: stimulation of [35S]GTPγS binding in CHOhNOP cell membranes (pA2 9.98), calcium mobilization in CHOhNOP cells expressing the Gαqi5 chimeric protein (pKB 8.73), inhibition of electrically evoked twitches in the mouse (pA2 8.44) and rat (pKB 8.28) vas deferens, and in the guinea pig ileum (pKB 9.12). In electrically stimulated tissues, Compound 24 up to 1 µM did not modify the effects of classical opioid receptor agonists. Finally in vivo, in the mouse tail withdrawal assay, Compound 24 at 10 mg/kg antagonized the pronociceptive and antinociceptive effects of 1 nmol N/OFQ given supraspinally and spinally, respectively. Under the same experimental conditions Compound 24 did not affect the antinociceptive action of 3 nmol endomorphin-1 injected intrathecally. The present study demonstrated that Compound 24 is a pure, competitive, and highly potent non-peptide NOP receptor selective antagonist.

Further studies on the pharmacological features of the nociceptin/orphanin FQ receptor ligand ZP120

ZP120 is a nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP) ligand. In previous studies, the effects of ZP120 were found to be sensitive to J-113397 in mouse tissues while resistant to UFP-101 in rat tissues. The aim of this study was to further investigate the ZP120 pharmacological profile using mouse and rat preparations, J-113397 and UFP-101, as well as NOP receptor knockout (NOP −/−) mice. Electrically stimulated mouse and rat vas deferens were used to characterize the pharmacology of ZP120 in vitro. For in vivo studies the tail-withdrawal assay was performed in wild type (NOP +/+) and NOP knockout (NOP −/−) mice. In the mouse and rat vas deferens ZP120 mimicked the effects of N/OFQ showing higher potency but lower maximal effects. In both preparations, J-113397 antagonized N/OFQ and ZP120 effects showing similar p K B values (≈7.8). UFP-101 antagonized the actions of N/OFQ (p K B values ≈7.3) but did not modify the effects of ZP120. The inhibitory effects of N/OFQ and ZP120 were no longer evident in vas deferens tissues taken from NOP −/− mice. In NOP +/+ mice subjected to the tail-withdrawal assay, ZP120 (1 nmol) mimicked the pronociceptive action of N/OFQ (10 nmol), producing longer lasting effects. The effects of both peptides were absent in NOP −/− animals. The NOP receptor ligand ZP120 is a high potency NOP selective partial agonist able to evoke long-lasting effects; its diverse antagonist sensitivity in comparison with N/OFQ may derive from different modality of binding to the NOP receptor.

The nociceptin/orphanin FQ-NOP receptor antagonist effects on an animal model of sepsis

The aim of this study was investigate the effects of nociceptin/orphanin FQ (N/OFQ) and ([Nphe(1),Arg(14),Lys(15)]N/OFQ-NH(2)) (UFP-101), the endogenous N/OFQ peptide receptor (NOP) ligand and a selective NOP antagonist, respectively, in the inflammatory response after cecal ligation and puncture (CLP) model of sepsis in rats. Prospective, controlled experiment. Animal basic science laboratory. Male Wistar rats, weighing 300-350 g. Rats subjected to CLP were treated with N/OFQ (0.001, 0.01 or 0.1 mg/kg) or UFP-101 (0.03, 0.03 or 0.3 mg/kg) subcutaneously administered immediately after surgery. Twelve hours after surgery, blood was collected by cardiac puncture and bronchoalveolar (BAL) and peritoneal lavage were performed. In a separate set of experiments mortality was evaluated monitoring CLP rats for 10 days. Our findings showed that UFP-101 (0.03 mg/kg, sc, but not 0.003 mg/kg) modified parameters related to the systemic inflammatory response by effectively preventing cells migration, bacterial dissemination, and by modulating the release of pro-inflammatory cytokines and chemokines, and reducing animal mortality in a clinically relevant model of sepsis. By contrast, N/OFQ (0.1 mg/kg, sc) increased mortality in the CLP model. Our findings point to a functional relationship between the N/OFQ-NOP receptor system and inflammatory response in the CLP model of sepsis and suggest that NOP receptor antagonists are worthy of testing as innovative drugs for the treatment of sepsis.

