Peptides (v.21, #7)

Preface: by Maurizio Massi; Carlo Polidori; Girolamo Calo’; Domenico Regoli (891).

The nociceptin (ORL1) receptor: molecular cloning and functional architecture by Jean-Claude Meunier; Lionel Mouledous; Christopher M Topham (893-900).
Nociceptin and the ORL1 receptor share high sequence similarity with opioid peptides, particularly dynorphin A, and their receptors. However, nociceptin and dynorphin A may use distinct molecular pathways to bind and activate their cognate receptors. Activation of the κ-opioid receptor by dynorphin A is thought to require interactions of its N-terminal hydrophobic domain (Y1GGF) with the receptor opioid binding pocket, located within the transmembrane helix bundle, while activation of the ORL1 receptor appears to require interactions of the positively charged core (R8KSARK) of nociceptin with the negatively charged second extracellular receptor loop.
Keywords: Neuropeptides; Nociceptin and dynorphin A; G protein-coupled receptors; ORL1 and opioid receptors; Ligand recognition and receptor activation; Molecular modeling;

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.
Keywords: Neuropeptide precursor; Processing; Dynorphin A; Gene regulation; Knockout mice; Analgesia; Stress; Anxiety;

Tissue distribution of the opioid receptor-like (ORL1) receptor by Catherine Mollereau; Lionel Mouledous (907-917).
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.).
Keywords: Neuropeptide; Nociceptin or Orphanin FQ; ORL1 receptor; G protein-coupled receptor; Anatomic distribution;

Nociceptin/orphanin FQ metabolism and bioactive metabolites by Lars Terenius; Johan Sandin; Tsukasa Sakurada (919-922).
The endogenous ligand for the orphan NOR receptor (earlier named ORL1) was recently discovered. This ligand, nociceptin/orphanin FQ is involved in a number of pharmacological actions in the CNS, including modulation of pain and cognition. However, its specific physiological role remains to be determined. Two major pathways of metabolism have been identified; the action of aminopeptidase(s) that prominently occurs in plasma, and endopeptidase activity that successively generates the N-terminal 1–13 and 1–9 fragments. Both pathways result in fragments that are inactive at the NOR receptor. However, short N-terminal fragments appear to be active in blocking the release of substance P from primary afferent C-fiber terminals in the dorsal spinal cord. The same endopeptidase(s) may also be involved in the fragmentation of dynorphin A since the inhibitor profile is similar. Enzyme activity is upregulated by morphine using either peptide as substrate that may lead to pharmacological interactions.

Structure-activity relationships of nociceptin and related peptides: comparison with dynorphin A by Remo Guerrini; Girolamo Calo’; Anna Rizzi; Raffaella Bigoni; Daniela Rizzi; Domenico Regoli; Severo Salvadori (923-933).
Nociceptin and its receptor (OP4) share sequence homologies with the opioid peptide ligand dynorphin A and its receptor OP2. Cationic residues in the C-terminal sequence of both peptides seem to be required for selective receptor occupation, but the number and the distribution of these basic residues are different and quite critical. Both receptors are presumably activated by the peptides N-terminal sequence (Xaa-Gly Gly-Phe, where Xaa = Phe or Tyr); however, although OP4 requires Phe4 as a determinant pharmacophore, OP2 requires Tyr1 as do the other opioid receptors. An extensive structure-activity analysis of the N-terminal tetrapeptide has led to conclude that the presence of aromatic residues in position one and four, preferably Phe, as well as the distance between Phe1 and Phe4 are extremely critical for occupation and activation of OP4 in contrast with other opioid receptors (e.g. OP1, OP3, OP2). Modification of distance between the side chains of Phe1 and Phe4 (as obtained with Nphe1 substitution in both NC and NC(1–13)-NH2) and/or conformational orientation of Phe1 (as in Phe1ψ(CH2-NH)-Gly2) has brought to discovery of pure antagonist ([Nphe1]-NC(1–13)-NH2) and a partial agonist ([Phe1 ψ(CH2-NH)-Gly2]-NC(1–13)-NH2), which have allowed us to characterize and classify the OP4 receptor in several species. Thus, although antagonist activities at the OP4 receptor are obtained by chemical modification of Phe1-Gly2 peptide bond or by a shift of Phe1 side chain of NC peptides, antagonism at the OP2 receptor requires the diallylation of the N-terminal amino function, for instance, of dynorphin A. These considerations support the interpretation that the two systems nociceptin/OP4 and dynorphin A/OP2 are distinct pharmacological entities that differs in both their active sites (Tyr1 for Dyn A and Phe4 for NC) and the number and position of cationic residues in the C-terminal portions of the molecules. The chemical features of novel OP4 receptor ligands either pseudopeptides obtained by combinatorial library screening or molecules of nonpeptide structure are reported and discussed in comparison with NC and NC related peptides.

