Peripheral orphanin FQ/nociceptin analgesia in the mouse.

Orphanin FQ/Nociceptin (OFQ/N) administered peripherally was an effective analgesic in the tailflick test in mice (ED50 16.3 microg). It had a peak effect at 5 min and lasted up to 30 min. The kappa3 analgesic naloxone benzoylhydrazone was also active peripherally (ED50 3.8 microg). The analgesic actions of both agents were blocked by naloxone. Neither OFQ/N(1-11) nor OFQ/N(1-7) had appreciable peripheral activity. Antisense mapping both compounds against the murine orphan opioid receptor (KOR-3) confirmed the importance of this clone in their actions. Antisense probes targeting the second and third coding exons significantly lowered the analgesic effects of both compounds. However, the antisense targeting the first coding exon blocked only the actions of OFQ/N and not kappa3 analgesia.     

Quantitative study of [Tyr10]nociceptin/orphanin FQ (1-11) at NOP receptors in rat periaqueductal gray and expressed NOP receptors in HEK293 cells

AimThe nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor was reported to be functionally heterogeneous. We investigated if [Tyr¹⁰]N/OFQ(1-11), a peptide ligand reported to selectively bind to the high affinity site of ¹²⁵I-[Tyr¹⁴]N/OFQ in rodent brains, can be a tool for revealing the NOP receptor heterogeneity. We have previously founded an NOP receptor subset insensitive to Ro 64-6198 and (+)-5a Compound, two non-peptide NOP agonists, in rat ventrolateral periaqueductal gray (vlPAG) neurons. Here, we examined if [Tyr¹⁰]N/OFQ(1-11) differentiated (+)-5a Compound-sensitive and -insensitive vlPAG neurons. Certain mu-opioid (MOP) receptor ligands highly competing with [Tyr¹⁰]N/OFQ(1-11) in binding studies also showed high affinity at expressed heteromeric NOP–MOP receptors. We also examined if [Tyr¹⁰]N/OFQ(1-11) distinguished heteromeric NOP–MOP receptors from homomeric NOP receptors.Main methodsThe NOP receptor activity was evaluated by G-protein coupled inwardly rectifying potassium (GIRK) currents in rat vlPAG slices, and by inhibition of cAMP accumulation in HEK293 cells expressing NOP receptors or co-expressing NOP and MOP receptors.Key findingsIn vlPAG neurons, [Tyr¹⁰]N/OFQ(1-11), like N/OFQ, induced GIRK currents through NOP receptors. It was less potent (EC₅₀: 8.98 μM) but equi-efficacious as N/OFQ. [Tyr¹⁰]N/OFQ(1-11) displayed different pharmacological profiles as (+)-5a Compound, and was effective in both (+)-5a Compound-sensitive and -insensitive neurons. In NOP-expressing HEK293 cells and NOP- and MOP-co-expressing cells, [Tyr¹⁰]N/OFQ(1-11) displayed similar concentration–response curves in decreasing cAMP accumulation.Significance[Tyr¹⁰]N/OFQ(1-11) is an NOP full agonist and less potent than N/OFQ. However, it can neither reveal the functional heterogeneity of NOP receptors in vlPAG neurons nor differentiate heteromeric NOP–MOP and homomeric NOP receptors.     

大鼠中脑导水管周围灰质微量注射孤啡肽对痛和针刺镇痛的影响

孤啡肽是新近发现的神经肽,结构与内阿片肽相似,但作用明显不同。本文采用脑核团内微量注射方法,在大鼠电刺激甩尾测痛模型上,观察ＯＦＱ在中脑导水管周围灰质内对痛和针刺镇痛的影响。结果表明：ＰＡＧ内微量注射ＯＦＱ可使大鼠痛阈降低,并明显对抗针刺镇痛;另外还发现,ＯＦＱ在ＰＡＧ内还可以对抗μ受体激动剂羟甲芬太尼加强针刺镇痛的效应。     