In vitro and in vivo studies on UFP-112, a novel potent and long lasting agonist selective for the nociceptin/orphanin FQ receptor

[(pF)Phe 4Aib 7Arg 14Lys 15]N/OFQ-NH 2 (UFP-112) has been designed as a novel ligand for the nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP) by combining into the same peptide different chemical modifications reported to increase N/OFQ potency. In vitro data obtained in the electrically stimulated mouse vas deferens demonstrated that UFP-112 behaved as a high potency (pEC 50 9.43) full agonist at the NOP receptor. UFP-112 effects were sensitive to the NOP antagonist UFP-101 but not to naloxone and no longer evident in tissues taken from NOP −/− mice. In vitro half life of UFP-112 in mouse plasma and brain homogenate was 2.6- and 3.5-fold higher than that of N/OFQ. In vivo, in the mouse tail withdrawal assay, UFP-112 (1–100 pmol, i.c.v.) mimicked the actions of N/OFQ producing pronociceptive effects after i.c.v. administration and antinociceptive effects when given i.t.; in both cases, UFP-112 was approximately 100-fold more potent than the natural peptide and produced longer lasting effects. UFP-112 also mimicked the hyperphagic effect of N/OFQ producing a bell shaped dose response curve with the maximum reached at 10 pmol. The hyperphagic effects of N/OFQ and UFP-112 were absent in NOP −/− mice. Equi-effective high doses of UFP-112 (0.1 nmol) and N/OFQ (10 nmol) were injected i.c.v. in mice and spontaneous locomotor activity recorded for 16 h. N/OFQ produced a clear inhibitory effect which lasted for 60 min while UFP-112 elicited longer lasting effects (>6 h). In conscious rats, UFP-112 (0.1 and 10 nmol/kg, i.v.) produced a marked and sustained decrease in heart rate, blood pressure, and urinary sodium excretion and a profound increase in urine flow. Collectively, these findings demonstrate that UFP-112 behaves in vitro and in vivo as a highly potent and selective ligand able to produce full and long lasting activation of NOP receptors.

Altered anxiety-related behavior in nociceptin/orphanin FQ receptor gene knockout mice

Studies showed that nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP) agonists produce anxiolytic-like actions, while little is known about the effects of blockade of NOP receptor signaling in anxiety. To this aim, we investigated the behavioral phenotype of NOP receptor gene knockout mice (NOP −/−) in different assays. In the elevated plus-maze and light-dark box, NOP −/− mice displayed increased anxiety-related behavior. In the novelty-suppressed feeding behavior and elevated T-maze, NOP −/− mice showed anxiolytic-like phenotype, while no differences were found in the open-field, hole-board, marble-burying, and stress-induced hyperthermia. Altogether, these findings suggest that the N/OFQ-NOP receptor system modulates anxiety-related behavior in a complex manner.

UFP-101 antagonizes the spinal antinociceptive effects of nociceptin/orphanin FQ: Behavioral and electrophysiological studies in mice

Nociceptin/orphanin FQ (N/OFQ) modulates various biological functions, including nociception, via selective stimulation of the N/OFQ peptide receptor (NOP). Here we used the NOP selective antagonist UFP-101 to characterize the receptor involved in the spinal antinociceptive effects of N/OFQ evaluated in the mouse tail withdrawal assay and to investigate the mechanism underlying this action by assessing excitatory postsynaptic currents (EPSC) in laminas I and II of the mouse spinal cord dorsal horn with patch-clamp techniques. Intrathecal (i.t.) injection of N/OFQ in the range of 0.1–10 nmol produced a dose dependent antinociceptive effect, which was prevented by UFP-101, but not by naloxone. In contrast the antinociceptive effect of the mu-opioid peptide receptor agonist endomorphin-1 was blocked by naloxone but not by UFP-101. Moreover, N/OFQ and endomorphin-1 induced a significant antinociceptive effect in wild type mice while in mice knockout for the NOP receptor gene only endomorphin-1 was found to be active. In mouse spinal cord slices 1 μM N/OFQ reduced EPSC to 60 ± 4% of control values. This inhibitory effect was reversed in a concentration dependent manner by UFP-101 (pA 2 value 6.44). The present results demonstrate that N/OFQ-induced spinal antinociception in vivo and inhibition of spinal excitatory transmission in vitro are mediated by receptors of the NOP type.