Nociceptin/orphanin FQ receptor ligands by Girolamo Calo’; Raffaella Bigoni; Anna Rizzi; Remo Guerrini; Severo Salvadori; Domenico Regoli (935-947).
Nociceptin (NC), alias Orphanin FQ (OFQ) is a heptadecapeptide structurally related to opioid peptides, especially Dynorphin A, which, however, does not interact with classic opioid receptors. NC selectively activates its own receptor (OP4), which has been shown to be insensitive to the naturally occurring opioid peptides as well as to a large number of non-peptide opioid receptor ligands, including naloxone. Thus, the NC/OP4 system represents a new peptide-based signaling pathway, which is pharmacologically distinct from the opioid systems. The pharmacological tools available for investigating NC actions are at present rather limited and include: 1) peptide ligands obtained from structure activity studies performed using NC(1–13)NH2 as a template or discovered by screening peptide combinatorial libraries; 2) nonpeptide ligands that are either molecules already known to interact with classic opioid receptors or novel molecules designed and synthesized as selective ligands of the OP4 receptor. In the present paper the functional data obtained from both in vitro and in vivo studies with each relevant OP4 receptor ligand will be analyzed and discussed comparing the advantages and disadvantages of each molecule. We hope that the present work will aid investigators, working in the NC/OP4 field, in the choice of the pharmacological tools suitable for their experiments.
Keywords: Nociceptin; Orphanin FQ; Nociceptin Orphanin FQ receptor; Receptor ligands; Agonists and antagonists; Pharmacological assays;

Orphanin FQ/Nociceptin receptor binding studies by Colette T. Dooley; Richard A. Houghten (949-960).
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.

Cellular actions of nociceptin: transduction mechanisms by Brian E. Hawes; Michael P. Graziano; David G. Lambert (961-967).
The recent identification of the nociceptin receptor-nociceptin system and the description of its role in nociceptive processing has produced numerous investigative studies. A fundamental part of this research is to understand the cellular signaling events (i.e. the building blocks) upon which the pharmacology of this intriguing system is based. As anticipated, nociceptin receptor activation inhibits the formation of cAMP formation via a pertussis toxin-sensitive G-protein. This indicates that nociceptin receptor couples to the Gi/Go class of G-protein(s). However, there is now growing evidence for nociceptin activation of additional signaling pathways, including MAP kinase and phospholipase C/[Ca2+]i. These signaling events are discussed in this review.
Keywords: Nociceptin; Nociceptin receptor; Signal transduction; cAMP; MAP kinase; Intracellular Ca2+; Desensitization;

Cellular neurophysiological actions of nociceptin/orphanin FQ by Timothy D. Moran; Fuad A. Abdulla; Peter A. Smith (969-976).
Cellular actions of nociceptin/orphanin FQ (N/OFQ) resemble those of μ-, δ-, and κ-opioids, i.e. activation of inwardly rectifying K+ conductance, inhibition of high-voltage-activated Ca2+ channel currents, and impediment of neurotransmitter release. Differences in ORL1 and μ-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 Ca2+ 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 μ-opioids are attenuated. Agonists at ORL1 receptors may therefore be of clinical interest in the management of neuropathic pain.
Keywords: Analgesic; Calcium channel; Dorsal root ganglion; Dorsal horn; Morphine; Neuropathic pain; Nociceptin; Opioid; Orphan opioid-like receptor; Potassium channel; Substantia gelatinosa;

Nociceptin and neurotransmitter release in the periphery by Sandro Giuliani; Alessandro Lecci; Carlo Alberto Maggi (977-984).
Nociceptin exerts a general modulatory effect on transmitter release from sympathetic, parasympathetic, NANC and sensory nerve endings in the peripheral nervous system in various species. This effect occurs at a prejunctional level and is independent from the activation of μ, δ and κ opioid receptors. Despite the growing evidence describing the peripheral activity of nociceptin since its discovery in 1995, the lack of selective and potent antagonists does not allow us to draw conclusions on the putative physiological role of this peptide at this level.
Keywords: Nociceptin; Orphanin FQ; ORL1 receptor; Presynaptic modulation; Neurotransmitter release; Peripheral nervous system;

Studies of the cardiovascular effects of nociceptin and related peptides by Maria Bonaria Salis; Costanza Emanueli; Anna Franca Milia; Remo Guerrini; Paolo Madeddu (985-993).
Nociceptin, a novel opioid peptide, and its ORL1 receptor share structural similarities with other opioid ligands and receptors. Although NC exerts evident cardiovascular effects at a central and peripheral level, its role in homeostatic mechanisms and disease states are just beginning to be understood, as only recently selective receptor antagonists became available. In this review, some of the new observations regarding the cardiovascular actions of NC, related peptides and newly synthesized receptor antagonists are discussed.

