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.     

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.     

Chronic intracerebroventricular infusion of nociceptin/orphanin FQ increases food and ethanol intake in alcohol-preferring rats

Central administration of low doses of nociceptin/orphanin FQ (N/OFQ), the endogenous ligand of the opioid-like orphan receptor NOP, have been shown to reduce ethanol consumption, ethanol-induced conditioned place preference and stress-induced reinstatement of alcohol-seeking behavior in alcohol preferring rats. The present study evaluated the effect of continuous (7 days) lateral brain ventricle infusions of N/OFQ (0, 0.25, 1, 4, and 8 microg/h), by means of osmotic mini-pumps, on 10% ethanol intake in Marchigian-Sardinian alcohol-preferring (msP) rats provided 2h or 24h access to it. N/OFQ dose-dependently increased food intake in msP rats. On the other hand, in contrast to previous studies with acute injections, continuous lateral brain ventricle infusion of high doses of N/OFQ increased ethanol consumption when the ethanol solution was available for 24h/day or 2h/day. The present study demonstrates that continuous activation of the opioidergic N/OFQ receptor does not blunt the reinforcing effects of ethanol. Moreover, the data suggest that continuous activation of the opioidergic N/OFQ receptor is not a suitable way to reduce alcohol abuse.     

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. '     

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.     

Functional studies using antibodies against orphanin FQ/nociceptin

Orphanin FQ/nociceptin (OFQ) is a recently discovered endogenous ligand for the novel opioid receptor-like receptor (ORL-1). There are numerous reports in the literature demonstrating paradoxical effects of exogenous OFQ on pain modulation. For example, OFQ produces a pronociceptive effect in the brain and an analgesic effect in the spinal cord. In order to better understand the physiological actions of OFQ, the present study focused on the pain-modulatory effect of endogenously released OFQ measured using antibody microinjection techniques. We found that electroacupuncture analgesia (EA) was increased by intracerebroventricular (i.c.v.) injection of an OFQ-antibody and decreased following intrathecal injection. Furthermore, i.c.v. OFQ-antibody partially reversed tolerance to both chronic morphine and chronic EA. These data suggest that endogenously released OFQ plays an important role in pain modulation, where pain sensitivity in the brain and spinal cord is increased and decreased, respectively.     

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.     

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.     

Biostable aptamers with antagonistic properties to the neuropeptide nociceptin/orphanin FQ

The neuropeptide nociceptin/orphanin FQ (N/OFQ), the endogenous ligand of the opioid receptor-like 1 (ORL1) receptor, has been shown to play a prominent role in the regulation of several biological functions such as pain and stress. Here we describe the isolation and characterization of N/OFQ binding biostable RNA aptamers (Spiegelmers) using a mirror-image in vitro selection approach. Spiegelmers are L-enantiomeric oligonucleotide ligands that display high affinity and specificity to their targets and high resistance to enzymatic degradation compared to D-oligonucleotides. A representative Spiegelmer from the selections performed was size-minimized to two distinct sequences capable of high affinity binding to N/OFQ. The Spiegelmers were shown to antagonize binding of N/OFQ to the ORL1 receptor in a binding-competition assay. The calculated IC(50) values for the Spiegelmers NOX 2149 and NOX 2137a/b were 110 nM and 330 nM, respectively. The competitive antagonistic properties of these Spiegelmers were further demonstrated by their effective and specific inhibition of G-protein activation in two additional models. The Spiegelmers antagonized the N/OFQ-induced GTPgammaS incorporation into cell membranes of a CHO-K1 cell line expressing the human ORL1 receptor. In oocytes from Xenopus laevis, NOX 2149 showed an antagonistic effect to the N/OFQ-ORL 1 receptor system that was functionally coupled with G-protein-regulated inwardly rectifying K(+) channels.     

Decreased plasma nociceptin/orphanin FQ levels after acute coronary syndromes

Foregoing researches made on the N/OFQ system brought up a possible role for this system in cardiovascular regulation. In this study we examined how N/OFQ levels of the blood plasma changed in acute cardiovascular diseases. Three cardiac patient groups were created: enzyme positive acute coronary syndrome (EPACS, n = 10), enzyme negative ACS (ENACS, n = 7) and ischemic heart disease (IHD, n = 11). We compared the patients to healthy control subjects (n = 31). We found significantly lower N/OFQ levels in the EPACS [6.86 (6.21-7.38) pg/ml], ENACS [6.97 (6.87-7.01) pg/ml and IHD groups [7.58 (7.23-8.20) pg/ml] compared to the control group [8.86 (7.27-9.83) pg/ml]. A significant correlation was detected between N/OFQ and white blood cell count (WBC), platelet count (PLT), creatine kinase (CK), glutamate oxaloacetate transaminase (GOT) and cholesterol levels in the EPACS group.Decreased plasma N/OFQ is closely associated with the presence of acute cardiovascular disease, and the severity of symptoms has a significant negative correlation with the N/OFQ levels. We believe that the rate of N/OFQ depression is in association with the level of ischemic stress and the following inflammatory response. Further investigations are needed to clarify the relevance and elucidate the exact effects of the ischemic stress on the N/OFQ system.     

