Traditional Opioid Receptors Research Articles

Oxytocin Receptor Activation Rescues Opioid-Induced Respiratory Depression by Systemic Fentanyl in the Rat.

Opioid overdose intervention by naloxone, a high affinity receptor antagonist, reverses opioid-induced respiratory depression (OIRD) and analgesia by displacing opioids. Systemic naloxone stimulates release of the hypothalamic neuropeptide oxytocin, which has analgesic properties and participates in cardiorespiratory homeostasis. To test the hypothesis that oxytocin can reverse OIRD, we assessed the rescue potential of graded doses (0, 0.1, 2, 5, 10, 50 nmol/kg, i.v.) of oxytocin to counter fentanyl (60 nmol/kg, i.v.)-induced depression of neural inspiration indexed by recording phrenic nerve activity (PNA) in anesthetized (urethane/α-chloralose), vagotomized, and artificially ventilated rats. Oxytocin dose-dependently rescued fentanyl OIRD by almost immediately reversing PNA burst arrest (P = 0.0057) and restoring baseline burst frequency (P = 0.0016) and amplitude (P = 0.0025) at low but not high doses, resulting in inverted bell-shaped dose-response curves. Oxytocin receptor antagonism (40 nmol/kg, i.v.) prevented oxytocin reversal of OIRD (arrest: P = 0.0066, frequency: P = 0.0207, amplitude: P = 0.0022). Vasopressin 1A receptor (V1aR) antagonism restored high-dose oxytocin efficacy to rescue OIRD (P = 0.0170 to P < 0.0001), resulting in classic sigmoidal dose-response curves, and prevented (P = 0.0135) transient hypertension from V1aR cross-activation (P = 0.0275). Alone, vasopressin (5 nmol/kg, i.v.) failed to reverse fentanyl respiratory arrest (P = 0.6184). The nonpeptide oxytocin receptor agonist WAY-267464 (75 nmol/kg, i.v.), which has V1aR antagonist properties, quickly reversed fentanyl OIRD (P < 0.0001), with rapid recovery of PNA frequency (P = 0.0011) and amplitude (P = 0.0044) without adverse hemodynamic consequences (P = 0.9991). Findings indicate that peptide and nonpeptide agonist activation of oxytocin receptors without V1aR cross-activation rescues fentanyl OIRD. Oxytocin receptor agonists could be lifesaving resuscitation agents that enhance rather than interrupt opioid analgesia. SIGNIFICANCE STATEMENT: Oxytocin receptor activation produces analgesia. Here, we demonstrate that activation by the US Food and Drug Administration-approved agonist oxytocin and the nonpeptide partial agonist WAY-267464 can each reverse fentanyl cardiorespiratory depression. Selective targeting of oxytocin receptors for resuscitation from opioid overdose, alone or in combination with an opioid antagonist, could eliminate or attenuate negative side effects associated with traditional opioid receptor antagonism.

Pharmacologic Evidence for a Putative Conserved Allosteric Site on Opioid Receptors

Allosteric modulators of G protein-coupled receptors, including opioid receptors, have been proposed as possible therapeutic agents with enhanced selectivity. BMS-986122 is a positive allosteric modulator (PAM) of the μ-opioid receptor (µ-OR). BMS-986187 is a structurally distinct PAM for the δ-opioid receptor (δ-OR) that has been reported to exhibit 100-fold selectivity in promoting δ-OR over μ-OR agonism. We used ligand binding and second-messenger assays to show that BMS-986187 is an effective PAM at the μ-OR and at the κ-opioid receptor (κ-OR), but it is ineffective at the nociceptin receptor. The affinity of BMS-986187 for δ-ORs and κ-ORs is approximately 20- to 30-fold higher than for μ-ORs, determined using an allosteric ternary complex model. Moreover, we provide evidence, using a silent allosteric modulator as an allosteric antagonist, that BMS-986187 and BMS-986122 bind to a similar region on all three traditional opioid receptor types (µ-OR, δ-OR, and κ-OR). In contrast to the dogma surrounding allosteric modulators, the results indicate a possible conserved allosteric binding site across the opioid receptor family that can accommodate structurally diverse molecules. These findings have implications for the development of selective allosteric modulators.

The Nociceptin Receptor as an Emerging Molecular Target for Cocaine Addiction.

