Designing bifunctional NOP receptor–mu opioid receptor ligands from NOP receptor-selective scaffolds. Part I.

The nociceptin receptor (NOP) and its endogenous agonist, nociceptin/orphanin FQ (N/OFQ), members of the opioid receptor and peptide families respectively, modulate the pharmacological effects of classical opioids, particularly opioid-induced reward and nociception. We hypothesized that compounds containing both NOP and opioid receptor activity in a single molecule may have useful pharmacological profiles as non-addicting analgesics or as drug abuse medications. We report here our forays into the structure–activity relationships for discovering ‘bifunctional’ NOP–mu opioid receptor (MOP) ligands, starting from our NOP-selective scaffolds. This initial SAR suggests pharmacophoric elements that may be modified to modulate/increase opioid affinity, while maintaining high affinity for the NOP receptor, to result in potent bifunctional small-molecule NOP/MOP ligands.     

Gastrointestinal effects of intracerebroventricularly injected nociceptin/orphaninFQ in rats

Nociceptin/orphanin FQ/(N/OFQ), a novel heptadecapeptide recently isolated from porcine and rat brain, is the endogenous ligand of the N/OFQ peptide receptor (NOP, previously known as ORL-1). In this study we examined the effects of intracerebroventricularly (icv) injected N/OFQ on gastric emptying, gastrointestinal transit, colonic propulsion and gastric acid secretion in rats. N/OFQ (0.01-10 nmol/rat) significantly delayed gastric emptying of a phenol red meal, inhibited transit of a non-absorbable charcoal marker through the small intestine and increased the mean colonic bead expulsion time. These N/OFQ-motor effects were abolished by the NOP receptor selective antagonist [NPhe(1)]N/OFQ(1-13)-NH(2) (50 nmol/rat), but were unaltered by the classical opioid receptor antagonist, naloxone (9.2 micromol/kg). Icv injected N/OFQ (10 nmol/rat) decreased gastric acid secretion in 2-h pylorus ligated rats in a naloxone sensitive manner. [NPhe(1)]N/OFQ(1-13)-NH(2) (100 nmol/rat) icv administered alone stimulated gastric acid secretion. These results indicate that N/OFQ activates via NOP receptor stimulation a central inhibitory pathway modulating gastrointestinal propulsive activity and gastric acid secretion in rats.     

Neuropeptide FF receptors antagonist, RF9, attenuates opioid-evoked hypothermia in mice

The present study used the endpoint of hypothermia to investigate opioid and neuropeptide FF (NPFF) interactions in conscious animals. Both opioid and NPFF systems played important roles in thermoregulation, which suggested a link between opioid receptors and NPFF receptors in the production of hypothermia. Therefore, we designed a study to investigate the relationship between opioid and NPFF in control of thermoregulation in mice. The selective NPFF receptors antagonist RF9 (30nmol) injected into the third ventricle failed to induce significant effect, but it completely antagonized the hypothermia of NPFF (45 nmol) after cerebral administration in mice. In addition, RF9 (30 nmol) co-injected i.c.v. in the third ventricle reduced the hypothermia induced by morphine (5nmol,) or nociceptin/orphanin FQ (N/OFQ) (2 nmol). Neither the classical opioid receptors antagonist naloxone (10 nmol) nor NOP receptor antagonist [Nphe(1)]NC(1-13)NH(2) (7.5 nmol) reduced the hypothermia induced by the central injection of NPFF at dose of 45 nmol. Co-injected with a low dose of NPFF (5 nmol), the hypothermia of morphine (5 nmol) or N/OFQ (2 nmol) was not modified. These results suggest that NPFF receptors activation is required for opioid to produce hypothermia. In contrast, NPFF-induced hypothermia is mainly mediated by its own receptors, independent of opioid receptors in the mouse brain. This interaction, quantitated in the present study, is the first evidence that NPFF receptors mediate opioid-induced hypothermia in conscious animals.     