Nociceptin effects in the airways by Christian Peiser; Bradley J Undem; Axel Fischer (995-998).
The opioid-like heptadecapeptide nociceptin (NC) has the following effects in the airways (investigated in isolated tracheae and bronchi from guinea pig or rat): the electric field stimulation (EFS)-induces release of acetylcholine (ACh), the tachykinin substance P (SP) and calcitonin gene-related peptide (CGRP) is reduced after pretreatment with NC, and EFS-induced tachykinergic nonadrenergic–noncholinergic (NANC) bronchoconstriction is inhibited by NC. Both the NC-mediated inhibition of neurotransmission and of smooth muscle contraction occurred in a concentration-dependent manner. Because these effects were naloxone-insensitive, were blocked by the NC receptor antagonist [F/G]NC(1–13)NH2, and could be mimicked by the NC analogs, NCNH2 and NC(1–13)NH2, it is thought that they are distinct from the classic opioid receptors. That these pharmacological actions of NC are of relevance for airway physiology is highly probable given the presence of NC-immunoreactivity in the nerve fibers of the airways and of opioid-like receptor (ORL-1) transcripts in the jugular ganglia, from where the tachykinin-containing afferents arise.

Orphanin FQ/nociceptin (OFQ/N) has been immunohistochemically localized in the rat enteric nervous system, and mRNA signals for its precursor (i.e. prepro-OFQ/-N) and cognate receptor ORL-1 are expressed in the intestinal tract. OFQ/N inhibits neurogenic contractions in a variety of stomach and small intestine preparations in vitro, but contracts rodent colon. In vivo, it acts at peripheral and central nervous system sites to stimulate or inhibit mechanical activity in the stomach and colon, respectively. Thus, OFQ/N acts as a neuromodulator of gastrointestinal motility and may have additional roles in regulation of intestinal blood flow, active ion transport, and immunity.
Keywords: Enteric nervous system; Opioid; Constipation; Diarrhea; Anion secretion; Peristalsis;

Nociceptin and the micturition reflex by Alessandro Lecci; Sandro Giuliani; Stefania Meini; Carlo A Maggi (1007-1021).
The i.v. administration of nociceptin (10–100 nmol/kg) inhibits the micturition reflex in a naloxone-resistant manner. The effects induced by i.v. nociceptin were not observed in capsaicin-pretreated animals indicating that i.v. nociceptin inhibits the micturition reflex by inhibiting afferent discharge from capsaicin-sensitive nerves. Supporting this interpretation, nociceptin also inhibited the reflex but not the local bladder contraction induced by topical capsaicin and protects this reflex (but not the local contraction) by desensitization. Intrathecal nociceptin (10 nmol/rat) produces urodynamic modifications similar to those induced by the i.v. administration. Intracerebroventricular (i.c.v.) administration of nociceptin (0.3–1 nmol/rat) also inhibited the micturition reflex in a naloxone-resistant manner suggesting a direct effect on supraspinal sites controlling the micturition. Beyond the inhibitory effects exerted by nociceptin on the micturition reflex, a peripheral excitatory effect mediated by capsaicin-sensitive fibers was also detected. The application of nociceptin (5–50 nmol/rat) onto the bladder serosa when the intravesical volume was subthreshold for the triggering of the micturition reflex, activated the reflex in a dose-dependent manner; the same treatment produced a biphasic effect on the ongoing reflex. In addition to the triggering of micturition reflex, topical nociceptin evokes a local tonic-type contraction that was abolished by the coadministration of tachykinin NK1 and NK2 receptor antagonists. Altogether these results indicate that ORL1 receptors are present at several sites for the integration of the micturition reflex, and that their activation may produce both excitatory or inhibitory effects, depending on the route of administration and the experimental conditions.
Keywords: Capsaicin; Central nervous system; ORL1 receptors; Peripheral nervous system; Sensory neurons; Rat; Tachykinins; Urinary bladder;