Cocaine addiction is a global public health and socioeconomic issue that requires pharmacological and cognitive therapies. Currently there are no FDA-approved medications to treat cocaine addiction. However, in preclinical studies, interventions ranging from herbal medicine to deep-brain stimulation have shown promise for the therapy of cocaine addiction. Recent developments in molecular biology, pharmacology, and medicinal chemistry have enabled scientists to identify novel molecular targets along the pathways involved in drug addiction. In 1994, a receptor that showed a great deal of homology to the traditional opioid receptors was characterized. However, endogenous and exogenous opioids failed to bind to this receptor, which led scientists to name it opioid receptor-like receptor, now referred to as the nociceptin receptor. The endogenous ligand of NOPr was identified a year later and named orphanin FQ/nociceptin. Nociceptin and NOPr are widely distributed throughout the CNS and are involved in many physiological responses, such as food intake, nociceptive processing, neurotransmitter release, etc. Furthermore, exogenous nociceptin has been shown to regulate the activity of mesolimbic dopaminergic neurons, glutamate, and opioid systems, and the stress circuit. Importantly, exogenous nociceptin has been shown to reduce the rewarding and addictive actions of a number of drugs of abuse, such as psychostimulants, alcohol, and opioids. This paper reviews the existing literature on the role of endogenous nociceptin in the rewarding and addictive actions of cocaine. The effect of exogenous nociceptin on these processes is also reviewed. Furthermore, the effects of novel small-molecule NOPr ligands on these actions of cocaine are discussed. Overall, a review of the literature suggests that NOPr could be an emerging target for cocaine addiction pharmacotherapy.

PC-FACS

PC-FACS

Functional Selectivity at GPCRs: New Opportunities in Psychiatric Drug Discovery

The superfamily of G-protein coupled receptors (GPCRs) is one of the largest gene families in the mammalian genome. GPCRs represent >50% of all current drug targets for treating numerous diseases that affect both peripheral and central nervous systems. Identification of novel GPCR drug targets and their promise for treatment in psychiatric disease has been recently slowed by the dearth of selective targets and changes in the pharmaceutical landscape. However, several recent advances in GPCR crystallization and in vivo pharmacology offer potentially exciting opportunities for therapeutics that target GPCRs in unique ways to treat psychiatric diseases (Kenakin and Christopoulos, 2013). Current mathematical and pharmacological data support the conclusion that GPCRs are pluridimensional proteins that occupy numerous structural conformations and signaling states. Depending on the ligand, the GPCR has the ability to engage a G-protein and arrestin state, or favor one over the other (Figure 1). These conformations are determined by the type of ligand, the receptor type, as well as interacting accessory proteins. This concept termed ‘functional selectivity' (also called, biased signaling, ligand-directed signaling) is the ability of a ligand to direct a GPCR toward a conformation that selectively evokes a particular stimulus–response and is now a well-accepted concept in the field of GPCR research. Some GPCR classes that have been studied in the context of functional selectivity in vitro and in vivo include: serotonin, opioid, adrenergic, cannabinoid, muscarinic, and metabotropic glutamate receptors (Luttrell and Gesty-Palmer, 2010). Figure 1 Schematic of functional selectivity at GPCRs. Cartoon model depicting various states of functional selectivity. Depending on the ligand and the induced receptor conformation, GPCRs are capable of signaling through the G-protein-mediated signaling pathway ... A recent and elegant development in this field has the potential to greatly advance our understanding of functional selectivity and to open new doors for drug discovery. The study uncovered a ‘snapshot' of the serotonin 5HT2B receptor bound to ergotamine, an arrestin-biased ligand at this receptor (Wacker et al, 2013). For the first time, this paper and its complementary study, elucidated the molecular signature of the G-protein vs arrestin-biased conformation at serotonin receptors using an X-ray crystallography approach. Together, these reports suggest that for a GPCR to occupy an arrestin-biased (alternate-agonist) state, the ligand-receptor conformation sits in an intermediate state that includes both active and inactivate components, yet also interferes with G-protein signaling. While the authors show convincing modeling for other GPCR types, whether this is indeed case for other biased ligands of other classes of GPCRs remains to be explored. However, this exciting result has the potential to accelerate our understanding of GPCR structure–function relationships, particularly as new ligands are developed for the treatment of psychiatric diseases. Having a clearer understanding of how various ligand types interact with and stabilize GPCR conformations will provide chemists with the necessary tools to rationally design more selective compounds with better delivery, higher affinity, and fewer unwanted side effects. Complimentary to these exciting molecular advances, contemporary reports have shown promising in vivo data indicating that functionally selective ligands may offer novel strategies to achieve pain relief without the traditional abuse liability, dysphorigenic properties, or psychomimetic components of traditional opioid receptor ligands (Bruchas and Chavkin, 2010; Bruchas et al, 2011) by selectively targeting G-protein vs arrestin-biased signaling states. Furthermore, advances in dopamine and serotonin receptor behavioral pharmacology have revealed that engaging D2R arrestin-biased signaling to GSK3β or 5HT2A arrestin-biased Src/Akt signaling may offer selective treatments for schizophrenia, psychosis, and other mood disorders (Schmid and Bohn, 2010; Urs et al, 2012). The combined efforts of molecular pharmacologists, biochemists, and behavioral pharmacologists to decipher these complex relationships between the molecular signatures of GPCRs and how various GPCR conformations ultimately transduce cellular responses into behavioral output are an active area of study for both Academia and Industry. These new discoveries are likely to provide novel therapeutic strategies for treating psychiatric diseases.

