Agonistic effects of the opioid buprenorphine on the nociceptin/OFQ receptor

The nociceptin/orphanin FQ (N/OFQ) receptor (e.g. the human ortholog ORL1) has been shown to be pharmacologically distinct from classic opioid receptors. Recently, we have identified buprenorphine as a full ORL1 agonist using a reporter gene assay. For further functional analysis, buprenorphine's effects on ORL1 receptors were investigated using a K(+) channel (GIRK1) assay in Xenopus oocytes and GTPgammaS assay in CHO-K1 membrane preparations. In both assays, buprenorphine behaved as a partial agonist compared to nociceptin itself. The N/OFQ agonism of buprenorphine might contribute to actions of buprenorphine in pain models in vivo beside its mu- or kappa-opioid receptor mediated effects.     

Nociceptin/Orphanin FQ Metabolism: Role of Aminopeptidase and Endopeptidase 24.15

Abstract: The endogenous opioid receptor-like₁ (ORL₁) ligand, nociceptin/orphanin FQ (FGGFTGARKSARKLANQ), a heptadecapeptide structurally resembling dynorphin A, has recently been identified. The wide distribution of ORL₁ mRNA and nociceptin/orphanin FQ precursor in the CNS, particularly in the limbic system regions and in several areas known to be involved in pain perception, suggests that nociceptin/orphanin FQ is potentially endowed with various central functions. In general, activation and/or inactivation of regulatory peptides occur through the action of cell surface peptidases. The physiological mechanisms under which nociceptin/orphanin FQ is metabolized should lead to a better understanding of its physiological functions. Mouse brain cortical slices were incubated in medium containing the heptadecapeptide in the presence or in the absence of peptidase inhibitors. The critical sites of enzymatic cleavage are Phe¹-Gly², Ala⁷-Arg⁸, Ala¹¹-Arg¹², and Arg¹²-Lys¹³ bonds. The major role played by metallopeptidases was confirmed by the complete protection of metabolism in the presence of EDTA. Aminopeptidase N and endopeptidase 24.15 are the two main enzymes involved in nociceptin/orphanin FQ metabolism, whereas endopeptidase 24.11 (involved in enkephalin [YGGFM(L)] catabolism) does not appear critically involved in nociceptin/orphanin FQ metabolism. The physiological relevance of aminopeptidase N and endopeptidase 24.15 in the heptadecapeptide metabolism remains to be determined.     

Design, synthesis, and biological evaluation of indole derivatives as novel nociceptin/orphanin FQ (N/OFQ) receptor antagonists

A novel series of 2-(1,2,4-oxadiazol-5-yl)-1H-indole derivatives as nociceptin/orphanin FQ (N/OFQ) receptor antagonists was discovered. Systematic modification of our original lead by changing the pendant functional groups, linker, heterocyclic core, and basic side chain revealed the structure-activity requirements for this novel template and resulted in the identification of more potent analog with improved potency as compared to the parent compound.     

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.     

Effect of orphanin FQ/nociceptin (OFQ/N) and isoflurane on the prolactin secretory response in OFQ/N knockout mice

The prolactin secretory response to subcutaneous injection of orphanin FQ/nociceptin (OFQ/N) was measured in wild-type and OFQ/N knockout mice. These injections were given with and without isoflurane anesthesia, to determine if isoflurane would affect the prolactin secretory response. OFQ/N injection significantly increased prolactin levels in males and females, regardless of genotype, with a more robust response in females. Isoflurane pretreatment did not affect prolactin levels in controls or in animals injected with OFQ/N. This is the first report that exogenously administered OFQ/N stimulates prolactin secretion in mice and that brief isoflurane exposure does not significantly affect this response.     

Tissue distribution of the opioid receptor-like (ORL1) receptor

The ORL1 receptor is a G protein-coupled receptor structurally related to the opioid receptors, whose endogenous ligand is the heptadecapeptide nociceptin/orphanin FQ. In this review, data which have contributed to the mapping of the anatomic distribution of the ORL1 receptor have been collated with an emphasis on their relation to physiological functions. The ORL1 receptor is widely expressed in the central nervous system, in particular in the forebrain (cortical areas, olfactory regions, limbic structures, thalamus), throughout the brainstem (central periaqueductal gray, substantia nigra, several sensory and motor nuclei), and in both the dorsal and ventral horns of the spinal cord. Regions almost devoid of ORL1 receptors are the caudate-putamen and the cerebellum. ORL1 mRNA and binding sites exhibit approximately the same distribution pattern, indicating that the ORL1 receptor is located on local neuronal circuits. The ORL1 receptor is also expressed at the periphery in smooth muscles, peripheral ganglia, and the immune system. The anatomic distribution of ORL1 receptor suggests a broad spectrum of action for the nociceptin/orphanin FQ system (sensory perception, memory process, emotional behavior, etc.).     