Morphine-induced overexpression of prepro-nociceptin/orphanin FQ in cultured astrocytes

The effects of morphine on the gene expression of prepro-nociceptin/orphanin FQ (ppN/OFQ) in various primary cultured brain cells from embryonic day 17, rats were studied by use of real-time RT-PCR method. The basal level of ppN/OFQ mRNA in terms of ratio to the β-actin in astrocytes was equivalent to that in neurons, but 10-times higher than that in microglia. The addition of 1 μM morphine significantly enhanced the ppN/OFQ mRNA levels in cultured astrocytes, but not neurons or microglia. The enhancement was observed as early as 1 h after the addition of morphine, reached maximum at 6 h. There was a concentration-dependency between 30 nM to 1 μM. The morphine-induced enhancement was abolished by naloxone, an antagonist of μ opioid peptide receptor (MOP), wortmannin, a phosphoinositide 3-kinase (PI3K) inhibitor, and PD98059, a MEK inhibitor, but not by 1,10-phenanthroline, a metalloprotease inhibitor and U73122, a phospholipase C inhibitor. These profiles contrast to the data with morphine-induced enhancement of brain-derived growth factor (BDNF) gene expression in microglia, where 1,10-phenanthroline abolished the expression. Furthermore, the ELISA analysis revealed that the immunoreactive ppN/OFQ or N/OFQ level was also increased by morphine. The present findings suggest that astrocytes could play roles in the neuronal plasticity during morphine chronic treatments by enhancing gene expression of anti-opioid peptide, N/OFQ.     

Effect of novel nociceptin/orphanin FQ-NOP receptor ligands on ethanol drinking in alcohol-preferring msP rats

Activation of the NOP receptor by the endogenous ligand nociceptin/orphanin FQ (N/OFQ) reduces alcohol consumption in genetically selected alcohol-preferring Marchigian Sardinian (msP) rats. The present study evaluated the effect of three newly synthesized peptidergic and one brain-penetrating heterocyclic NOP receptor agonists on alcohol drinking in the two bottle choice paradigm. MsP rats were intracerebroventricularly (ICV) injected with the NOP receptor agonists OS-462 (0.5 and 1.0 μg), UFP-102 (0.25 and 1.0 μg) or UFP-112 (0.01 and 0.05 μg), or with Ro 64-6198 (0.3 and 1.0 mg/kg) given intraperitoneally (i.p.) and tested for 10% alcohol consumption. Results showed decreased alcohol consumption after treatment with all three peptidergic NOP receptor agonists (OS-462, UFP-102 and UFP-112). OS-462 (at the 1.0 μg dose) and UFP-102 (at the 0.25 μg dose) induced a significant increase in food intake as well. Surprisingly, Ro 64-6198 was ineffective at the 0.3 mg/kg dose, whereas it increased ethanol and food consumption at the 1.0 mg/kg dose. Pre-treatment with the selective μ-receptor antagonist naloxone (0.5 mg/kg, i.p.) reduced these effects of 1.0 mg/kg of Ro 64-6198. These findings confirm that activation of brain NOP receptors reduces alcohol drinking in msP rats and demonstrate that OS-462, UFP-102 and UFP-112 act as potent NOP receptor agonists. On the other hand, Ro 64-6198 increased alcohol drinking, an effect probably induced by a residual agonist activity of this compound at μ-opioid receptors. Overall, the results indicate that OS-462, UFP-102 and UFP-112 may represent valuable pharmacological tools to investigate the functional role of the brain N/OFQ system.     

Antinociceptive effects of spinally administered nociceptin/orphanin FQ and its N-terminal fragments on capsaicin-induced nociception