Near-fatal tramadol cardiotoxicity in a CYP2D6 ultrarapid metabolizer

Tramadol is a synthetic, centrally acting analgesic for the treatment of moderate to severe pain. The marketed tramadol is a racemic mixture containing 50% (+)tramadol and 50% (-)tramadol and is mainly metabolized to O-desmethyltramadol (M1) by the cytochrome P450 CYP2D6. Tramadol is generally considered to be devoid of any serious adverse effects of traditional opioid receptor agonists, such as respiratory depression and drug dependence. A 22-year-old Caucasian female patient was admitted to our ICU in refractory cardiac arrest requiring extracorporeal membrane oxygenation. This aggressive support allowed resolution of multi-organ dysfunction syndrome. Repeated blood analyses using liquid chromatography-tandem mass spectrometry confirmed high concentrations of both tramadol and its main metabolite O-desmethyltramadol. Genotyping of CYP2D6 revealed the patient to be heterozygous for a duplicated wild-type allele, predictive of a CYP2D6 ultrarapid metabolizer (UM) phenotype, confirmed by calculation of the tramadol/M1 (MR1) metabolic ratio at all time points. We here report a case of near-fatal isolated tramadol cardiotoxicity. Because of the inhibition of norepinephrine reuptake, excessive blood epinephrine levels in this CYP2D6R UM patient following excessive tramadol ingestion could explain the observed strong myocardial stunning. This patient admitted intermittent tramadol consumption to gain a "high" sensation. In patients with excessive morphinomimetic effects, levels of tramadol and its main metabolite M1could be measured, ideally combined with CYP2D6 genotyping, to identify individuals at risk of tramadol-related cardiotoxicity. Tramadol treatment could be optimized in these at-risk individuals, consequently improving patient outcome and safety.

Quantitative study of the antagonistic effect of (−)- cis-1-Methyl-7-[[4-(2,6-dichlorophenyl)piperidin-1-yl]methyl]-6,7,8,9-tetrahydro-5 H-benzocyclohepten-5-ol (SB-612111) on nociceptin/orphanin FQ-mediated potassium channel activation in rat periaqueductal gray slices

Nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor, a non-opioid branch of the opioid receptor family, shows structural similarities to traditional opioid receptors but binds opioid with very poor affinity. This receptor has been implicated in many physiological functions including pain regulation. This study quantitatively investigated the effect of (−)- cis-1-Methyl-7-[[4-(2,6-dichlorophenyl)piperidin-1 -yl]methyl]-6,7,8,9-tetrahydro-5 H-benzocyclohepten-5-ol (SB-612111), a novel non-peptide ligand of NOP receptor, on the native NOP receptors in the midbrain ventrolateral periaqueductal gray (vlPAG), a crucial region for pain regulation. SB-612111 concentration-dependently antagonized N/OFQ-induced G-protein coupled inwardly rectifying K + (GIRK) current in vlPAG neurons. The IC 50 value of SB-612111 estimated from dose–response curves is 87.7 ± 1.2 nM. SB-612111 had no intrinsic agonistic activity and did not affect the GIRK current induced by [D-Ala 2, N-Me-Phe 4, Gly 5-ol]-enkephalin, a mu-opioid receptor agonist, when tested at concentrations of up to 1 μM. It is concluded that SB-612111 is a pure, potent and selective antagonist of NOP receptors that mediate GIRK channel activation in the vlPAG neurons.

