Orphanin FQ/nociceptin blocks methamphetamine place preference in rats

Orphanin FQ/nociceptin (NOC) has been reported to regulate dopaminergic neurotransmission in rewarding pathway, and to suppress the development of conditioned place preference (CPP) induced by certain addictive drugs. In this study, we investigated the effect of NOC on CPP induced by repeated administration of methamphetamine (MAP) in rats. Repeated administration of MAP (1 mg/kg, i.p.) induced substantial CPP. MAP-induced CPP was completely suppressed by NOC (10 nmol, i.c.v.). Pretreatment with [Nphe1]nociceptin(1-13)NH2 (50 nmol, i.c.v.), an antagonist of the NOC receptor, antagonized the suppressive effect of NOC on MAP-induced CPP. These results suggest that NOC blocks MAP-induced CPP by activation of the NOC receptor.     

Orphanin FQ/nociceptin analgesia in the rat

The heptadecapeptide orphanin FQ or nociceptin (OFQ/N), the endogenous ligand for the orphan opioid receptor, has a complex pharmacology in mice, eliciting either an anti-opioid/hyperalgesic action or analgesia depending upon the dose and testing paradigm. Unlike mice, orphanin FQ/nociceptin fails to elicit hyperalgesia in the rat following intracerebroventricular injection. Both OFQ/N and a truncated version, OFQ/N(1-11), produce a robust analgesic response. OFQ/N analgesia is readily antagonized by the opioid antagonists naloxone or diprenorphine, despite their very poor affinity for the cloned orphan opioid receptor. Antisense studies revealed that probes targeting the second and third coding exon of the orphan clone significantly attenuate OFQ/N analgesia, while the exon 1 probe was inactive. These results indicate that OFQ/N elicits a naloxone-sensitive analgesia in rats similar to that previously reported in mice.     

Orphanin FQ/nociceptin blocks chronic morphine-induced tyrosine hydroxylase upregulation

The recently discovered endogenous peptide orphanin FQ/nociceptin (OFQ/N) activates the opioid receptor-like 1 (ORL1) receptor and produces diverse effects on pain perception. In addition to producing spinal analgesia, OFQ/N also exhibits an 'anti-opioid activity' against functional (supraspinal analgesia) and behavioral (conditioned place preference and withdrawal) properties of morphine. One manifestation of the behavioral changes resulting from chronic use of morphine is the upregulation of tyrosine hydroxylase (TH, the rate-limiting enzyme in catecholamine biosynthesis), which contributes to the dramatic increases in catecholamine release in the target regions of the locus coeruleus (LC) and the ventral tegmental area (VTA). The present study sought to determine the molecular mechanism(s) by which OFQ/N modulates the chronic actions of morphine by utilizing human neuroblastoma cell lines [BE(2)-C and SH-SY5Y] that endogenously express TH, and mu and ORL1 receptors. Activation of mu or ORL1 receptors in these cells in turn activates extracellular signal-regulated protein kinases (ERKs), ERK1 and ERK2. Chronic activation of mu, but not ORL1, receptors upregulated TH levels in these cells as previously reported in rat brain. Morphine-induced TH upregulation was blocked upon inclusion of a MEK-1 (mitogen-activated protein kinase kinase-1) inhibitor (PD98059), confirming the role for ERKs in this adaptive response to morphine. Inclusion of OFQ/N during chronic morphine exposure also blocked morphine-induced TH upregulation. Furthermore, chronic OFQ/N exposure increased levels of the TH gene repressor, Oct-2, irrespective of the presence or absence of morphine. This report suggests a potential role for Oct-2 in mediating the anti-opioid actions of OFQ/N against the behavioral manifestations resulting from chronic use of morphine.     

Orphanin FQ/nociceptin blocks acquisition of morphine place preference.

Orphanin FQ/nociceptin (OFQ/N) suppresses the activity of the dopaminergic mesolimbic reward pathway yet reportedly fails to produce conditioned place aversion or preference. The present study sought to determine if this peptide could attenuate the development of morphine place preference. Male rats were administered OFQ/N (3 to 30 nmol intracerebroventricularly) during the induction of morphine (3 mg/kg subcutaneously) place preference. Animals receiving 3 or 10 nmol (but not 30 nmol) OFQ/N showed significant reductions in the development of place preference to morphine.     