Nociceptin/orphanin FQ (N/OFQ), the endogenous ligand for the N/OFQ peptide (NOP) receptors, has been shown to be metabolized into some fragments. We examined to determine whether intrathecal (i.t.) N/OFQ (1-13), (1-11) and (1-7) have antinociceptive activity in the pain-related behavior after intraplantar injection of capsaicin. The i.t. administration of N/OFQ (0.3-1.2 nmol) produced an appreciable and dose-dependent inhibition of capsaicin-induced paw-licking/biting response. The N-terminal fragments of N/OFQ, (1-13) and (1-11), were antinociceptive with a potency lower than N/OFQ. Calculated ID₅₀ values (nmol, i.t.) were 0.83 for N/OFQ, 2.5 for N/OFQ (1-13) and 4.75 for N/OFQ (1-11), respectively. The time-course effect revealed that the antinociceptive effects of these N-terminal fragments lasted longer than those of N/OFQ. Removal of amino acids down to N/OFQ (1-7) led to be less potent than N/OFQ and its fragments, (1-13) and (1-11). Antinociception induced by N/OFQ or N/OFQ (1-13) was reversed significantly by i.t. co-injection of [Nphe¹]N/OFQ (1-13)NH₂, a peptidergic antagonist for NOP receptors, whereas i.t. injection of the antagonist did not interfere with the action of N/OFQ (1-11) and (1-7). Pretreatment with the opioid receptor antagonist naloxone hydrochloride did not affect the antinociception induced by N/OFQ and its N-terminal fragments. These results suggest that N-terminal fragments of N/OFQ are active metabolites and may modulate the antinociceptive effect of N/OFQ in the spinal cord. The results also indicate that N/OFQ (1-13) still possess antinociceptive activity through NOP receptors.     

Activation of nociceptin/orphanin FQ-NOP receptor system inhibits tyrosine hydroxylase phosphorylation, dopamine synthesis, and dopamine D(1) receptor signaling in rat nucleus accumbens and dorsal striatum

Nociceptin/orphanin FQ (N/OFQ) has been reported to inhibit dopamine (DA) release in basal ganglia mainly by acting on NOP receptors in substantia nigra and ventral tegmental area. We investigated whether N/OFQ could affect DA transmission by acting at either DA nerve endings or DA-targeted post-synaptic neurons. In synaptosomes of rat nucleus accumbens and striatum N/OFQ inhibited DA synthesis and tyrosine hydroxylase (TH) phosphorylation at Ser40 via NOP receptors coupled to inhibition of the cAMP/protein kinase A pathway. Immunofluorescence studies showed that N/OFQ preferentially inhibited phospho-Ser40-TH in nucleus accumbens shell and that in this subregion NOP receptors partly colocalized with either TH or DA D(1) receptor positive structures. In accumbens and striatum N/OFQ inhibited DA D(1) receptor-stimulated cAMP formation, but failed to affect either adenosine A(2A) or DA D(2) receptor regulation of cAMP. In accumbens slices, N/OFQ inhibited DA D(1)-induced phosphorylation of NMDA and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate glutamate receptors, whereas in primary cultures of accumbal cells, which were found to coexpress NOP and DA D(1) receptors, N/OFQ curtailed DA D(1) receptor-induced cAMP-response element-binding protein phosphorylation. Thus, in accumbens and striatum N/OFQ exerts an inhibitory constraint on DA transmission by acting on either pre-synaptic NOP receptors inhibiting TH phosphorylation and DA synthesis or post-synaptic NOP receptors selectively down-regulating DA D(1) receptor signaling.     

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.     

A novel and facile synthesis of tetra branched derivatives of nociceptin/orphanin FQ

Branched peptides have been found to be useful in several research fields however their synthesis and purification is complicated. Here we present a novel and facile synthesis of tetra branched derivatives of nociceptin/orphanin FQ (N/OFQ). Three N/OFQ tetra branched derivatives were prepared using novel cores (PWT1, PWT2 and PWT3) containing a maleimido moiety. [Cys¹⁸]N/OFQ-NH₂ was linked to the cores via thiol-Michael reaction characterized by high yield and purity of the desired final product. In the electrically stimulated mouse vas deferens PWT-N/OFQ derivatives mimicked the inhibitory action of the natural sequence showing similar maximal effects and 3 fold higher potencies. The NOP selective antagonist SB-612111 antagonized the effects of N/OFQ and PWT derivatives with similar pK_B values (8.02–8.48). In vivo after supraspinal administration PWT2-N/OFQ stimulated food intake in mice mimicking the action of N/OFQ. Compared to the natural peptide PWT2-N/OFQ was 40 fold more potent and elicited larger effects. These findings suggest that the PWT chemical strategy can be successfully applied to biologically active peptides to generate, with unprecedented high purity and yield, tetra branched derivatives displaying an in vitro pharmacological profile similar to that of the natural sequence associated, in vivo, to increased potency and effectiveness.     