An opioid system in connective tissue: a study of achilles tendon in the rat.

The occurrence of endogenous opioids and their receptors in rat achilles tendon was analyzed by immunohistochemistry (IHC), radioimmunoassay (RIA), and in vitro binding assays. The investigation focused on four enkephalins, dynorphin B, and nociceptin/orphanin FQ. Nerve fibers immunoreactive to all enkephalins (Met-enkephalin, Leu-enkephalin, Met-enkephalin-Arg-Gly-Lys, Met-enkephalin-Arg-Phe) were consistently found in the loose connective tissue and the paratenon, whereas dynorphin B and nociceptin/orphanin FQ could not be detected. The majority of enkephalin-positive nerve fibers exhibited varicosities predominantly seen in blood vessel walls. Measurable levels of Met-enkephalin-Arg-Phe and nociceptin/orphanin FQ were found in tendon tissue using RIA, whereas dynorphin B could not be detected. In addition to the endogenous opioids identified, delta-opioid receptors on nerve fibers were also detected by IHC. Binding assays to characterize the opioid binding sites showed that they were specific and saturable for [3H]-naloxone (Kd 7.01 +/- 0.98 nM; Bmax 23.52 +/- 2.23 fmol/mg protein). Our study demonstrates the occurrence of an opioid system in rat achilles tendon, which may be assumed to be present also in other connective tissues of the locomotor apparatus. This system may prove to be a useful target for pharmacological therapy in painful and inflammatory conditions by new drugs acting selectively in the periphery.     

The relaxant effect of nociceptin on porcine coronary arterial ring segments

Nociceptin (NC), alias orphanin FQ, has been identified as the endogenous ligand of the opioid receptor-like 1 receptor (ORL1). The purpose of this study was to assess the effect of nociceptin on porcine coronary arteries and to investigate the mechanism of its action, if any. Rings of coronary arteries from porcine hearts were suspended in baths containing Krebs solution, and isometric tension was measured. The response to nociceptin (10(-1)2-10(-5) mol/L) was investigated in porcine coronary arterial rings and also in such rings contracted with prostaglandin F2alpha (PGF2alpha). The effects of endothelium, nitroxide (NO), methylene blue, cyclic GMP (cGMP), naloxone, [Nphe1]NC(1-13)NH2, and propranolol on nociceptin-induced relaxation were also assessed. Our study showed nociceptin relaxed the porcine coronary arterial rings and inhibited the vasocontractivity to PGF2alpha. The relaxing response of nociceptin in coronary arteries was significantly reduced by removal of endothelium and by the presence of L-NNA, cGMP, and [Nphe1]NC(1-13)NH2, the selective nociceptin receptor antagonist, but not by naloxone, the nonselestive opioid receptor blocker or propranolol, which blocks the adrenergic beta-receptor. Our results suggest that nociceptin induces relaxation of isolated coronary artery through NO, cGMP, and ORL1.     

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.     

Intracerebroventricular Injection of the Glutamatergic Receptors Antagonist Affects N/OFQ-Induced Hyperphagia in Neonatal Broilers: Role of NMDA and AMPA Receptors

International Journal of Peptide Research and Therapeutics - This study was designed to determine possible interaction of the central nociceptin/orphanin FQ (N/OFQ) and glutamatergic system on...     

Nociceptin/Orphanin FQ (N/OFQ) Receptors are Involved in Adrenaline-Induced Feeding Behavior in Broiler Cockerels

Nociceptin/orphanin FQ (N/OFQ) is an endogenous ligand of a G protein-coupled receptornamed NOP. This neuropeptide has been identified as an orexigenic stimulus in the brain of birds and mammals. The purpose of the present study was to clarify whether blockade or stimulation of nociceptin receptors affects adrenaline-induced feeding behaviour in broilers. In Experiment 1, birds received intracerebroventricular (ICV) injection of Nociceptin (1–13) NH2 (potent NOP receptor agonist, 16 nmol) followed by adrenaline (80 nmol). In Experiment 2, the birds received UFP-101, (NOP receptor antagonist, 10 nmol) prior to injection of adrenaline (80 nmol). Cumulative food and water intake was measured at 2 h post-injection. When administrated alone, adrenaline significantly increased food and water intake. The ICV injection of Nociceptin (1–13) NH2 significantly increased food intake but not water intake. Pre-injection of Nociceptin (1–13) NH2 significantly increased the adrenaline-induced feeding response. The effect of adrenaline on food intake was transiently blocked by microinjection of UFP-101. UFP-101-induced anorexia was accompanied by a transient increase in water intake. The transient dipsogenic effect of UFP-101 suggests a role of endogenous N/OFQ-NOP receptor pathways in the regulation of water intake in chickens, which is food intake-independent. These results also provide further evidence for a reciprocal interaction between adrenergic receptors and N/OFQ on feeding behavior.     