Effects of supraspinal orphanin FQ/nociceptin

The first reported behavioral action of the endogenous ligand for the "orphan" opioid receptor was a seemingly paradoxical increased sensitivity to nociception (i.e. hyperalgesia) after supraspinal injection into the cerebral ventricles of mice. In the continuing absence of an appropriate in vivo receptor antagonist, studies attempting to define the role of orphanin FQ/nociceptin (OFQ/N) in pain modulation and other behaviors have also featured central injection of peptide. This article reviews the findings of such studies. There appears to be concordance around the observation of anti-opioid actions of supraspinally injected OFQ/N, whereas the observations of hyperalgesia and/or analgesia are much less clear. A portion of the discrepant data may be explained in terms of methodological issues, stress-induced analgesia accompanying experimental protocols, and genotypic variation among subjects. Clarification of OFQ/N's role in nociception, as with other putative biologic functions, will probably depend upon the availability of a selective receptor antagonist.     

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 (O2-) production from neutrophils. fMLP stimulated all the leukocyte functions examined. N/OFQ stimulated monocyte (pEC50 12.15) but not neutrophil chemotaxis. The production of O2- from neutrophils was not affected by N/OFQ while the release of lysozyme was increased in a concentration dependent manner (pEC50 11.00) although the maximal effects evoked by N/OFQ were about half of those of fMLP. The NOP ligands [Arg14, Lys15]N/OFQ, N/OFQ(1-13)NH2, 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. [Arg14, Lys15]N/OFQ, N/OFQ(1-13)NH2, and Ro 64-6198 mimicked the action of N/OFQ showing similar maximal effects and the following order of potency: [Arg14, Lys15]N/OFQ (pEC50 13.22)>Ro 64-6198 (pEC50 12.96)>N/OFQ(1-13)NH2 (pEC50 12.67)>N/OFQ (pEC50 12.15). Moreover, the monocyte chemoattractant action of N/OFQ was not modified by naloxone 1 microM while antagonized by UFP-101 10 microM (pA2 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.     

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.     

Activation of the nociceptin/orphanin FQ system is unable to reverse CRF2 receptor mediated anorexia in the rat

Central injection of Nociceptin/Orphanin FQ (N/OFQ), inhibits the anorectic effect of corticotropin-relasing factor (CRF) and stress in rats. Recently, Urocortin II (Ucn II) and Urocortin III (Ucn III), two selective CRF₂ receptor agonists, have been identified. Here, we investigated the effect of intracerebroventricular (ICV) injection of 0.25, 0.75, 1.50 or 3 nmol/rat of Ucn II or Ucn III on food and water intake in food deprived rats. The effect of N/OFQ on Ucn II and UCNIII-induced anorexia was also studied. Results showed a greater inhibition of food consumption by Ucn II than Ucn III. Pretreatment with N/OFQ (0.25–2.0 nmol/rat) did not block the effects of Ucn II and UCNIII. Conversely, injection of N/OFQ (0.25–2.0 nmol/rat) blocked the anorectic effect of CRF (0.1 nmol/rat). These findings suggest that N/OFQ selectively prevent the anorectic effect mediated by activation of the CRF₁ receptor system.     

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.     

Cellular neurophysiological actions of nociceptin/orphanin FQ

Cellular actions of nociceptin/orphanin FQ (N/OFQ) resemble those of micro-, delta-, and kappa-opioids, i.e. activation of inwardly rectifying K(+) conductance, inhibition of high-voltage-activated Ca(2+) channel currents, and impediment of neurotransmitter release. Differences in ORL(1) and micro-receptor distribution lead to: 1) more widespread actions of N/OFQ on periaqueductal gray neurons than opioids and 2) differential effects of N/OFQ and opioids in the brainstem. Also, unlike opioids, N/OFQ inhibits T-type Ca(2+) channel current in sensory neurons. Opioids and N/OFQ may modulate glutamate responses in different ways, and certain actions of N/OFQ are potentiated following nerve injury whereas those of micro-opioids are attenuated. Agonists at ORL(1) receptors may therefore be of clinical interest in the management of neuropathic pain.     