Characterisation of the non-peptide nociceptin receptor agonist,Ro64-6198 in Chinese hamster ovary cells expressing recombinant human nociceptin receptors

Nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand for the opioid receptor-like receptor or nociceptin receptor (NOP). We have compared a novel non-peptide NOP agonist Ro64-6198 with N/OFQ in a series of GTPgamma35S binding and inhibition of forskolin stimulated cAMP formation assays. GTPgamma35S binding assays were performed in membranes prepared from Chinese hamster ovary cells expressing the recombinant human NOP (CHOhNOP). cAMP inhibition studies were performed in whole CHOhNOP cells. Both Ro64-6198 and N/OFQ stimulated GTPgamma35S binding with pEC50 values(95%CL) of 7.61(0.18) and 8.58(0.21) respectively. Both Ro64-6198 and N/OFQ inhibited cAMP formation with pEC50 values of 8.45(0.9) and 9.28(028) respectively. In each assay Ro64-6198 and N/OFQ were full agonists. Ro64-6198 stimulation of GTPgamma35S binding and inhibition of cAMP formation was competitively antagonised by the NOP antagonists [Nphe1]NC(1 - 13)NH2 (10microM), J-113397 (100nM) and III-BTD (1microM) with pKB values of 7.04(0.34) and 6.29(0.10), 8.65(0.34) and 7.90(0.30) and 7.59(0.22) and 7.60(0.22) respectively. Despite the slightly reduced potency of Ro64-6198 compared with N/OFQ, by virtue of high selectivity and relative metabolic stability this molecule will be of considerable use in studies of the actions of the NOP.     

Effects of PKC on inhibition of delayed rectifier potassium currents by N/OFQ

Nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand for the opioid receptor-like-1 (ORL-1) orphan receptor, which is responsible for inhibition of delayed rectifier potassium current (I(K)). But its mechanism of N/OFQ acting on I(K) is not clear and whether PKC is involved in the modulation of this processing is still unknown. Whole-cell patch-clamp recordings were performed in acutely dissociated rat parietal cortical neurons. Bath application of N/OFQ (10 nM-10 microM) resulted in a dose-dependent depression of I(K) with partially recovery on washout. Furthermore, we investigated the role of PKC in the inhibition of I(K) by N/OFQ. Chelerythrine, an inhibitor of PKC, attenuated the inhibition of N/OFQ on I(K). On the contrary, PDBu, an activator of PKC, augmented N/OFQ-evoked responses. The present study suggested that N/OFQ inhibited I(K) and PKC was involved in N/OFQ-evoked response in acutely dissociated rat cerebral parietal cortical neurons.     

Nonpeptide/peptide chimeric ligands for the nociceptin/orphanin FQ receptor: design, synthesis and in vitro pharmacological activity.

Nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand for the G-protein coupled receptor referred to as N/OFQ peptide (NOP) receptor. NOP receptor activation by N/OFQ modulates several biological functions both at central and peripheral level. Structure activity relationship (SAR) studies demonstrated that the N/OFQ sequence can be divided into a N-terminal tetrapeptide 'message' crucial for receptor activation and a C-terminal 'address' important for receptor binding. On the basis of this message/address concept we synthesized some chimeric compounds in which we substituted the natural message domain with the nonselective nonpeptide NOP ligand (8-Naphthalen-1-yl-methyl-4-oxo-1-phenyl-1,3,8-triaza-spiro[4,5]dec-3-yl)-aceticacid methyl ester (NNC 63-0532) and used as address domain the peptide sequences Thr-NH2, N/OFQ(5-9)-NH2, N/OFQ(5-13)-NH2 and N/OFQ(5-17)-NH2. All the compounds were pharmacologically evaluated in the electrically stimulated guinea-pig ileum. NNC 63-0532 produced a concentration-dependent inhibition of the electrically induced twitches showing, in comparison with N/OFQ, lower potency and higher maximal effects. In addition, contrary to N/OFQ, the effects of NNC 63-0532 were insensitive to the NOP selective antagonist [Nphe1, Arg14, Lys15]N/OFQ-NH2 (UFP-101) while prevented by naloxone. Similar results were obtained with NNC 63-0532/Thr-NH2 and NNC 63-0532/N/OFQ(1-9)-NH2. On the contrary, the inhibitory effects of NNC 63-0532/N/OFQ(5-13)-NH2 and NNC 63-0532/N/OFQ(5-17)-NH2 were slightly antagonized by UFP-101 while naloxone prevented the effects of the high but not of the low concentrations of the two ligands. These data indicate that it is possible to functionalize with the N/OFQ address sequence a nonpeptide NOP ligand for increasing its binding to the NOP receptor. Moreover, these results corroborate the idea that the 5-13 sequence represents the crucial core of the N/OFQ address domain.     