Orphanin FQ/nociceptin modulates glutamate- and kainic acid-induced currents in acutely isolated rat spinal dorsal horn neurons

The heptadecapeptide orphanin FQ (OFQ), also known as nociceptin (NOC), is a newly discovered endogenous ligand for the opioid-like G-protein coupled receptor, ORL1. In the present study, the effects of OFQ/NOC on glutamate (Glu), kainic acid (KA) and quisqualic acid (QA) induced currents were examined in isolated rat spinal dorsal horn neurons of young rats using whole-cell patch-clamp techniques. Glu, KA and QA elicited rapid inward currents in 90%, 69%, 83% of tested neurons. OFQ/NOC(0.03300 nM) failed to induce any changes of membrane currents, but modulated Glu-, KA- and QA-elicited currents. OFQ/NOC inhibited and potentiated Glu-induced currents in 40.6% and 27.3% of examined cells (n=106) respectively. In about one third examined neurons, OFQ/NOC had no detectable effects on Glu responses. OFQ/NOC also inhibited and enhanced KA- and QA-induced currents (inhibition: KA, 67.1%, N=76; QA, 50%, N=36. Potentiation: KA, 23.7%, N=76; QA, 16.7%, N=36). In about 10% of tested cells, Gluinduced currents were potentiated after the application of OFQ/NOC, and lasted for 2030 min. The inhibitory effects of OFQ/NOC on KA and QA responses were naloxone-insensitive. The C-terminal fragment OFQ(8–17) presented same effects on EAA-induced responses. Taken together, OFQ/NOC primarily inhibited Glu-, KA- and QA-induced currents in isolated rat spinal dorsal horn neurons via non-opioid mechanism, which might contribute to nociceptive transmission in the spinal level.     

Inhibition of nociceptin/orphanin FQ on penicillin-induced seizures in rats

The opioid peptides were thought to be involved in specific types of seizures. Nociceptin/Orphanin FQ (NC) is the endogenous ligand of the nociceptin opioid peptide (NOP) receptor and may play a role in epilepsy. However, accumulated evidences indicated that NC had both anti- and pro-convulsive effects, and the direct effect of NC in modulating epilepsy in the hippocampus still remained unclear. In this study, we investigated the effect of NC on penicillin-induced seizures (PIS) in rats. Seizure model was produced by intra-hippocampus injection of penicillin in anesthetized rats. The electroencephalography (EEG) was then observed and estimated by power spectrum analysis. Pretreatment of NC (intracerebroventricular, i.c.v.) depressed PIS in a dose-dependent manner at doses of 0.055, 0.55 and 5.5 nmol in 2 microl saline, respectively. [Nphe1]Nociceptin(1-13)NH2, a selective NOP receptor antagonist reversed the effect of NC against PIS, and this antagonist was inactive to PIS itself. These results indicated that NC had a receptor-specific preventive effect against PIS.     

Orphanin FQ stimulates prolactin and growth hormone release in male and female rats

Intracerebroventricular administration of Orphanin FQ (5.5, 55 or 550 pmol) caused a dose-related increase in prolactin secretion in both male and female rats and stimulated GH secretion in males. The magnitude of the prolactin secretory response was greater in females than in males. These effects of OFQ on prolactin and growth hormone release are the same as the stimulatory effects of the endogenous opioid peptides.     

Nociceptin/Orphanin FQ Microinjected into Hippocampus Impairs Spatial Learning in Rats

The newly discovered peptide nociceptin/orphanin FQ has been found to increase reactivity to pain and to influence locomotor activity after intracerebroventricular administration. This study investigated the possible role of hippocampal nociceptin/orphanin FQ in spatial learning and in spontaneous locomotion. Male rats were trained in the Morris water task after microinjection of 10 nmol nociceptin/orphanin FQ or artificial cerebrospinal fluid (as control) into the CA3 region of the dorsal hippocampus. Nociceptin/orphanin FQ was found to severely impair spatial learning without interfering with swimming performance. Itrahippocampal injection of nociceptin/orphanin FQ markedly decreased exploratory locomotor activity including vertical movements (rearing). The data suggest that nociceptin/orphanin FQ is a potent modulator of synaptic plasticity within the hippocampus.     

Distribution of nociceptin/orphanin FQ in adult human brain

Nociceptin/orphanin FQ (N/OFQ), the endogenous agonist for the opioid receptor-like receptor 1 (ORL1), shows significant similarities to dynorphin A in structure and distribution in rat central nervous system. The distribution of N/OFQ in human brain has not been studied. We measured the concentrations of N/OFQ in 47 microdissected areas of the central nervous system of adult human brain using radioimmunoassay (RIA). Significant heterogeneity was found in the levels of N/OFQ concentration in the various analyzed regions. The highest concentrations were measured in the dorsal central gray matter (periaqueductal gray), the locus coeruleus, the ventromedial nucleus of hypothalamus, the septum and the dorsal horn of the spinal cord. High concentrations were also detected in other hypothamamic nuclei, the inferior colliculus, the ventral central gray matter, the pontine tegmentum, the amygdala, the reticular formation and the spinal trigeminal nucleus. Considerable similarity with the distribution of N/OFQ in rat CNS was observed. The widespread distribution in CNS predicts multifaceted functions for N/OFQ.     