Proinflammatory and vasodilator effects of nociceptin/orphanin FQ in the rat mesenteric microcirculation are mediated by histamine

Nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand for the N/OFQ peptide receptor (NOP). N/OFQ causes hypotension and vasodilation, and we aimed to determine the role of histamine in inflammatory microvascular responses to N/OFQ. Male Wistar rats (220-300 g, n = 72) were anesthetized with thiopental (30 mg/kg bolus, 40-90 mg x kg(-1) x h(-1) iv), and the mesentery was prepared for fluorescent intravital microscopy using fluorescein isothiocyanate-conjugated BSA (FITC-BSA, 0.25 ml/100 g iv) or 1 microm fluorescently labeled microspheres. N/OFQ (0.6-60 nmol/kg iv) caused hypotension (SAP, baseline: 154 +/- 11 mmHg, 15 nmol/kg N/OFQ: 112 +/- 10 mmHg, P = 0.009), vasodilation (venules: 23.9 +/- 1.2 microm, 26.7 +/- 1.2 microm, P = 0.006), macromolecular leak (interstitial gray level FITC-BSA: 103.7 +/- 3.4, 123.5 +/- 11.8, P = 0.009), and leukocyte adhesion (2.0 +/- 0.9, 15.2 +/- 0.9/100 microm, P = 0.036). Microsphere velocity also decreased (venules: 1,230 +/- 370 microm/s, P = 0.037), but there were no significant changes in blood flow. Flow cytometry measured a concurrent increase in neutrophil expression of cd11b with N/OFQ vs. controls (Geo mean fluorescence: 4.19 +/- 0.13 vs. 2.06 +/- 0.38, P < 0.05). The NOP antagonist [Nphe(1),Arg(14),Lys(15)]N/OFQ-NH(2) (UFP-101; 60 and 150 nmol/kg iv), H(1) and H(2)antagonists pyrilamine (mepyramine, 1 mg/kg iv) and ranitidine (1 mg/kg iv), and mast cell stabilizer cromolyn (1 mg x kg(-1) x min(-1)) also abolished vasodilation and macromolecular leak to N/OFQ in vivo (P < 0.05), but did not affect hypotension. Isolated mesenteric arteries (approximately 200 microm, n = 25) preconstricted with U-46619 were also mounted on a pressure myograph (60 mmHg), and both intraluminally and extraluminally administered N/OFQ (10(-5) M) caused dilation, inhibited by pyrilamine in the extraluminal but not the intraluminal (control: -6.9 +/- 3.8%; N/OFQ: 32.6 +/- 8.4%; pyrilamine: 31.5 +/- 6.8%, n = 18, P < 0.05) experiments. We conclude that, in vivo, mesenteric microvascular dilation and macromolecular leak occur via N/OFQ-NOP-mediated release of histamine from mast cells. Therefore, N/OFQ-NOP has an important role in microvascular inflammation, and this may be targeted during disease, particularly as we have proven that UFP-101 is an effective antagonist of microvascular responses in vivo.     

Kainate seizures increase nociceptin/orphanin FQ release in the rat hippocampus and thalamus: a microdialysis study

The neuropeptide nociceptin/orphanin FQ (N/OFQ) has been suggested to play a facilitatory role in kainate seizure expression. Furthermore, mRNA levels for the N/OFQ precursor are increased following kainate seizures, while its receptor (NOP) density is decreased. These data suggest increased N/OFQ release. To obtain direct evidence that this is the case, we have developed a microdialysis technique, coupled with a sensitive radioimmunoassay, that allows measurement of N/OFQ release from the hippocampus and thalamus of awake, freely moving animals. In both these brain areas, the spontaneous N/OFQ efflux decreased by approximately 50% and 65% when Ca2+ was omitted and when tetrodotoxin was added to the perfusion medium, respectively. Perfusion of the dialysis probe with high K+ increased N/OFQ release (approximately threefold) in a Ca2+-dependent and tetrodotoxin-sensitive manner. Kainate seizures caused a twofold increase in N/OFQ release followed, within 3 h, by a return to baseline levels. Approximately 5 h after kainate, a late increase in N/OFQ release was observed. On the following day, when animals were having only low grade seizures, N/OFQ release was not significantly different from normal. These phenomena were observed with similar patterns in the hippocampus and in the thalamus. The present data indicate that acute limbic seizures are associated with increased N/OFQ release, which may prime the molecular changes described above, i.e. cause down-regulation of NOP receptors and activation of N/OFQ biosynthesis.     