Designing bifunctional NOP receptor–mu opioid receptor ligands from NOP-receptor selective scaffolds. Part II

The nociceptin opioid receptor (NOP) and its endogenous peptide ligand nociceptin/orphanin FQ have been shown to modulate the pharmacological effects of the classical opioid receptor system. Suppression of opioid-induced reward associated with mu-opioid receptor (MOP)-mediated analgesia, without decreasing anti-nociceptive efficacy, can potentially be achieved with NOP agonists having bifunctional agonist activity at MOP, to afford ‘non-addicting’ analgesics. In Part II of this series, we describe a continuing structure–activity relationship (SAR) study of the NOP-selective piperidin-4-yl-1,3-dihydroindol-2-one scaffold, to obtain bifunctional activity at MOP, and a suitable ratio of NOP/MOP agonist activity that produces a non-addicting analgesic profile. The SAR reported here is focused on the influence of various piperidine nitrogen aromatic substituents on the ratio of binding affinity and intrinsic activity at both the NOP and MOP receptors.     

Structure-activity studies on different modifications of nociceptin/orphanin FQ: identification of highly potent agonists and antagonists of its receptor

Nociceptin/orphanin FQ (N/OFQ) and its receptor system modulate a variety of biological functions and further understandings of physiological and pathological roles of this system require new potent agonists and antagonists of its receptor. Two series of N/OFQ related analogues were synthesized to investigate the relationship of different modifications. We combined modifications including: (a) Phe⁴→(pF)Phe⁴; (b) Ala⁷, Ala¹¹→Aib⁷, Aib¹¹; (c) Leu¹⁴, Ala¹⁵→Arg¹⁴, Lys¹⁵. Compared with the first series, N-terminus of the second series was changed from Phe¹ to Nphe¹. All the analogues were amidated at C-terminus. These compounds were tested in binding studies on rat brain membranes and mouse vas deferens assay. Results indicated that the compounds of the first series showed higher affinity and potency than N/OFQ (pK_i = 9.33; pEC₅₀ = 7.50). In particular, [(pF)Phe⁴, Aib⁷, Aib¹¹, Arg¹⁴, Lys¹⁵] N/OFQ-NH₂ was found to be a highly potent agonist with pK_i = 10.78 in binding studies and pEC₅₀ = 9.37 in mouse vas deferens assay. The second series all competitively antagonized the effects of N/OFQ in mouse vas deferens assay. [Nphe¹, (pF)Phe⁴, Aib⁷, Aib¹¹, Arg¹⁴, Lys¹⁵] N/OFQ-NH₂ was the best antagonist with pA₂ = 8.39 and showed high binding affinity with pK_i = 9.99. Thus modifications which increase the potency of agonist have synergistic effect on biological activity and a replacement of N-terminus leads to shift of analogues from agonist to antagonist.     