In this article, the effect of nociceptin (orphanin FQ) on transmitter release in the central nervous system in vitro and in vivo is reviewed. Nociceptin inhibits the electrically or K+-evoked noradrenaline, dopamine, serotonin, and glutamate release in brain slices from guinea-pig, rat, and mouse. This effect is usually naloxone-resistant but antagonized by OP4 receptor antagonists like [Phe1ψ(CH2-NH)Gly2]-nociceptin(1–13)NH2. In the rat in vivo, nociceptin diminishes acetylcholine release in the striatum, reduces dopamine release, and prevents the stimulatory effect of morphine on this transmitter in the nucleus accumbens and also elevates extracellular glutamate and γ-aminobutyric acid levels in mesencephalic dopaminergic areas. The effect of nociceptin on the mesencephalic dopaminergic system might explain its actions on motor behavior.
Keywords: Acetylcholine; Glutamate; 5-Hydroxytryptamine (serotonin; 5-HT); Naloxone benzoylhydrazone; Nociceptin; Noradrenaline; [Nphe1]Nociceptin-(1–13)NH2; Orphanin FQ; [Phe1ψ(CH2-NH)Gly2]Nociceptin(1–13)NH2;

Nociceptin/orphanin FQ in spinal nociceptive mechanisms under normal and pathological conditions by Xiao-Jun Xu; Stefan Grass; Jing-Xia Hao; Isabella Shi Xu; Zsuzsanna Wiesenfeld-Hallin (1031-1036).
Nociceptin and its receptor are present in dorsal spinal cord, indicating a possible role for this peptide in pain transmission. The majority of functional studies using behavioral and electrophysiological studies have shown that nociceptin applied at spinal level produces antinociception through pre- and post-synaptic mechanisms. The spinal inhibitory effect of nociceptin is not sensitive to antagonists of opioid receptors such as naloxone. Thus, nociceptin-induced antinociception is mediated by a novel mechanism independent of activation of classic opioid receptors. This has raised the possibility that agonists of the nociceptin receptor may represent a novel class of analgesics. Supporting this hypothesis, several groups have shown that intrathecal nociceptin alleviated hyperalgesic and allodynic responses in rats after inflammation or partial peripheral nerve injury. Electrophysiological studies have also indicated that the antinociceptive potency of spinal nociceptin is maintained or enhanced after nerve injury. It is concluded that the predominant action of nociceptin in the spinal cord appears to be inhibitory. The physiological role of nociceptin in spinal nociceptive mechanisms remains to be defined. Moreover, further evaluation of nociceptin as a new analgesic calls the development of non-peptide brain penetrating agents.
Keywords: Analgesia; Antinociception; Inflammation; Morphine; Nerve injury; Neuropathic pain; Opioids;

Effects of supraspinal orphanin FQ/nociceptin by Judith E. Grisel; Jeffrey S. Mogil (1037-1045).
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.

Functional studies using antibodies against orphanin FQ/nociceptin by Jin-Hua Tian; Ji-Sheng Han (1047-1050).
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.
Keywords: Orphanin FQ; Nociceptin; Antibody; Pain; Tolerance;

The hyperphagic effect of nociceptin/orphanin FQ in rats. by Carlo Polidori; Giuseppe de Caro; Maurizio Massi (1051-1062).
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.
Keywords: Nociceptin; Orphanin FQ; Nocistatin; Food intake; Water intake; ORL1 receptor agonists; ORL1 receptor antagonists;

Role of nociceptin systems in learning and memory by Yukihiro Noda; Takayoshi Mamiya; Toshiya Manabe; Miyuki Nishi; Hiroshi Takeshima; Toshitaka Nabeshima (1063-1069).
This article summarizes our recent finding that the nociceptin system is involved in the regulation of learning and memory. The nociceptin-knockout mice show greater learning ability in the water maze task, an enhanced latent learning in the water finding task, better memory in the passive avoidance task, and further, larger long-term potentiation in the hippocampal CA1 region than wild-type mice. Nociceptin itself induces an impairment of passive avoidance task in wild-type mice, which is reversed by naloxone benzoylhydrazone (NalBzoH). Thus, the nociceptin system seems to play negative roles in learning and memory, and NalBzoH may act as a potent antagonist for the nociceptin receptor.
Keywords: Nociceptin (ORL-1) receptor; Knockout mice; Learning and memory; κ3-Opioid receptor; NalBzoH;