Antidepressant- and anxiolytic-like effects of nociceptin/orphanin FQ receptor ligands

Many studies point toward the nociceptin/orphanin FQ (N/OFQ) and the N/OFQ peptide receptor (NOP) as targets for the development of innovative drugs for treating affective disorders. It has been reported that the activation of NOP receptors produces anxiolytic-like effects in rodents in a large series of behavioral assays, i.e., elevated plus maze, light-dark aversion, operant conflict, fear-potentiated startle, pup ultrasonic vocalizations, and hole board tests. In contrast, the blockade of N/OFQ signaling obtained with NOP-selective antagonists promotes antidepressant-like effects in the forced swimming and tail suspension tests. In these assays, N/OFQ is inactive per se, but reverses the antidepressant-like effects of NOP antagonists. NOP receptor knockout mice show an antidepressant-like phenotype, and NOP antagonists are inactive in these animals. Thus, the activation of the NOP receptor seems to evoke anxiolytic-like effects while its blockade antidepressant-like effects. This appears to be a rather unique behavioral profile since the activation or the blockade of a given neuropeptide receptor produces, in most of the cases, both antidepressant- and anxiolytic-like effects. This particular behavioral profile, the possible mechanisms of action, and the therapeutic potential of NOP receptor ligands for the treatment of depression and anxiety disorders are discussed in this review article.

In vitro Binding and Functional Studies of Ac-RYYRIK-ol and Its Derivatives, Novel Partial Agonists of the Nociceptin/Orphanin F/Q Receptor

Following the discovery of nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP) and its endogenous ligand, an extensive search has started to find selective agonists and antagonists targeting this novel receptor-ligand system due to their therapeutic potentials. By the help of the combinatorial chemistry a series of hexapeptides with a general formula of Ac-RYY-R/K-W/I-R/K-NH₂ having high NOP receptor affinity and selectivity were identified. On the basis of this information we developed a number of novel compounds. The detailed structure-activity studies on the partial agonist Ac-RYYRIK-NH₂ are reported in this communication. Besides the modifications on N- and C-terminal, Arg-Cit exchange was performed on the template structure. The novel hexapeptides were analyzed in radioligand binding, functional biochemical [³⁵S]GTPγS binding assays by using membranes from rat brains and Chinese hamster ovary cells expressing human NOP receptor. The agonist/antagonist properties were also tested on in the mouse vas deferens bioassay. C-terminal modification yielded a high affinity, selective and potent NOP ligand (Ac-RYYRIK-ol) with a partial agonist property. Several analogs of this compound were synthesized. The presence of the positively charged arginine residue at the first position turned out to be crucial for the biological activity of the hexapeptide. The N-terminal modifications with various acyl groups (ClAc, pivaloyl, formyl, benzoyl, mesyl) decreased the affinity of the ligand towards the receptor and the intrinsic activity for stimulating the G-protein activation was also decreased. The structure-activity studies on the hexapeptide derivatives provided some basic information on the structural requirements for receptor binding and activation.

The nociceptin receptor antagonist [Nphe1,Arg14,Lys15]nociceptin/orphanin FQ-NH2 blocks the stimulatory effects of nociceptin/orphanin FQ on the HPA axis in rats

Nociceptin/orphanin FQ (N/OFQ) is an opioid-related peptide that stimulates corticosterone release after i.c.v. administration in non-stressed rats. We employed in situ hybridization histochemistry to investigate N/OFQ–stimulated activation of the HPA axis at the hypothalamic and pituitary level. We have demonstrated that N/OFQ–induced activation of the HPA axis is mediated via the central N/OFQ peptide receptor (NOP) using the recently described selective NOP antagonist [Nphe1,Arg14,Lys15]nociceptin/orphanin FQ-NH2 (UFP-101).We found that, at 30 min post–i.c.v. injection, N/OFQ dose-dependently increased plasma adrenocorticotrophin hormone and corticosterone compared with the vehicle-injected controls. N/OFQ (1.0 μg) significantly increased CRF mRNA but not AVP mRNA within the parvocellular hypothalamic paraventricular nucleus compared with the control group, and significantly increased pro-opiomelanocortin (POMC) mRNA in the anterior pituitary. While UFP-101 (1.0 μg) alone had no significant effect on plasma corticosterone concentration it blocked the effect of N/OFQ (1.0 μg) on plasma corticosterone levels when compared with N/OFQ administered alone. UFP-101 also blocked the N/OFQ-induced increase in CRF mRNA and POMC mRNA. These results demonstrate that centrally administered N/OFQ activates the HPA axis via up-regulation of CRF and POMC mRNA and stimulation of corticosterone release in rats. Further, we have demonstrated for the first time that the selective NOP receptor antagonist UFP-101 blocks these effects indicating that N/OFQ–induced HPA axis activation is mediated via central NOP receptors.