Nociceptin/orphanin FQ and related peptides reduce the increase in plasma corticosterone elicited in mice by an intracerebroventricular injection

Nociceptin/orphanin FQ (=N/OFQ), the endogenous ligand of ORL1 receptor (=NOP), has been reported to induce, in rodents, after intracerebroventricular (i.c.v.) administration, anti-stress and anxiolytic effects. We have observed that the handling of mice followed by an i.c.v. injection of saline, induced a marked increase in the plasma corticosterone level (+250%) measured 30 minutes later. When N/OFQ was injected intracerebroventricularly, using a 1 microg dose, the increase in plasma corticosterone was significantly lower than in saline injected mice. N/OFQ(1-13)NH(2), known as a NOP receptor agonist, at the same 1 microg dose, also induced a lesser increase in plasma corticosterone level than a saline i.c.v. injection. The pseudopeptide [Phe(1)-psi(CH(2)-NH)Gly(2)]N/OFQ(1-13)NH(2), defined either as an agonist or an antagonist of NOP receptor, at the 0.1 microg dose, behaved in a similar manner as N/OFQ, by decreasing the plasma corticosterone level. Finally, [Nphe(1)]N/OFQ(1-13)NH(2), although presumed to be a selective NOP receptor antagonist, also decreased the corticosterone level at the 0.1 microg dose. These observations suggest the implication of N/OFQ in the regulation of response to stress, through an action on the hypothalamo-pituitary-adrenocortical axis. Moreover, they evidence a similar effect of N/OFQ and N/OFQ(1-13)NH(2), but also of two other related peptides displaying antagonist properties on NOP receptors. These data suggest that several subtypes of N/OFQ receptors could exist.     

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.     

Further evidence for an involvement of nociceptin/orphanin FQ in the pathophysiology of Parkinson's disease: a behavioral and neurochemical study in reserpinized mice

The contribution of nociceptin/orphanin FQ (N/OFQ) to reserpine-induced Parkinsonism was evaluated in mice. A battery of motor tests revealed that reserpine caused dose-dependent and long-lasting motor impairment. Endogenous N/OFQ sustained this response because N/OFQ peptide (NOP) receptor knockout (NOP(-/-) ) mice were less susceptible to the hypokinetic action of reserpine than wild-type (NOP(+/+) ) animals. Microdialysis revealed that reserpine elevated glutamate and reduced GABA levels in substantia nigra reticulata, and that resistance to reserpine in NOP(-/-) mice was accompanied by a milder increase in glutamate and lack of inhibition of GABA levels. To substantiate this genetic evidence, the NOP receptor antagonist 1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H benzimidazol-2-one (J-113397) simultaneously reduced akinesia and nigral glutamate levels in reserpinized NOP(+/+) mice, being ineffective in NOP(-/-) mice. Moreover, repeated J-113397 administration in reserpinized mice resulted in faster recovery of baseline motor performance which was, however, accompanied by a loss of acute antiakinetic response. The short-term beneficial effect of J-113397 was paralleled by normalization of nigral glutamate levels, whereas loss of acute response was paralleled by loss of the ability of J-113397 to inhibit glutamate levels. We conclude that endogenous N/OFQ contributes to reserpine-induced Parkinsonism, and that sustained NOP receptor blockade produces short-term motor improvement accompanied by normalization of nigral glutamate release.     

Exploring LPS-induced sepsis in rats and mice as a model to study potential protective effects of the nociceptin/orphanin FQ system

The nociceptin receptor (NOP) and its ligand nociceptin/orphanin FQ (N/OFQ) have been shown to exert a modulatory effect on immune cells during sepsis. We evaluated the suitability of an experimental lipopolysaccharide (LPS)-induced sepsis model for studying changes in the nociceptin system. C57BL/6 mice BALB/c mice and Wistar rats were inoculated with different doses of LPS with or without a nociceptin receptor antagonist (UFP-101 or SB-612111). In C57BL/6 mice LPS 0.85 mg/kg injection produced no septic response, whereas 1.2 mg/kg produced a profound response within 5 h. In BALB/c mice, LPS 4 mg/kg produced no response, whereas 7 mg/kg resulted in a profound response within 24 h. In Wistar rats LPS 15 mg/kg caused no septic response in 6/10 animals, whereas 25 mg/kg resulted in marked lethargy before 24 h. Splenic interleukin-1β mRNA in BALB/c mice, and serum TNF-α concentrations in Wistar rats increased after LPS injection in a dose-dependent manner, but were undetectable in control animals, indicating that LPS had stimulated an inflammatory reaction. IL-1β and TNF-α concentrations in LPS-treated animals were unaffected by administration of a NOP antagonist. Similarly NOP antagonists had no effect on survival or expression of mRNA for NOP or ppN/OFQ (the N/OFQ precursor) in a variety of tissues. In these animal models, the dose–response curve for LPS was too steep to allow use in survival studies and no changes in the N/OFQ system occurred within 24 h. We conclude that LPS-inoculation in rodents is an unsuitable model for studying possible changes in the NOP-N/OFQ system in sepsis.     