Orphanin FQ/nociceptin binds to functionally coupled ORL1 receptors on human immune cell lines and alters peripheral blood mononuclear cell proliferation

Orphanin FQ/nociceptin (OFQ/N) has been shown to modulate nociception, responses to stress and anxiety. We investigated OFQ/N function in human immune cells. We find that monocytic U937, T lymphocytic CEM, and MOLT-4 cell lines express OFQ/N binding sites at levels comparable to that of human SH-SY5Y neuroblastoma cells. We show that OFQ/N receptors are functionally coupled to G proteins in these cells. Finally OFQ/N decreases proliferation of phytohemagglutinin-stimulated peripheral blood mononuclear cells in vitro at doses ranging from 10(-13) to 10(-8) M. Thus, our data suggest that OFQ/N and OFQ/N receptor may act as an immunomodulatory system.     

Brain Interstitial Nociceptin/Orphanin FQ Levels are Elevated in Parkinson's Disease

Expression and release of nociceptin/orphanin FQ (N/OFQ) are elevated in the substantia nigra reticulata of 6‐hydroxydopamine‐hemilesioned rats, suggesting a pathogenic role for N/OFQ in Parkinson's disease. In this study, we investigated whether elevation of N/OFQ expression in 6‐hydroxydopamine‐hemilesioned rats selectively occurs in substantia nigra and whether hypomotility following acute haloperidol administration is accompanied by a rise in nigral N/OFQ levels. Moreover, to prove a link between N/OFQ and idiopathic Parkinson's disease in humans, we measured N/OFQ levels in the cerebrospinal fluid of parkinsonian patients undergoing surgery for deep brain stimulation. In situ hybridization demonstrated that dopamine depletion was associated with increase of N/OFQ expression in substantia nigra (compacta +160%, reticulata +105%) and subthalamic nucleus (+45%), as well as reduction in caudate putamen (?20%). No change was observed in globus pallidus, nucleus accumbens, thalamus, and motor cortex. Microdialysis coupled to the bar test allowed to demonstrate that acute administration of haloperidol (0.8 and 3 mg/kg) increased nigral N/OFQ levels (maximally of +47% and +53%, respectively) in parallel with akinesia. A correlation with preclinical studies was found by analyzing N/OFQ levels in humans. Indeed, N/OFQ levels were found to be ～3.5‐fold elevated in the cerebrospinal fluid of parkinsonian patients (148 fmol/ml) compared with nonparkinsonian neurologic controls (41 fmol/ml). These data represent the first clinical evidence linking N/OFQ to idiopathic Parkinson's disease in humans. They strengthen the pathogenic role of N/OFQ in the modulation of parkinsonism across species and provide a rationale for developing N/OFQ receptor antagonists as antiparkinsonian drugs. © 2010 Movement Disorder Society     

In vivo metabolism of nociceptin/orphanin FQ in rat hippocampus

The in vivo metabolism of the newly identified endogenous ligand for the ORL1 receptor, the opioid-like peptide nociceptin (orphanin FQ) in rat hippocampus was studied using size-exclusion chromatography linked to electrospray ionization mass spectrometry. The results show that nociceptin is metabolized step-wise in vivo into fragments (1-13) and (14-17) as well as (1-9) and (10-13), respectively. Interestingly, the (1-13) and (1-9) fragments have the same C-terminus, Arg-Ala-Lys, suggesting that this is a motif recognized by an enzyme which fragments the peptide in two consecutive steps. Injection of the (1-13) fragment into rat hippocampus had no effect on spatial learning or motor function under conditions where nociceptin is active, showing that this metabolic conversion reduces affinity for the ORL-1-receptor.     