Nociceptin/orphanin FQ stimulates human monocyte chemotaxis via NOP receptor activation

Nociceptin/orphanin FQ (N/OFQ) produces several biological actions by activating the N/OFQ peptide receptor (NOP). It has been previously shown that N/OFQ stimulates leukocyte chemotaxis both in vitro and in vivo. In the present study we investigated the ability of N/OFQ, in comparison with the proinflammatory peptide formyl-Met-Leu-Phe (fMLP), to stimulate human neutrophil and monocyte chemotaxis and the release of lysozyme and superoxide anion (O 2 −) production from neutrophils. fMLP stimulated all the leukocyte functions examined. N/OFQ stimulated monocyte (pEC 50 12.15) but not neutrophil chemotaxis. The production of O 2 − from neutrophils was not affected by N/OFQ while the release of lysozyme was increased in a concentration dependent manner (pEC 50 11.00) although the maximal effects evoked by N/OFQ were about half of those of fMLP. The NOP ligands [Arg 14, Lys 15]N/OFQ, N/OFQ(1-13)NH 2, Ro 64-6198, UFP-101 and the opioid antagonist naloxone were used for pharmacologically characterizing the receptor involved in the monocyte chemoattractant action of N/OFQ. [Arg 14, Lys 15]N/OFQ, N/OFQ(1-13)NH 2, and Ro 64-6198 mimicked the action of N/OFQ showing similar maximal effects and the following order of potency: [Arg 14, Lys 15]N/OFQ (pEC 50 13.22) > Ro 64-6198 (pEC 50 12.96) > N/OFQ(1-13)NH 2 (pEC 50 12.67) > N/OFQ (pEC 50 12.15). Moreover, the monocyte chemoattractant action of N/OFQ was not modified by naloxone 1 μM while antagonized by UFP-101 10 μM (pA 2 7.00). Thus, the order of potency of agonists and the antagonist selectivity demonstrated that N/OFQ stimulates human monocyte chemotaxis via NOP receptor activation.

N- and C-Terminal Modifications of Nociceptin/Orphanin FQ Generate Highly Potent NOP Receptor Ligands

Previous structure-activity studies on nociceptin/orphanin FQ (N/OFQ) identified [Phe(1)Psi(CH(2)NH)Gly(2)]N/OFQ(1-13)-NH(2) and [Nphe(1)]N/OFQ(1-13)-NH(2) as a N/OFQ peptide receptor (NOP) partial agonist and pure antagonist, respectively. The addition of fluorine to the Phe(4) or the insertion of a further pair of basic amino acids Arg(14)-Lys(15) generate potent agonists. On the basis of these findings, we combined in the N/OFQ-NH(2) template the chemical modifications Arg(14)-Lys(15) and (pF)Phe(4) that increase the agonist potency with those conferring partial agonist (Phe(1)Psi(CH(2)NH)Gly(2)) or pure antagonist (Nphe(1)) properties. Twelve peptides were synthesized and pharmacologically evaluated in Chinese hamster ovary cells expressing the human recombinant NOP and in electrically stimulated mouse vas deferens and guinea pig ileum assays. All peptides behaved as NOP ligands; the chemical modifications Arg(14)-Lys(15) and (pF)Phe(4) increased ligand affinity/potency. Peptides with the normal Phe(1)-Gly(2) peptide bond behaved as full agonists, and those with the Phe(1)Psi(CH(2)NH)Gly(2) modification behaved as partial agonists, while those with the Nphe(1) modification behaved as partial agonists or pure antagonists depending on the presence or absence of the (pF)Phe(4) modification, respectively. The full agonist [(pF)Phe(4),Arg(14),Lys(15)]N/OFQ-NH(2), the partial agonist [Phe(1)Psi(CH(2)NH)Gly(2),(pF)Phe(4),Arg(14),Lys(15)]N/OFQ-NH(2), and the pure antagonist [Nphe(1),Arg(14),Lys(15)]N/OFQ-NH(2) represent the most potent peptide ligands for NOP.