Opiate modulating properties of nociceptin/orphanin FQ

The recently discovered peptide nociceptin/orphanin FQ (N/OFQ) and its receptor NOR share many structural similarities with the opioid peptides and their receptors. The anatomical distributions of N/OFQ and NOR are similar to those of opioid peptides and receptors. In addition, NOR and opiate receptors couple via the same G-proteins to similar effectors, such as Ca(2+) channels, K(+) channels, adenylyl cyclase, and several protein kinases. Thus, the behavioral effects of N/OFQ have been investigated in the context of known opiate effects, and a possible connection has been sought between the effects of these two homologous signaling systems. Originally characterized as a nociception-producing peptide, N/OFQ has now been shown to have diverse effects on nociception, as well as effects on many other behaviors. With regard to nociception, the peptide has been reported to produce hyperalgesia, reversal of opioid-mediated analgesia, analgesia, and allodynia. N/OFQ also has effects on other behaviors, such as locomotion, feeding, anxiety, spatial attention, reproductive behaviors, and opiate tolerance. The relationship between opiates and N/OFQ is strengthened by the fact that opiates also affect these behaviors. However, the exact nature of the relationship of N/OFQ with opiates-opiate-like versus antiopiate-remains controversial. This review will detail the diverse effects of N/OFQ and suggest that this peptide, like other putative antiopiate peptides, can be described as 'opiate modulating. '     

Blockade of nociceptin/orphanin FQ transmission in rat substantia nigra reverses haloperidol-induced akinesia and normalizes nigral glutamate release

We recently showed that pharmacological blockade of nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptors located in the substantia nigra stimulates the nigrostriatal dopaminergic pathway and motor behavior (Marti et al. J. Neurosci. 2004, 24, 6659-6666). To investigate whether such motor-stimulating action was dependent on functional dopaminergic transmission, the selective NOP receptor peptide antagonist [Nphe1,Arg14,Lys15]N/OFQ-NH2 (UFP-101) was microinjected into the substantia nigra reticulata of rats made cataleptic by systemic haloperidol administration. UFP-101 reduced haloperidol-induced akinesia as measured by immobility time in the bar test. UFP-101 also induced contralateral turning in cataleptic rats. To investigate the mechanisms involved in the anti-akinetic action of UFP-101, nigral glutamate release was monitored by microdialysis technique. The anti-akinetic action of UFP-101 correlated with normalization of nigral glutamate release, previously elevated by haloperidol injection. We conclude that endogenous N/OFQ in the substantia nigra sustains akinesia generated by impaired DA transmission and subthalamic nucleus overactivation. NOP receptor antagonists may be beneficial in the symptomatic therapy of parkinsonism, via normalization of subthalamonigral glutamatergic transmission.     

The hyperphagic effect of nociceptin/orphanin FQ in rats

Nociceptin/orphanin FQ (NC), the endogenous ligand of the opioid receptor-like1 (ORL1) receptor, has been reported to stimulate feeding in rats. The present article reviews the studies so far published on the effect of NC on food intake and reports new findings concerning the sensitivity of brain regions to the hyperphagic effect of NC in rats. The results obtained indicate that the hypothalamic arcuate nucleus is the most sensitive site among the brain regions so far investigated. On the basis of these findings and of the neurochemical and electrophysiological effects of NC, possible mechanisms of action and possible interactions with other neurotransmitter systems affecting feeding are discussed.     

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.     

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 microM) or atropine (1 microM) and concentration-dependently (10 nM-1 microM) inhibited by N/OFQ (Emax, 11.5+/-1.8% inhibition). The inhibitory effects of N/OFQ were mimicked by the N/OFQ receptor (NOP) ligand [Arg14, Lys15]N/OFQ which displayed however, higher significant maximal effects (17.7+/-2.9% inhibition, P<0.05). The actions of N/OFQ and [Arg14, Lys15]N/OFQ were not affected by naloxone (1 microM) while prevented by the selective NOP receptor antagonist UFP-101 (10 microM). Moreover, the inhibitory effects of NOP agonists were no longer evident in tissues treated with tertiapin (10 microM), 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.     

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 pK(B) values ( approximately 7.8). UFP-101 antagonized the actions of N/OFQ (pK(B) values approximately 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.     