Nociceptin/Orphanin FQ(1-13)NH2 analogues modified in the Phe1-Gly2 peptide bond

The synthesis and pharmacological activity of novel nociceptin/orphanin FQ (N/OFQ) analogues modified in the Phe(1)-Gly(2) peptide bond are reported. The aim of the present work was to elucidate the importance of this peptide bond for the N/OFQ receptor (NOP) interaction. Our study indicates that the first peptide bond in N/OFQ is important but not crucial for interaction with the N/OFQ receptor; for instance, substitution with a methyleneoxy bond generates an agonist derivative just 3-fold less potent than the reference compound.     

The effects of nociceptin peptide (N/OFQ)–receptor (NOP) system activation in the airways

The heptadecapeptide nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand for the N/OFQ peptide (NOP) receptor. It is cleaved from a larger precursor identified as prepronociceptin (ppN/OFQ). NOP is a member of the seven transmembrane-spanning G-protein coupled receptor (GPCR) family. ppN/OFQ and NOP receptors are widely distributed in different human tissues. Asthma is a complex heterogeneous disease characterized by variable airflow obstruction, bronchial hyper-responsiveness and chronic airway inflammation. Limited therapeutic effectiveness of currently available asthma therapies warrants identification of new drug compounds. Evidence from animal studies suggests that N/OFQ modulates airway contraction and inflammation. Interestingly up regulation of the N/OFQ–NOP system reduces airway hyper-responsiveness. In contrast, inflammatory cells central to the inflammatory response in asthma may be both sources of N/OFQ and respond to NOP activation. Hence paradoxical dysregulation of the N/OFQ–NOP system may potentially play an important role in regulating airway inflammation and airway tone. To date there is no data on N/OFQ–NOP expression in the human airways. Therefore, the potential role of N/OFQ–NOP system in asthma is unknown. This review focuses on its physiological effects within airways and potential value as a novel asthma therapy.     

Opioid and nociceptin receptors regulate cytokine and cytokine receptor expression

Opioids were originally discovered because of their ability to induce analgesia, but further investigation has shown that the opioids regulate the function of cells involved in the immune response. We suggest that the regulation of cytokine, chemokine, and cytokine receptor expression is a critical component of the immunomodulatory activity of the opioids. In this paper we review the literature dealing with the regulation of cytokine and cytokine receptor expression by agonists for the three major opioid receptor types (mu, kappa, and delta), and nociceptin, the natural agonist for the orphanin FQ/nociceptin receptor. Although the opioid receptors share a high degree of sequence homology, opposing roles between the kappa opioid receptor (KOR) and the mu opioid receptor (MOR) have become apparent. We suggest that activation of the KOR induces an anti-inflammatory response through the down-regulation of cytokine, chemokine and chemokine receptor expression, while activation of the MOR favors a pro-inflammatory response. Investigation into the opioid receptor-like (ORL1)/nociceptin system also suggests a role for this receptor as a down-regulator of immune function. These effects suggest a broad role for opioids in the modulation of the function of the immune system, and suggest possible targets for the development of new therapeutics for inflammatory and infectious diseases.     

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.     

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.     

Cellular uptake and biological activity of peptide nucleic acids conjugated with peptides with and without cell‐penetrating ability