Identification of central nervous system sites involved in the water diuresis response elicited by central microinjection of nociceptin/ Orphanin FQ in conscious rats via c-Fos and inducible cAMP early repressor immunocytochemistry

Intracerebroventricular (i.c.v.) administration of the opioid-like peptide, nociceptin/Orphanin (nociceptin), in conscious rats produces diuretic and antinatriuretic effects. The present study utilised changes in Fos and inducible cAMP early repressor (ICER) immunocytochemistry expression to examine the central nervous (CNS) sites activated or inhibited, respectively, by central administration of nociceptin. Urine samples were collected during control (15 min) and after i.c.v. vehicle (5 microl, n = 12) or nociceptin (10 microg/5 microl; n = 12). Four additional urine samples (15-min) were collected after the i.c.v. injection. The brain was processed for Fos using a commercially available antibody (Oncogene AB-5) and for ICER using a polyclonal anti-ICER antibody raised in rabbits. In vehicle-injected conscious rats, renal excretion of water or sodium was not altered. However, nociceptin produced a rapid and marked increase in urine flow (V) and a decrease in urinary sodium excretion rate. In addition, i.c.v. nociceptin produced a significant increase in Fos staining in the dorsomedial nucleus of the hypothalamus, the perinuclear zone of the supraoptic nucleus, the organum vasculosum of the lamina terminalis (OVLT), the lateral preoptic area and the lateral hypothalamic area compared to control. By contrast, Fos expression decreased in the area postrema and locus coeruleus compared to controls. Furthermore, ICER staining was significantly increased in the perinuclear zone of the supraoptic nucleus, supraoptic nucleus, median preoptic nucleus, OVLT, medial preoptic area, central nucleus of the amygdala, and medial nucleus of the solitary tract. Together, central opioid receptor-like type 1 activation in these CNS regions may participate in the neural pathways involved in the diuretic and antinatriuretic effects of nociceptin.     

Involvement of nociceptin/orphanin FQ and its receptor in electroacupuncture-produced anti-hyperalgesia in rats with peripheral inflammation

The neuropeptide nociceptin/orphanin FQ (N/OFQ), the endogenous agonist of the N/OFQ peptide receptor (NOP receptor), has been demonstrated to be involved in many physiological and pathological functions including pain regulation. In the present study, the involvement of N/OFQ-NOP receptor system in electroacupuncture (EA)-produced anti-hyperalgesia was investigated in rats with peripheral inflammation. Intrathecal (i.t.) administration of N/OFQ (15 nmol) or EA at acupoints GB30 and GB34 could significantly attenuate hyperalgesia which was induced by subcutaneously injecting complete Freund's adjuvant (CFA) into one hindpaw of rats, manifesting as decreased paw withdrawal latency (PWL) to the noxious thermal stimulus. The anti-nociceptive effect of N/OFQ or EA was significantly blocked by intrathecal injection of [Nphe(1)]nociceptin(1-13)NH(2) (20 nmol), a selective antagonist of the NOP receptor, indicating the NOP-receptor-mediated mechanism. Additionally, the combination of N/OFQ injection with EA treatment could enhance anti-hyperalgesia compared to that produced by each component alone. These findings suggested that the spinal N/OFQ-NOP system might be involved in EA analgesia, which may be one of the mechanisms underlying the anti-nociceptive effect of EA in rat's peripheral inflammatory pain.     

Biotransformation of nociceptin/orphanin FQ by enzyme activity from morphine-naive and morphine-treated cell cultures

The biotransformation of nociceptin/orphanin FQ (NOFQ) by enzyme activity isolated from U1690 human lung carcinoma and SH-SY5Y human neuroblastoma cell lines, and from rat brain cortex cells in primary culture was investigated. The identification and quantification of the cleavage products were performed using electrospray ionization mass spectrometry linked to size-exclusion chromatography. The effect of chronic morphine treatment of the cells (5 days) on NOFQ biotransformation was also studied. It was found that major products generated from NOFQ were the amino-terminal peptides N1-9 and N1-13. The pattern of NOFQ biotransformation was quite similar for all three cell cultures. However, different proportions of the formed peptides were noted. The cleavage was inhibited by EDTA, PMSF, Hg2+, Cu2+ and Zn2+. Dynorphin A2-13 inhibited NOFQ cleavage in a manner suggesting competition of the two peptides for the same enzyme. Chronic morphine treatment of the cell cultures resulted in a substantial increase in the enzyme activity, leading to higher levels of the major fragments and accumulation of N1-12 and the shorter peptides N1-5, N1-6. Since the effect of morphine treatment of the cells was blocked by naloxone, it is likely that it was receptor specific. Taken together, the findings suggest that a metallosensitive endopeptidase, the activity of which is increased by chronic morphine treatment of the cells, is responsible for the biotransformation of NOFQ with fragments N1-9 and N1-13 being the major products.     