Possible involvement of endogenous nociceptin/orphanin FQ in the pain-related behavioral responses induced by its own metabolite, nociceptin/orphanin FQ(14-17)

Nociceptin/orphanin FQ(14-17) (N/OFQ(14-17)) is one of the major fragments that are released from N/OFQ, an endogenous ligand for the opioid receptor like-1 (ORL-1) receptor by endopeptidase 24.11. In the present study, we determined the pharmacological profiles of N/OFQ(14-17) on pain-related behavioral responses in the mouse. Intrathecal (i.t.) administration of N/OFQ(14-17) (5-160 pmol) evoked pain-related behaviors, and these behavioral responses were reduced by i.t. co-administration of an ORL-1 receptor antagonist, [Nphe(1)]N/OFQ(1-13)NH2 (4 pmol). However, in the ligand-binding receptor assay, N/OFQ(14-17) had no affinity for the ORL-1 receptor. Furthermore, i.t. pretreatment with an antiserum against N/OFQ (1:50) diminished the N/OFQ(14-17)-induced pain-related behaviors, suggesting that endogenous N/OFQ is involved in their expression. Therefore, N/OFQ(14-17)-induced pain-related behaviors may be mediated through the release of endogenous N/OFQ in the mouse spinal cord.     

Inhibitory activity of nociceptin/orphanin FQ on capsaicin-induced bronchoconstriction in the guinea-pig

In vivo studies were conducted in the guinea-pig to investigate the activity of the selective ORL1 receptor agonist nociceptin/orphanin FQ against capsaicin-induced bronchoconstriction, a response mediated by the release of tachykinins from pulmonary sensory nerves. Anesthetized guinea-pigs were ventilated with a rodent ventilator and placed in a whole-body plethysmograph, and pulmonary resistance (R(L)) and dynamic lung compliance (C(Dyn)) were monitored. Intravenous administration of nociceptin/orphanin FQ (0.3 mg/kg) inhibited the capsaicin-induced bronchoconstriction. The new nonpeptide ORL1 receptor antagonist 1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one (J-113397) administered intravenously (1 mg/kg) produced a significant blockade of the inhibitory effect of nociceptin/orphanin FQ (0.3 mg/kg) on capsaicin-induced bronchoconstriction, whereas the nonselective opioid receptor antagonist naloxone (1 mg/kg) had no effect. Nociceptin/orphanin FQ (0.3 mg/kg) did not affect the bronchoconstriction induced exogenously by the tachykinin NK2 receptor agonist [beta-ala8]-neurokinin A (4-10). We conclude that nociceptin inhibits in vivo capsaicin-evoked tachykinin release from sensory nerve terminals in the guinea-pig by a prejunctional mechanism. This inhibitory action does not involve activation of opioid receptors.     

Identification of an achiral analogue of J-113397 as potent nociceptin/orphanin FQ receptor antagonist

To date, J-113397 represents the most potent and selective non peptide NOP receptor antagonist widely used in pharmacological studies. However, the synthesis, purification, and enantiomer separation of this molecule, which contains two chiral centers, is rather difficult and low-yielding. Here, we synthesized and tested a series of simplified J-113397 analogues to investigate the importance of the stereochemistry and the influence of the substituents at position 3 of the piperidine nucleus and on the nitrogen atom of the benzimidazolidinone nucleus. The compound coded as Trap-101, an achiral analogue of J-113397, combines a pharmacological profile similar to that of the parent compound with a practical, high-yielding preparation.     

Distinct effect of intracerebroventricular and intrathecal injections of nociceptin/orphanin FQ in the rat formalin test

Nociceptin/orphanin FQ (nociceptin/OFQ), a newly discovered heptadecapeptide has been regarded as an endogenous ligand for orphan opioid receptor. The present study was designed to investigate the effect of nociceptin/OFQ on pain response and opioid analgesia in the rat formalin test. The results showed that intracerebroventricular injection of 1 microg nociceptin/OFQ enhanced the pain response, and 0.1 or 0.5 microg nociceptin/OFQ had no effect on formalin-induced pain. When 0.1 or 1 microg nociceptin/OFQ were used together with mu-, delta-, or kappa-opioid receptor agonists, endomorphin-1, DSLET or U50488H, respectively, it attenuated mu- and kappa- but not delta-receptor mediated analgesia. On the other hand, intrathecal injection of nociceptin/OFQ (0.1, 1 and 5 microg) reduced the pain response in the formalin test. In conclusion, nociceptin/OFQ potentiated formalin-induced pain response and antagonized opioid analgesia in the rat brain but inhibited pain response in the spinal cord.