A 12‐mer peptide nucleic acid (PNA) directed against the nociceptin/orphanin FQ receptor mRNA was disulfide bridged with various peptides without and with cell‐penetrating features. The cellular uptake and the antisense activity of these conjugates were assessed in parallel. Quantitation of the internalized PNA was performed by using an approach based on capillary electrophoresis with laser‐induced fluorescence detection (CE‐LIF). This approach enabled a selective assessment of the PNA moiety liberated from the conjugate in the reducing intracellular environment, thus avoiding bias of the results by surface adsorption. The biological activity of the conjugates was studied by an assay based on the downregulation of the nociceptin/orphanin FQ receptor in neonatal rat cardiomyocytes (CM). Comparable cellular uptake was found for all conjugates and for the naked PNA, irrespective of the cell‐penetrating properties of the peptide components. All conjugates exhibited a comparable biological activity in the 100 nM range. The naked PNA also exhibited extensive antisense activity, which, however, proved about five times lower than that of the conjugates. The found results suggest cellular uptake and the bioactivity of PNA‐peptide conjugates to be not primarily related to the cell‐penetrating ability of their peptide components. Likewise from these results it can be inferred that the superior bioactivity of the PNA‐peptide conjugates in comparison with that of naked PNA rely on as yet unknown factors rather than on higher membrane permeability. Several hints point to the resistance against cellular export and the aggregation propensity combined with the endocytosis rate to be candidates for such factors. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Anxiolytic-like effects of nociceptin/orphanin FQ in the elevated plus maze and in the conditioned defensive burying test in rats

Different reports suggest that nociceptin/orphanin FQ (N/OFQ) may have either anxiolytic- or anxiogenic-like effect in rodents. Since N/OFQ elicits hypolocomotion, which undergoes rapid tolerance, and hypolocomotion may be associated to emotional consequences, the present study was designed to investigate the effect of N/OFQ on anxiety after development of tolerance to its hypolocomotor effect. The effect of single or double intracerebroventricular (i.c.v.) injection of N/OFQ was evaluated on anxiety-related behaviors in rats, in the elevated plus maze (EPM) and conditioned defensive burying (CDB) tests. After single administration, N/OFQ displayed an anxiogenic-like pattern of response on the elevated plus maze but hypolocomotion was also observed. Conversely, in the CDB test, N/OFQ induced a clear-cut anxiolytic pattern. To produce tolerance to N/OFQ-induced hypolocomotion the peptide was administered by two i.c.v. injections separated by 120 min; in these conditions it decreased the expression of anxiety-related behaviors in both tests without affecting locomotor activity. The nociceptin/orphanin FQ peptide (NOP) receptor antagonist UFP-101 significantly reduced the effects of N/OFQ to control values in either tests. Corticosterone levels were significantly increased after a single N/OFQ administration (not in a dose-dependent manner) but this increase did not reach significance after double administration (1 nmol/rat). Our results support the idea that N/OFQ may act as an anxiolytic-like agent in the rat; the apparent anxiogenic-like effect observed following its single administration in the EPM may be consequent to its effect on locomotion.     

N/OFQ system in brain areas of nerve‐injured mice: its role in different aspects of neuropathic pain

Several studies showed that chronic pain causes reorganization and functional alterations of supraspinal brain regions. The nociceptin‐NOP receptor system is one of the major systems involved in pain control and much evidence also suggested its implication in stress, anxiety and depression. Therefore, we investigated the nociceptin‐NOP system alterations in selected brain regions in a neuropathic pain murine model. Fourteen days after the common sciatic nerve ligature, polymerase chain reaction (PCR) analysis indicated a significant decrease of pronociceptin and NOP receptor mRNA levels in the thalamus; these alterations could contribute to the decrease of the thalamic inhibitory function reported in neuropathic pain condition. Nociceptin peptide and NOP mRNA increased in the anterior cingulate cortex (ACC) and not in the somatosensory cortex, suggesting a peculiar involvement of this system in pain regulating circuitry. Similarly to the ACC, an increase of nociceptin peptide levels was observed in the amygdala. Finally, the pronociceptin and NOP mRNAs decrease observed in the hypothalamus reflects the lack of hypothalamus‐pituitary‐adrenal axis activation, already reported in neuropathic pain models. Our data indicate that neuropathic pain conditions affect the supraspinal nociceptin‐NOP system which is also altered in regions known to play a role in emotional aspects of pain.     

Orphanin FQ/nociceptin inhibits morphine withdrawal

The influence of orphanin FQ/nociceptin (OFQ/N) on the morphine-withdrawal symptom was investigated. Withdrawal syndrome was induced in the morphine-dependent rats by an intraperitoneal (i.p.) injection of 2 mg/kg naloxone hydrochloride--an opioid receptors antagonist. Wet-dog shakes were used as a measure of the abstinence syndrome. Intraventricular injections of OFQ/N (5-20 microg/animal) caused significant inhibition of the withdrawal signs at doses between 15-20 microg, in the morphine-dependent rats. OFQ/N alone did not change behavior of the morphine-dependent animals. The obtained results indicate that OFQ/N can inhibit the morphine withdrawal symptoms induced by naloxone.