Nociceptin/orphanin FQ and drugs of abuse by Roberto Ciccocioppo; Stefania Angeletti; Izabela Panocka; Maurizio Massi (1071-1080).
Nociceptin/orphanin FQ (NC) binds with high affinity to the opioid receptor-like1 (ORL1) receptor. NC has been reported to block opioid-induced supraspinal analgesia, and it has been proposed that it may represent a functional antiopioid peptide in the control of brain nociceptive processes. The wide distribution of NC and of its receptors in the central nervous system suggests, however, that it may be involved in the control of a variety of biologic functions. Increasing evidence indicates that it may influence the rewarding and reinforcing properties of drugs of abuse. NC has been shown to abolish the rewarding properties of ethanol and morphine in the place conditioning paradigm, to reduce ethanol consumption in alcohol-preferring rats and to inhibit stress-induced alcohol-seeking behavior. These findings suggest that drugs directed at central NC receptors may represent an interesting approach to the treatment of ethanol and opiate abuse.
Keywords: Nociceptin; Orphanin FQ; ORL1 receptors; Opioids; Brain reward system; Drug abuse; Reward; Reinforcement;

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.
Keywords: ORL1; Nociceptin; Orphanin FQ; Endorphins; Opioids; Sympathetic; Parasympathetic; Autonomic nervous system; Blood pressure; Heart rate; Hypertension; Renal hemodynamics; Renal function; Kidney; Renal nerves; Vasopressin; Antidiuretic hormone; Central nervous system; Baroreflex; Vagus; Vasculature; Hormone; Neurotransmission; Conscious; Anesthetized Sprague–Dawley rats; Mice; Sheep; Anesthesia; Intravenous; Intracerebroventricular; Microinjection; Rostral ventrolateral medulla;

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 E2 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.

Integrity of tritiated orphanin FQ/Nociceptin: implications for establishing a reliable binding assay by Denise Irene Quigley; Seksiri Arttamangkul; Richard Gordon Allen; David Kilgore Grandy (1111-1118).
In the course of establishing a reliable and reproducible binding assay for the orphanin FQ/nociceptin (OFQ/N) ligand-receptor system we used reversed phase-high-performance liquid chromatography (HPLC) (RP-HPLC) to monitor the integrity of [3H]OFQ/N obtained from three different manufacturers. This means of analysis revealed that the stability of [3H]OFQ/N during storage varied considerably depending on the manufacturer. Furthermore, the integrity of [3H]OFQ/N was significantly compromised in the presence of COS-7 cell membranes. Interestingly, if the peptide was added to COS-7 membranes after they had been exposed to low pH it remained intact, suggesting that the peptide’s breakdown during binding is, in part, enzymatically mediated. Although a variety of protease inhibitors were tested, none proved completely effective at protecting the tritiated peptide. The intention of the studies presented here was to evaluate OFQ/N binding components, namely the available [3H]OFQ/N ligands, in an effort to standardize the binding conditions for this receptor ligand system. Consequently, this study underscores the importance of monitoring the integrity of the trace ligand being used in a given binding assay.
Keywords: Orphanin FQ; Nociceptin; Radioligand binding; Nociceptin; OFQ receptor; Peptide degradation;

To further characterize the anti-opioid action of the neuropeptide nociceptin, we examined the effects of the repeated intracerebroventricular (i.c.v.) treatment (once daily for 4 days) with an antisense oligodeoxynucleotide complementary to pronociceptin mRNA, in the rat. We also investigated possible changes of the antinociceptive and hyperthermic effects induced by the i.c.v. administration of morphine, in rats i.c.v. pretreated with nociceptin 3 h before. The pretreatment with the antisense oligodeoxynucleotide, but not with a mismatched sequence (used as a control), caused an increase in spontaneous locomotor activity and produced a potentiation of the antinociceptive effect of a submaximal dose of i.c.v. morphine (1 μg/rat). The i.c.v. pretreatment with nociceptin (2 nmol/rat, 3 h before) prevented both the antinociceptive and the hyperthermic effects of morphine (10 μg/rat i.c.v.). These results strengthen the hypothesis of an anti-opioid action of nociceptin at supraspinal level and suggest that the neuropeptide may exert long-term modulatory effects.
Keywords: Nociceptin; Morphine; Antisense oligodeoxynucleotide; Tail-flick; Body temperature; Rat;