1-Benzyl-N-[3-[spiroisobenzofuran-1(3H),4′-piperidin-1-yl]propyl]pyrrolidine-2-carboxamide (Compound 24) antagonizes NOP receptor-mediated potassium channel activation in rat periaqueductal gray slices

Nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor, the fourth member of opioid receptor family, shares 60–70% homology with traditional opioid receptors but displays little affinity for opioids. This receptor was implicated in many neurological functions and its functional heterogeneity has been proposed. Therefore, it is imperative to develop and characterize new ligands for NOP receptors. 1-Benzyl-N-[3-[spiroisobenzofuran-1(3H),4′-piperidin-1-yl]propyl]pyrrolidine-2-carboxamide (Compound 24) is a new non-peptide ligand of NOP receptor having antagonistic actions in cloned and peripheral NOP receptors. In this study, we quantitatively characterized its effect on the native NOP receptors in the midbrain slices containing ventrolateral periaqueductal gray (vlPAG), a region with dense NOP receptors and involved in pain regulation. In vlPAG neurons, N/OFQ induced G-protein-coupled inwardly rectifying potassium (GIRK) current through NOP receptors. Compound 24, at 0.3–10 μM, attenuated N/OFQ-induced GIRK current concentration-dependently. The antagonistic potency of Compound 24 in vlPAG neurons (IC 50: 2.6 ± 0.6 μM) was, however, lower than that obtained in mouse vas deferens preparations or expressed human NOP receptors. The action kinetic of Compound 24 was slower than [Nphe 1, Arg 14, Lys 15]N/OFQ-NH 2 (UFP-101), a peptide antagonist, in the same preparation. Compound 24 had no intrinsic agonistic activity at NOP receptors at the concentration up to 10 μM. However, at concentrations higher than 3 μM, it also attenuated the GIRK current induced by [D-Ala 2, N-Me-Phe 4, Gly 5-ol]-enkephalin, a mu-opioid receptor agonist. It is concluded that Compound 24 acts as a pure antagonist at the native NOP receptors in the vlPAG with moderate potency and selectivity.

Effects of melatonin on orphanin FQ/nociceptin-induced hyperalgesia in mice

The pain modulatory properties of melatonin (MT) are generally recognized but the detail of the interaction between melatonin and opioid system in pain regulation is not fully understood. The present study was undertaken to investigate the modulatory effect of melatonin (MT) on the hyperalgesic effect of Orphanin FQ/Nociceptin (OFQ/NC, NC), a member of opioid peptide family. Intracerebroventricular (i.c.v.) administration of NC (10 μg/mouse) induced significant hyperalgesic effect in tail-flick test in mice; i.c.v. (5, 10, 50 μg/mouse) or intraperitoneal (i.p.) (5, 10, 50 mg/kg) co-injection of melatonin dose-dependently reversed NC-induced hyperalgesia and showed a profound analgesic effect. The antihyperalgesia effect of MT could be significantly antagonized by i.c.v. co-injection of luzindole (10 μg/mouse) (an antagonist of MT receptor) or naloxone (10 μg/mouse) (antagonist of traditional opioid receptor). Taken together, all the results suggested that MT could produce a luzindole and naloxone sensitive reversing effect on NC-induced hyperalgesia at supraspinal and peripheral level in mice. The augmentation effect of MT on the traditional opioid system may be one of the mechanisms of this antihyperalgesia action induced by MT. The present work will help to elucidate the mechanism of the pain modulation effect of MT, and also will help to represent new interesting modulating therapeutic targets for the relief of pain.

Cardiovascular responses to microinjection of nociceptin and endomorphin-1 into the nucleus tractus solitarii in conscious rats

