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.     

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. '     

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.     

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.     

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.     

Nocistatin: a novel neuropeptide encoded by the gene for the nociceptin/orphanin FQ precursor

We identified a novel neuropeptide and named it "nocistatin." Its presence was expected by analysis of the precursor for the neuropeptide nociceptin or orphanin FQ (Noc/OFQ), previously identified as an endogenous ligand for the orphan opioid receptor-like receptor. The precursor prepronociceptin/orphanin FQ (ppNoc/OFQ) comprises at least two bioactive peptides, nocistatin and Noc/OFQ. Noc/OFQ is involved in a broad range of pharmacological actions in various tissues from the central nervous system to the periphery. In pain transmission, Noc/OFQ is reported to have different effects including nociception, no effect, and analgesia, depending on the animal species tested, doses, route of administration, and so on. We found that intrathecal administration of Noc/OFQ induced pain responses including allodynia and hyperalgesia. Simultaneous administration of nocistatin blocked the allodynia and hyperalgesia induced by Noc/OFQ, whereas anti-nocistatin antibody decreased the threshold for the Noc/OFQ-induced allodynia. The endogenous heptadecapeptide nocistatin was isolated from bovine brains and recently identified in mouse, rat, and human brain and in human cerebrospinal fluid. Although human, rat and mouse ppNoc/OFQ produced larger respective counterparts with 30, 35, and 41 amino acid residues, all peptides showed the antinociceptive activity. This activity was ascribed to the carboxyl-terminal hexapeptide of nocistatin, Glu-Gln-Lys-Gln-Leu-Gln, which is conserved beyond species. Nocistatin also attenuated the allodynia and hyperalgesia evoked by prostaglandin E(2) and the inflammatory hyperalgesia induced by formalin or carrageenan/kaolin, and reversed the Noc/OFQ-induced inhibition of morphine analgesia at picogram doses. Furthermore, nocistatin counteracted the impairment of learning and memory induced by Noc/OFQ or scopolamine. Nocistatin is widely present in the spinal cord and brain. Although nocistatin did not bind to the Noc/OFQ receptor, it bound to the membrane of mouse brain and spinal cord with a high affinity. Nocistatin is a novel bioactive peptide produced from the same precursor as Noc/OFQ, and it plays important roles in the regulation of pain transmission and learning and memory processes in the central nervous system.     

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.     

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.     

Orphanin FQ/nociceptin receptor binding studies

A review of the binding studies performed on the receptor (ORL) for Orphanin FQ/Nociceptin is presented. Binding studies have been conducted using a variety of receptor sources: cell lines expressing the cloned receptor, cell lines endogenously expressing the receptor, and brain and other tissue from several different species. Binding studies of opioids, new ligands and antagonists at the ORL receptor are briefly discussed. Saturation, competition and binding kinetic experiments, and the effects of buffer composition are reviewed. There are numerous instances of conflicting data in published reports on OFQ; the basis for these disparities is as yet undetermined. This review endeavors to compile the results and conditions employed in binding studies as an aid to current and new researchers in this field. In an attempt to explain binding disparities, we have determined that Orphanin/Nociceptin binds to glass fiber filtermats in a "specific" manner; these new data are presented.     

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.     

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.     

Neurohumoral effects of orphanin FQ/nociceptin: relevance to cardiovascular and renal function

Orphanin FQ/Nociceptin (OFQ/N) is a peptide whose structure resembles that of the endogenous opioid peptides (endorphins). OFQ/N and its receptor are distributed in neural tissue and brain regions involved in the regulation of pituitary hormone release. Functional studies have shown that this peptide evokes a unique pattern of cardiovascular and renal excretory responses. This review will focus on the neural and humoral effects of OFQ/N and how this peptide may participate in the regulation of cardiovascular and renal function.     

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.     

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.     

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.     

Effects of naloxone benzoylhydrazone on native and recombinant nociceptin/orphanin FQ receptors

We have studied the effects of naloxone benzoylhydrazone (NalBzoH) at recombinant human OP4 receptors expressed in Chinese hamster ovary (CHO) cells (CHOhOP4) and native OP4 sites in isolated tissues from various species. In CHOhOP4 membranes, nociceptin (NC) and NalBzoH displaced [125I]Tyr14-NC with pKi values of 10.1 and 7.3. In the presence of 100 microM GDP, NC stimulated GTPgamma35S binding (pEC50 = 8.5). NalBzoH was ineffective but antagonized the effects of NC (pA2 = 6.9). At 5 microM GDP, there was an increase in potency (pEC50 = 9.3) and efficacy (4.3-fold) of NC. NalBzOH was a partial agonist (pEC50 = 7.0, Emax = 13% relative to NC). In CHOhOP4 cells, NC and NalBzoH inhibited cAMP formation with pEC50 and Emax values of 9.8 and 100% and 6.0 and 44%, respectively. In the rat vas deferens, NalBzoH (10 microM) did not modify electrically induced twitches but competitively antagonized the inhibitory action of NC (pA2 = 6.2). In the mouse vas deferens (mVD) and guinea pig ileum (gpI), NalBzoH inhibited twitches with pEC50 and Emax values of 7.6 and 78% and 8.5 and 77%, respectively. The effect of 3 microM NalBzoH was fully inhibited by 3 microM naloxone in mVD and 30 microM in gpI. Under these conditions, NalBzoH antagonized the actions of NC in both preparations with pA2 values of 6.3 and 6.8, respectively. Collectively, these data demonstrate that NalBzoH is a nonselective OP4 ligand with system-dependent behaviour.     

The orphanin FQ/nociceptin gene: structure, tissue distribution of expression and functional implications obtained from knockout mice

Like other neuropeptides, orphanin FQ/nociceptin (OFQ/N) is encoded by a larger precursor protein. The cDNA for the OFQ/N precursor has been cloned from human, rat, mouse and bovine tissue demonstrating that this peptidergic system serves important functions that have been conserved during evolution. The structural organization of the precursor protein is similar to opioid peptide precursors, supporting the view of a common origin for the opioid systems and the OFQ/N system. In addition to OFQ/N, the precursor may encode two other biologically active peptides. Anatomic studies have revealed high levels of expression of the OFQ/N messenger RNA in brain structures involved in sensory, emotional and cognitive processing. In particular, high levels of OFQ/N mRNA were detected in the limbic system, underlining the stress attenuating activities that have been described as an important function of OFQ/N. Recently, mutant mice have been generated that lack the precursor protein of OFQ/N to further define the physiological functions of the OFQ/N system. The OFQ/N-deficient mice are characterized by an increased sensitivity to stressful stimuli and a lack of habituation to chronic and repeated stress. This review will summarize recent findings on the molecular biology of the OFQ/N precursor and relate it to possible physiological functions of this newly discovered neuropeptide system.