The effects induced by nociceptin on morphine-induced release of dopamine (DA), 3,4-dihydroxyphenilacetic acid (DOPAC) and homovanillic acid (HVA) in the nucleus accumbens and nucleus caudate were studied in rats by microdialysis with electrochemical detection. Nociceptin administered intracerebroventricularly (i.c.v.) at doses of 2, 5 and 10 nmol/rat changed neither DA nor metabolites release in the shell of the nucleus accumbens or in the nucleus caudate. Morphine administered intraperitoneally (i.p.) (2, 5, and 10 mg/kg) increased DA and metabolites release more in the shell of the nucleus accumbens than in the nucleus caudate. When nociceptin (5 or 10 nmol) was administered 15 min before morphine (5 or 10 mg/kg), it significantly reduced morphine-induced DA and metabolites release in the shell of the nucleus accumbens, whereas only a slight, nonsignificant reduction was observed in the nucleus caudate. Our data indicate that nociceptin may regulate the stimulating action associated with morphine-induced DA release more in the nucleus accumbens than in the nucleus caudate, and are consistent with recent observations that nociceptin reversed ethanol- and morphine-induced conditioned place preference. Therefore, the nociceptin-induced reduction of DA release stimulated by morphine in the nucleus accumbens, and the results obtained with nociceptin in the conditioned place preference procedure suggest a role for nociceptin in the modulation of the behavioral and neurochemical effects of abuse drugs.
Keywords: Nociceptin; Orphanin; Dopamine; Striatum; Accumbens; Caudate; Microdialysis; Rats;

The nociceptin/orphanin FQ receptor ligand acetyl-RYYRIK-amide exhibits antagonistic and agonistic properties by Hartmut Berger; Raffaella Bigoni; Erika Albrecht; Regina M Richter; Eberhard Krause; Michael Bienert; Girolamo Calo’ (1131-1139).
The hexapeptide acetyl-RYYRIK-amide (Ac-RYYRIK-NH2) 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 GTPγ35S 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-NH2 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-NH2 inhibited spontaneous locomotor activity in mice with ID50 of 1.1 and 0.07 nmol, respectively. Co-administration of both peptides lead to additive effects. The higher potency of Ac-RYYRIK-NH2 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-NH2, [Phe1psi(CH2-NH)Gly2]noc/OFQ(1–13)-NH2 ([F/G]NC(1–13)NH2) inhibited the noc/OFQ-stimulated GTPγ35S 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-NH2 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-NH2 and of [F/G]NC(1–13)NH2, 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)NH2 and Ac-RYYRIK-NH2 were reported to exhibit antagonist, partial agonist, or even full agonist properties, depending on the system studied.
Keywords: Nociceptin; Orphanin FQ; Brain receptor; GTPγS binding; Acetyl-RYYRIK-amide; Agonism; Antagonism; Locomotor activity; Microdialysis; Metabolism;

Agonistic effects of the opioid buprenorphine on the nociceptin/OFQ receptor by Petra Bloms–Funke; Clemens Gillen; Achim J Schuettler; Stephan Wnendt (1141-1146).
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 GTPγS 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 μ- or κ-opioid receptor mediated effects.
Keywords: Opioids; ORL1; Nociceptin/Orphanin FQ; Xenopus oocytes; Potassium currents; GTPγS; CHO-K1 cells; Analgesia;

Species differences in the efficacy of compounds at the nociceptin receptor (ORL1) by Jacquelyn L. Burnside; Lizel Rodriguez; Lawrence Toll (1147-1154).
Recent studies have identified compounds with reduced efficacy relative to nociceptin/orphanin FQ at the opioid-like receptor ORL1. Utilizing stimulation of [35S]GTPγS binding as in vitro assays, it was determined that both [Phe1ψ(CH2-NH)Gly2]N/OFQ(1–13)NH2 and the hexapeptide Ac-RYYRIK-NH2 act as partial agonists in CHO cells transfected with either human or mouse ORL1. Maximal activity for both [Phe1ψ(CH2-NH)Gly2]N/OFQ(1–13)NH2 and Ac-RYYRIK-NH2 was significantly greater in cells transfected with the human receptor (90% and 73% in a high expressing clone, 76% and 68% in low expressing clone) rather than the mouse receptor (37.5 and 33%), regardless of receptor number in individual clones. In vitro studies in cells transfected with exaggerated receptor numbers can lead to unreliable estimates of agonist and antagonist activity, however, these studies suggest that animal experiments on the activity of novel compounds may not always be better predictors of the ultimate activity in humans.
Keywords: Nociceptin; Orphanin FQ; ORL1; Efficacy; CHO cells; [35S]GTPγS binding;