Increasing evidence suggests an active participation of nociceptinergic transmission in the central control of cardiovascular activity and reflex. In this study, the role of the classic opioid μ receptor and the nociceptin/orphanin FQ receptor, a novel opioid receptor, in the nucleus tractus solitarii (NTS) in the regulation of cardiovascular activity was investigated and compared in chronically cannulated and freely moving conscious rats. Microinjections of nociceptin, an endogenous ligand for the nociceptin receptor, into the relatively rostral NTS produced dose-related (0.04, 0.2, and 1 nmol) increases in blood pressure and heart rate. Intra-NTS injection of the selective nociceptin receptor antagonist [Nphe 1]Nociceptin(1–13)NH 2 (NOR-AN) at 1 nmol blocked the increases in blood pressure and heart rate induced by nociceptin. In contrast, pretreatment with the nonselective opioid receptor antagonist naloxone (5 nmol) had no effects on the cardiovascular responses to nociceptin. Like nociceptin, microinjection of endomorphin-1 (EM-1), an endogenous ligand for the opioid μ receptor, into the rostral NTS increased blood pressure and heart rate in a dose-dependent manner (0.04, 0.2, and 1 nmol). Pretreatment with naloxone (5 nmol), but not NOR-AN, blocked cardiovascular responses elicited by EM-1. Neither NOR-AN nor naloxone alone had significant effects on the baseline blood pressure and heart rate. Injection of excitatory amino acid l-glutamate (1 nmol) into the same sites caused the typical depressor and bradycardic responses. In the caudal NTS areas, nociceptin and EM-1 seemed to induce opposite responses: hypotension and bradycardia. These results suggest that the novel nociceptin receptors and traditional opioid receptors in the NTS may be independently involved in the regulation of cardiovascular activity.

A new cytokine: the possible effect pathway of methionine enkephalin.

To investigate experimentally the effects of methionine enkephalin on signal transduction of mouse myeloma NS-1 cells. The antigen determinate of delta opioid receptor was designed in this lab and the polypeptide fragment of antigen determinate with 12 amino acids residues was synthesized. Monoclonal antibody against this peptide fragment was prepared. Proliferation of Mouse NS-1 cells treated with methionine enkephalin of 1 x 10(-6) mol x L(-1) was observed. The activities of protein kinase A (PKA) and protein kinase C (PKC) were measured and thereby the mechanism of effect of methionine enkephalin was postulated. The results demonstrated that methionine enkephalin could enhance the proliferation of NS-1 cells and the effect of methionine enkephalin could be particularly blocked by monoclonal antibody. The activity of PKA was increased in both cytosol and cell membrane. With reference to PKC, the intracellular activity of PKC in NS-1 cells was elevated at 1 x 10(-7) mol x L(-1) and then declined gradually as the concentration of methionine enkephalin was raised. The effects of methionine enkephalin might be reversed by both naloxone and monoclonal antibody. Coupled with the findings, it indicates that the signal transduction systems via PKA and PKC are involved in the effects of methionine enkephalin by binding with the traditional opioid receptors,and therefore resulting in different biological effects.

Nociceptin und der ORL1-Rezeptor

Molecular biological investigations led to the discovery of the ORL1 receptor ( opioid receptor like-1 receptor) and its endogenous ligand nociceptin. Although its sequence and structure are closely related to traditional opioid receptors, the ORL1 receptor shows low binding affinities for selective opioid agonists and antagonists. On the other hand, the ORL1 ligand nociceptin does not bind to the three traditional opioid receptors. The activation of the G protein-coupled ORL1 receptor inhibits adenlylate cyclase activity, reduces the intracellular concentration of the second messenger cAMP and regulates ion channels. The supraspinal administration of nociceptin produces hyperalgesia. unlike opioids. Spinal intrathecal and peripheral administration of nociceptin causes hyperalgesia in low doses and analgesia in high doses. The physiological role and detailed mechanisms of these dose-dependent nociceptin effects in opposite directions are not yet known. In addition, nociceptin modulates other biological phenomena such as feeding, locomotion, gastrointestinal function,memory, cardiovascular function,immunity, renal function, anxiety,dependence and tolerance.Future research on the physiological and pathophysiological importance of the nociceptin/ORL1 receptor systems may provide a target for novel therapeutics.

Proenkephalin A gene products activate a new family of sensory neuron--specific GPCRs.

Several peptide fragments are produced by proteolytic cleavage of the opioid peptide precursor proenkephalin A, and among these are a number of enkephalin fragments, in particular bovine adrenal medulla peptide 22 (BAM22). These peptide products have been implicated in diverse biological functions, including analgesia. We have cloned a newly identified family of 'orphan' G protein--coupled receptors (GPCRs) and demonstrate that BAM22 and a number of its fragments bind to and activate these receptors with nanomolar affinities. This family of GPCRs is uniquely localized in the human and rat small sensory neuron, and we called this family the sensory neuron--specific G protein--coupled receptors (SNSRs). Receptors of the SNSR family are distinct from the traditional opioid receptors in their insensitivity to the classical opioid antagonist naloxone and poor activation by opioid ligands. The unique localization of SNSRs and their activation by proenkephalin A peptide fragments indicate a possible function for SNSRs in sensory neuron regulation and in the modulation of nociception.