Enhancement of spatial attention in nociceptin/orphanin FQ receptor-knockout mice

We isolated genes for the opioid receptor homologue MOR-C, namely nociceptin receptor (designated alternatively as orphanin FQ receptor) and generated nociceptin receptor-knockout mice. Previously, we have reported that the nociceptin system appears to participate in the regulation of the auditory system. However, the behavior of the nociceptin receptor-knockout mice has yet to be fully characterized. In the present study, we investigated changes in several behavioral performances in mice which lack nociceptin receptor. Nociceptive thresholds of nociceptin receptor-knockout mice were unchanged in the hot-plate and electric foot-shock tests as well as tail-flick and acetic acid-induced writhing tests compared to those of wild-type mice. The nociceptin receptor-knockout mice did not show any behavioral changes in the elevated plus-maze task. Surprisingly, in the water-finding test, the nociceptin receptor-knockout mice showed an enhanced retention of spatial attention (latent learning) compared to wild-type mice. In a biochemical study, dopamine content in the frontal cortex was lower in nociceptin receptor-knockout mice than wild-type mice. These results suggest that nociceptin receptor plays an important role in spatial attention by regulating the dopaminergic system in the brain.     

Nocistatin reverses the effect of orphanin FQ/nociceptin in antagonizing morphine analgesia

Nocistatin is a recently characterized neuropeptide derived from the preprohormone containing nociceptin (Orphanin FQ, OFQ). Nocistatin was reported to antagonize OFQ induced allodynia, hyperalgesia and prostaglandin E2-elicited pain responses. The aim of the present study was to determine whether nocistatin, injected intracerebroventricularly (i.c.v.), would reverse the anti-morphine effect of OFQ in rats using the tail-flick latency (TFL) as the nociceptive index. I.c.v. injection of nocistatin at doses of 0.005, 0.05, 0.5, 5, 50, and 500 ng produced no significant changes in the basal TFL, nor did it affect morphine analgesia. However, it significantly reversed the antagonistic effect of OFQ on morphine analgesia when co-injected i.c.v. at doses of 0.05, 0.5, 5, 50 and 500 ng per rat with OFQ. The dose-response curve was bell-shaped and the most effective dose was 0.5 ng. The results suggest that nocistatin can reverse the anti-morphine effect of OFQ in rat brain.     

Functional coupling of the nociceptin/orphanin FQ receptor in dog brain membranes

Nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand for the N/OFQ receptor (NOP) which is yet to be functionally characterized in dog brain. Ligand binding data reports low NOP density (29 fmol mg(-1) protein) in dog. In this study using dog brain membranes, we have examined the effects of N/OFQ on [leucyl-(3)H]N/OFQ(1-17)OH ([leucyl-(3)H]N/OFQ) binding in the presence and absence of 120 mM NaCl and 100 microM GTPgammaS. Data from standard [(35)S]GTPgammaS binding and immunoprecipitation (G(alphai1-3)) assays are also presented, along with data from a limited number of control experiments with human NOP expressed in Chinese hamster ovary (CHO(hNOP)) cells. N/OFQ displaced [leucyl-(3)H]N/OFQ binding with pK(i) and slope values of 9.62+/-0.07 and 0.38+/-0.05, respectively. Addition of NaCl/GTPgammaS produced a steepening (slope 0.95+/-0.06, n=3) of the curve. N/OFQ stimulated [(35)S]GTPgammaS binding with pEC(50) and E(max) values of 8.21+/-0.17 and 1.17+/-0.01, respectively (in CHO(hNOP), pEC(50) and E(max) values were 8.47+/-0.01 and 7.01+/-0.63). N/OFQ stimulated [(35)S]GTPgammaS binding in dog and CHO(hNOP) cell membranes could be immunoprecipitated with an anti-G(alphai1-3) antibody, indicating coupling to a pertussis toxin (PTx)-sensitive G-protein. N/OFQ actions were competitively antagonized by the selective NOP antagonists, 100 nM J-113397, 1 microM [Nphe(1)]N/OFQ(1-13)NH(2) and 1 microM [Phe(1)Psi(CH(2)-NH)Gly(2)]N/OFQ(1-13)NH(2) (partial agonist) yielding pK(B) values of 8.58+/-0.21, 7.06+/-0.59 and 7.32+/-0.41, respectively (in CHO(hNOP), a pK(B) for J-113397 of 8.33+/-0.02 was obtained). Despite relatively low receptor density, we were able to detect functional activity of native dog NOP, with pharmacology consistent with reports for other species.