Release of orphanin FQ/nociceptin in the medial preoptic nucleus and ventromedial nucleus of the hypothalamus facilitates lordosis

Opioid regulation of reproduction has been widely studied. However, the role of opioid receptor-like 1 receptor (NOP; also referred to as ORL-1 and OP4) and its endogenous ligand orphanin FQ/nociceptin (OFQ/N) have received less attention despite their extensive distribution throughout nuclei of the limbic-hypothalamic system, a circuit that regulates reproductive behavior in the female rat. Significantly, the expression of both receptor and ligand is regulated in a number of these nuclei by estradiol and progesterone. Activation of NOP in the ventromedial nucleus of the hypothalamus (VMH) of estradiol-primed nonreceptive female rats facilitates lordosis. NOPs are also expressed in the medial preoptic nucleus (MPN), however, their roles in reproductive behavior have not been studied. The present experiments examined the role of NOP in the regulation of lordosis in the MPN and tested whether endogenous OFQ/N in the MPN and VMH mediates reproductive behavior. Activation of NOP by microinfusion of OFQ/N in the MPN facilitated lordosis in estradiol-primed sexually nonreceptive female rats. Passive immunoneutralization of OFQ/N in either the MPN or the VMH reduced lordosis in estradiol-primed females, but had no effect on lordosis in estradiol+progesterone-primed sexually receptive rats. These studies suggest that OFQ/N has a central role in estradiol-only induced sexual receptivity, and that progesterone appears to involve additional circuits that mediate estradiol+progesterone sexual receptivity.     

GABA(A) signalling is involved in N/OFQ anxiolytic-like effects but not in nocistatin anxiogenic-like action as evaluated in the mouse elevated plus maze

Nociceptin/orphanin FQ (N/OFQ) and nocistatin are two neuropeptides originated from the same precursor prepronociceptin/orphanin FQ (ppN/OFQ). N/OFQ is the endogenous ligand of the NOP receptor, while the target of action of nocistatin is still unknown. N/OFQ modulates various biological functions, including anxiety. Conversely, nocistatin either behaves as a functional N/OFQ antagonist or evokes per se effects opposite to those of N/OFQ. Here we investigated the interaction between the anxiolytic-like effects of N/OFQ and the anxiogenic-like action of nocistatin with those evoked by GABA_A receptor ligands in the mouse elevated plus maze. The anxiogenic-like effects of the GABA_A receptor antagonist pentylenetetrazol (20 mg/kg; intraperitoneal, i.p.) were abolished by the co-treatment with N/OFQ (10 pmol; intracerebroventricular, i.c.v.) while potentiated by the administration of nocistatin (0.01 pmol; i.c.v.). The anxiolytic-like effects of the benzodiazepine receptor agonist diazepam (0.75 mg/kg, i.p.) were reversed by nocistatin (0.1 pmol; i.c.v.), whereas signs of sedation were observed when mice were co-treated with diazepam and N/OFQ (3 pmol). Interesting enough, the i.p. treatment with flumazenil (1 mg/kg) blocked the anxiolytic-like effects of N/OFQ (10 pmol; i.c.v.), but not the anxiogenic effect elicited by nocistatin. Collectively, our findings suggest that the effects on anxiety elicited by pentylenetetrazol and diazepam can be counteracted or potentiated in the presence of N/OFQ and nocistatin. In addition, the effects on anxiety of N/OFQ, but not nocistatin, appear to be dependent on the benzodiazepine site of the GABA_A receptor.