Analgesia elicited by OFQ/nociceptin and its fragments from the amygdala in rats

The heptadecapeptide, orphanin FQ/nociceptin (OFQ/N), binds with high affinity to the ORL-1/KOR-3 opioid receptor clone, yet binds poorly with traditional opioid receptors. OFQ/N has a complex functional profile with relation to nociceptive processing, displaying pro-nociceptive properties in some studies, acting as an inhibitor of stress-induced analgesia in others, yet producing both spinal and supraspinal antinociceptive actions in other studies. Among the intracerebral sites at which OFQ/N might produce one or more of these actions is the amygdala which has been intimately implicated in both antinociceptive and stress-related responses. Therefore, the present study assessed whether microinjections into the amygdala of equimolar doses of OFQ/N 1–17 or its shorter-chained active fragments, OFQ/N 1–11 or OFQ/N 1–7, would produce analgesia as measured by either reactivity to high-intensity radiant heat or reactivity to electric shock, and produce hyperalgesia as measured by reactivity to lower-intensity radiant heat. OFQ/N 1–17 in the amygdala produced a dose-dependent and time-dependent increase in high-intensity tail-flick latencies with maximal effects observed at a dose range of 0.75–3 nmol, and lesser effects at lower (0.015–0.15 nmol) and higher (5.5–30 nmol) doses. Both OFQ/N 1–11 and OFQ/N 1–7 in the amygdala displayed lower magnitudes of analgesia than OFQ/N 1–17 on this measure, with OFQ/N 1–11 displaying maximal effects at higher (15–30 nmol) doses and OFQ/N 1–7 displaying maximal effects at lower (0.15–1.5 nmol) doses. In contrast to traditional μ and κ opioids and β-endorphin, none of the OFQ/N fragments in the amygdala exhibited any analgesic responses on the jump test. Finally, using a low-intensity radiant heat assay capable of detecting hyperalgesic responses, each of the OFQ/N fragments in the amygdala increased tail-flick latencies on this measure. Therefore, OFQ/N fragments appear to exert only analgesic responses in the amygdala with quantitative and qualitative differences relative to traditional opioid agonists.

Identification of a high-affinity orphanin FQ/nociceptin(1-11) binding site in mouse brain.

The presence of pairs of basic amino acids within the orphanin FQ/Nociceptin (OFQ/N) sequence has raised the possibility that truncated versions of the peptide might be physiologically important. OFQ/N(1-11) is pharmacologically active in mice, despite its poor affinity in binding assays (K(i) > 250 nM) for the OFQ/N receptor. Using an analog of OFQ/N(1-11), [(125)I][Tyr(10)]OFQ/N(1-11), we identified a high-affinity binding site (K(D) 234 pM; B(max) 43 fmol/mg protein) with a selectivity profile distinct from the OFQ/N receptor and all the traditional opioid receptors. This site had very high affinity for OFQ/N and its related peptides. The most striking differences between the new site and the OFQ/N receptor previously observed in brain were seen with traditional opioids. Dynorphin A analogs and alpha-neoendorphin competed with [(125)I][Tyr(10)]OFQ/N(1-11) binding in mouse brain with K(i) values below 10 nM, while naloxone benzoylhydrazone (K(i) 3.9 nM) labeled the [(125)I][Tyr(10)]OFQ/N(1-11) binding site as potently as many traditional opioid receptors. Several other opioids, including fentanyl, (-)cyclazocine, levallorphan, naltrindole, and diprenorphine, also displayed moderate affinities for this site. Finally, the [(125)I][Tyr(10)]OFQ/N(1-11) site had a unique regional distribution consistent with a distinct receptor. Thus, [(125)I][Tyr(10)]OFQ/N(1-11) labels a novel site in brain with a selectivity profile intermediate between that of either opioid or OFQ/N receptors.