Relationship between nociceptin/orphanin FQ and cerebral hemodynamics after hypoxia-ischemia in piglets

This study was designed to characterize the role of the newly described endogenous opioid nociceptin/orphanin FQ (NOC/oFQ) in reduced cerebral blood flow (CBF) observed after ischemia-reperfusion (I/R) and combined hypoxia and ischemia-reperfusion (H-I/R), as a function of time after onset of reperfusion in newborn pigs equipped with a closed cranial window. Global cerebral ischemia (20 min) was induced via elevation of intracranial pressure, whereas hypoxia (10 min) decreased PO(2) to 35 +/- 3 mmHg with unchanged PCO(2). I/R elevated cerebrospinal fluid (CSF) NOC/oFQ from 67 +/- 4 to 266 +/- 29 pg/ml within 1 h, whereas values returned to control level within 4 h of reperfusion. H-I/R elevated CSF NOC/oFQ to 483 +/- 67 pg/ml within 1 h, and such values returned slowly to control level within 12 h of reperfusion. Topical NOC/oFQ (10(-8) M, 10(-6) M)-induced vasodilation was attenuated by I/R and reversed to vasoconstriction by H-I/R at 1 h of reperfusion (control, 9 +/- 1 and 16 +/- 1%; I/R, 3 +/- 1 and 6 +/- 1%; H-I/R, -6 +/- 1 and -11 +/- 1%). Such altered dilation returned to control values within 4 h in I/R animals and within 12 h in H-I/R animals. Blood flow in the cerebrum was reduced from 58 +/- 4 to 33 +/- 2 ml x min(-1) x 100 g(-1) within 1 h and returned to control value within 4 h in I/R animals. In animals pretreated with [F/G]NOC/oFQ(1-13)-NH(2) (1 mg/kg iv), an NOC/oFQ antagonist, however, CBF only fell to 43 +/- 3 ml x min(-1) x 100 g(-1) at 1 h of reperfusion. Similar observations were made in H-I/R animals. These data suggest that an elevated CSF NOC/oFQ concentration and altered vascular responsiveness to this opioid contribute to reductions in CBF observed after either I/R or H-I/R.     

Functional interaction between nociceptin/orphanin FQ and alpha-melanocyte-stimulating hormone in the regulation of feeding

Nociceptin/orphanin FQ (N/OFQ), an endogenous agonist of the opioid N/OFQ (NOP) receptor, increases food intake when administered centrally. As N/OFQ is part of a larger neural network that governs consummatory behavior, presumably its orexigenic properties stem from interplay with other neuropeptidergic components of the feeding-related circuitry. One such peptide may be the ligand of the melanocortin-3 and -4 receptors, alpha-melanocyte-stimulating hormone (alpha-MSH), which is known to inhibit food intake. The aim of the present study was to establish whether there is a functional "interaction" between N/OFQ and alpha-MSH in the regulation of feeding. By using double immunostaining for c-Fos and alpha-MSH, we found that intracerebroventricular (i.c.v.) injection of N/OFQ at a 10nmol dose that moderately prolongs deprivation-induced food intake in rats, decreases activation of alpha-MSH neurons involved in feeding termination. However, i.c.v. injections of alpha-MSH at doses previously established to reduce deprivation-induced feeding, do not decrease hyperphagia generated by N/OFQ in ad libitum-fed animals. Our results suggest that while alpha-MSH does not appear to modify the orexigenic response to N/OFQ in sated rats, the NOP receptor ligand promotes a decrease in activation of neurons synthesizing the anorexigenic peptide, alpha-MSH, at the time of re-feeding. Thus, to some degree, the stimulatory effect of N/OFQ on consumption may arise from this peptide's inhibitory influence on activity of anorexigenic pathways containing alpha-MSH.     

Blockade of the nociceptin/orphanin FQ/NOP receptor system in the rat ventrolateral periaqueductal gray potentiates DAMGO analgesia

Nociceptin/orphanin FQ (N/OFQ) and its receptor (NOP) are involved in various biological functions including pain. High density of NOP receptor has been found in the ventrolateral periaqueductal gray (vlPAG), the main output pathway involved in descending pain-control system. The aim of our work was to evaluate the involvement of the N/OFQ/NOP system in the modulation of MOP analgesia in the rat vlPAG using UFP-101, a selective NOP antagonist. N/OFQ significantly blocked DAMGO (a selective MOP agonist) analgesia, while UFP-101 enhanced the effect of the opioid given at a subanalgesic dose. These results confirm our hypothesis of an antiopioid role for N/OFQ in the vlPAG.     

A cellular mechanism for the bidirectional pain-modulating actions of orphanin FQ/nociceptin

Orphanin FQ/nociceptin (OFQ/N) and its receptor share substantial structural features and cellular actions with classic opioid peptides and receptors, but have distinct pharmacological profiles and behavioral effects. Currently there is an active debate about whether OFQ/N produces hyperalgesia or analgesia. Using a well-defined brainstem pain-modulating circuit, we show that OFQ/N can cause either an apparent hyperalgesia by antagonizing mu opioid-induced analgesia or a net analgesic effect by reducing the hyperalgesia during opioid abstinence. It presumably produces these two opposite actions by inhibiting two distinct groups of neurons whose activation mediates the two effects of opioid administration. OFQ/N antagonism of the hyperalgesia may have significance for the treatment of opioid withdrawal and sensitized pain.