Nociceptin, a novel endogenous ligand for the ORL 1 receptor, dilates isolated resistance arteries from the rat

The heptadecapeptide nociceptin, also known as Orphanin FQ, is a recently discovered endogenous ligand for the opioid-like G-protein coupled receptor, ORL 1. In the present study, responses to nociceptin were investigated in isolated pressurized resistance arteries from the rat mesenteric vascular bed. Nociceptin in bath concentrations of 10 −9–10 −6 M induced concentration-dependent increases in arterial diameter when the artery was precontracted with U46619; and administration of the structurally related opioid agonists, dynorphin A and met-enkephalin, had no effect on arterial diameter. Vasodilator responses to nociceptin were not altered by the opioid receptor antagonist naloxone or by the nitric oxide synthase inhibitor N ω-nitro- l-arginine. Responses to nociceptin were not altered by the muscarinic receptor blocking agent atropine or phentolamine, or the calcitonin gene-related peptide (CGRP) receptor antagonist CGRP-(8-37). These data suggest that nociceptin has direct vasodilator activity that is not dependent upon the activation of a traditional opioid receptor, muscarinic or CGRP receptors, an inhibitory effect on the adrenergic nervous system, or the release of nitric oxide in isolated resistance arteries from the rat mesentery.

Profound inhibition of cardiomotor neurons in the rat rostral ventrolateral medulla by nociceptin (orphanin FQ).

Nociceptin (orphanin FQ), the newly discovered endogenous ligand for the opioid receptor like-1 receptor, profoundly inhibited spontaneous discharges of neurons in the rostral ventrolateral medulla (RVLM) in rat brain slices. This inhibition was concentration-dependent (0.3, 1, 3 and 10 nM) and insensitive to pharmacological blockade of traditional opioid receptors by naloxone. Moreover, nociceptin injected into the RVLM (10 nM, 0.1 microliter) in anesthetized rats decreased arterial blood pressure and heart rate by 31% and 15%, respectively. The data obtained in vitro and in vivo suggest that nociceptin has powerful effects on the RVLM neurons involving central control of cardiovascular activity. The negative regulation of cardiovascular activity by nociceptin is not mediated through typical naloxone-sensitive opioid receptors.

Blockade of Morphine-Induced Hindlimb Myoclonic Seizures in Mice by Ketamine

Morphine administration can lead to a variety of side-effects, including myoclonus. In an animal model, high morphine doses given intrathecally elicit hindlimb myoclonic seizures which are not influenced by traditional opioid receptor antagonists, such as naloxone. Ketamine prevents this seizure-like activity in a dose-dependent manner. The response is stereoselective, with S-ketamine far more potent than R-ketamine. A competitive NMDA antagonist, NPC17742, also prevents the seizures, although less potently than ketamine. Dextromethorphan has limited activity in this model, while haloperidol and pentothal are without any effect.

Biochemical Evidence for Orphanin FQ/Nociceptin Receptor Heterogeneity in Mouse Brain

A recently identified novel peptide, orphanin FQ/nociceptin (OFQ/N), is an endogenous ligand for a unique member of the cloned opioid receptor family. Saturation studies in mouse brain membranes reveal curvilinear Scatchard plots with both a higher (KD3.8 pM, Bmax31.6 fmol/mg protein) and a lower (KD896 pM, Bmax233 fmol/mg protein) affinity site in brain tissue, compared to only a single site in transfected CHO cells (KD36 pM). Competition studies confirm the high affinity of OFQ/N for this site, but shallow Hill slopes suggest heterogeneity. Traditional opioids have poor affinity for this receptor and OFQ/N and its fragments do not label traditional opioid receptors. In brain homogenates, both OFQ/N and OFQ/N(1-11) inhibit forskolin-stimulated camp accumulation with IC50values of 1 nM or less, an action which is readily reversed by opioid antagonists. OFQ/N(1-7) shows little activity. Together, these studies suggest the presence of heterogeneous, functionally active OFQ/N receptors in mouse brain.

Naloxone sensitive orphanin FQ-induced analgesia in mice

Orphanin FQ, also known as nociceptin, is a heptadecapeptide with very high affinity for a novel member of the cloned opioid receptor family which produces hyperalgesia in mice. In addition to hyperalgesia, which is observed soon after administration of orphanin FQ, we now describe a delayed analgesic response. Unlike orphanin FQ-induced hyperalgesia, orphanin FQ-induced analgesia is readily reversed by the opioid antagonist naloxone, implying an opioid mechanism of action. In view of the very poor affinity of orphanin FQ for all the known traditional opioid receptors and the low affinity of opioids for the 125I[Tyr 14]orphanin FQ binding site, orphanin FQ-induced analgesia is probably mediated through a novel orphanin FQ receptor subtype.