Opioid Receptor-like Receptor Research Articles

Caenorhabditis Elegans Exhibits Morphine Addiction-like Behavior via the Opioid-like Receptor NPR-17.

Addiction has become a profound societal problem worldwide, and few effective treatments are available. The nematode Caenorhabditis elegans (C. elegans) is an excellent invertebrate model to study neurobiological disease states. C. elegans reportedly developed a preference for cues that had previously been paired with addictive drugs, similar to place conditioning findings in rodents. Moreover, several recent studies discovered and reported the existence of an opioid-like system in C. elegans. Still unclear, however, is whether C. elegans exhibits addictive-like behaviors for opioids, such as morphine. In the present study, we found that C. elegans exhibited dose-dependent preference for morphine using the conditioned chemosensory-cue preference (CCP) test. This preference was blocked by co-treatment with the opioid receptor antagonist naloxone. C. elegans also exhibited aversion to naloxone-precipitated withdrawal from chronic morphine exposure. The expression of morphine-induced CCP and morphine withdrawal were abolished in worms that lacked the opioid-like receptor NPR-17. Dopamine-deficient mutant (cat-2 (e1112)) worms also did not exhibit morphine-induced CCP. These results indicate that the addictive function of the opioid system exists in C. elegans, which may serve as a useful model of opioid addiction.

Возможный механизм лиганд-рецепторного связывания синтетического трипептида Ac-RRR-NH2 с мембраной ноцицептивного нейрона

The article reports the discovery of a novel signaling cascade opioid-like receptor → Na,K-ATPase/Src → NaV1.8 channel in the nociceptive neuron membrane. Triggering this cascade results in the modulation of its effector unit—the NaV1.8 channel activation gating device, whereas the Na,K-ATPase/Src complex performs the signal transducer function. The cascade has three targets. Their modulation by the attacking molecules may evoke an antinociceptive response at the peripheral level. The first target is the opioid-like receptor activated by a number of gamma-pyrone derivatives. The second target is the Na,K-ATPase/Src complex, with its transducer function controlled by ouabain at nanomolar (endogenous) concentrations. The third target is the NaV1.8 channel activation gating device modulated by arginine-containing short peptides. The article discusses a possible mechanism of ligand-receptor binding of the arginine-containing tripeptide Ac-RRR-NH2 to the NaV1.8 channel in the primary sensory neuron membrane. Extracellular application of the tripeptide is shown by the patch-clamp method to decrease the voltage sensitivity of NaV1.8 channels. Positively charged guanidinium groups of arginine side chains are supposed to play the key role in the ligand-receptor complex formation. The results of conformational analysis demonstrate that the distances between the guanidinium groups in the tripeptide molecule exceed 10 Å. The obtained data lead us to conclude that the studied tripeptide can bind to the NaV1.8 channel using the mechanism described earlier for a range of other short arginine-containing peptides. In view of the foregoing, the tripeptide Ac-RRR-NH2 is a promising analgesic.

Role of Nociceptin/Orphanin FQ-NOP Receptor System in the Regulation of Stress-Related Disorders

Nociceptin/orphanin FQ (N/OFQ) is a 17-residue neuropeptide that binds the nociceptin opioid-like receptor (NOP). N/OFQ exhibits nucleotidic and aminoacidics sequence homology with the precursors of other opioid neuropeptides but it does not activate either MOP, KOP or DOP receptors. Furthermore, opioid neuropeptides do not activate the NOP receptor. Generally, activation of N/OFQ system exerts anti-opioids effects, for instance toward opioid-induced reward and analgesia. The NOP receptor is widely expressed throughout the brain, whereas N/OFQ localization is confined to brain nuclei that are involved in stress response such as amygdala, BNST and hypothalamus. Decades of studies have delineated the biological role of this system demonstrating its involvement in significant physiological processes such as pain, learning and memory, anxiety, depression, feeding, drug and alcohol dependence. This review discusses the role of this peptidergic system in the modulation of stress and stress-associated psychiatric disorders in particular drug addiction, mood, anxiety and food-related associated-disorders. Emerging preclinical evidence suggests that both NOP agonists and antagonists may represent a effective therapeutic approaches for substances use disorder. Moreover, the current literature suggests that NOP antagonists can be useful to treat depression and feeding-related diseases, such as obesity and binge eating behavior, whereas the activation of NOP receptor by agonists could be a promising tool for anxiety.

New approaches to the design of analgesic medicinal substances.

A gamma-pyrone derivative, comenic acid, activates the opioid-like receptor-mediated signaling pathway that modulates the NaV1.8 channels in the primary sensory neuron membrane. These channels are responsible for the generation of the nociceptive signal; therefore, gamma-pyrones have great therapeutic potential as analgesics, and this effect deserves a deeper understanding. The novelty of our approach to the design of a medicinal substance is based on a combination of the data obtained from living neurons using very sensitive physiological methods and the results of quantum chemical calculations. This approach allows the correlation of the molecular structure of gamma-pyrones with their ability to evoke a physiological response of the neuron. Comenic acid can bind to two calcium cations. One of them is chelated by the carbonyl and hydroxyl functional groups, while the other forms a salt bond with the carboxylate anion. Calcium-bound gamma-pyrones have fundamentally different electrostatic properties from free gamma-pyrone molecules. These two calcium ions are key elements involved in ligand-receptor binding. It is very likely that ion-ionic interactions between these cations and anionic functional groups of the opioid-like receptor activate the latter. The calculated intercationic distance of 9.5Å is a structural criterion for effective ligand-receptor binding of calcium-bound gamma-pyrones.

A comprehensive review of partial opioid agonists for the treatment of chronic pain.

Chronic pain is a common condition that is being increasingly recognized, diagnosed, and treated in a variety of settings. Opioids can be used to treat chronic pain but at the cost of adverse effects and risk of dependence. Recently, there has been a movement to improve analgesic care in the setting of the opioid epidemic and the overprescribing of opioids, causing over-accessibility, dependence, and large numbers of overdose deaths. Opioid-specific receptors, including the μ, δ, κ, and opioid receptor like-1 (ORL-1) receptors, are each 7-transmembrane spanning proteins, which affect the G-protein and β-arrestin cascades. Each opioid class can act differently on the receptors, resulting in full, partial, or antagonizing effects. This comprehensive review looks at different agents in major classes, nonselective and mixed/partial agonists/antagonists, including the nonselective partial agonists, levorphanol and tramadol. Mixed partial agonists/antagonists include buprenorphine, pentazocine, nalbuphine, and butorphanol. Oliceridine is the only current selective partial agonist that agonizes specific pathways to promote analgesic effects and discourage adverse effects.

P4441Serum activity against specific G-protein coupled receptors is associated with the severity of orthostatic symptoms in patients with POTS

Abstract Background Postural orthostatic tachycardia syndrome (POTS) is characterised by an excessive heart rate increase upon standing and orthostatic intolerance. Previous studies indicate autoimmune involvement mediated via G-protein coupled receptors (GPCRs) as a possible aetiology. Purpose To test if serum from POTS patients and controls activates selected GPCRs and if the activation associates with the severity of symptoms. Methods A total of 34 POTS patients (age 28.9 [9.9] years; 29 women) and a population-based control group of 25 healthy subjects (age 30.7 [8.6] years; 21 women) were included. The subjects performed an active standing test and completed the orthostatic hypotension questionnaire (OHQ) including ten items related to orthostatic intolerance. The OHQ composite score was calculated (range 0–10). Sera were analysed by a FRET-based reporter system (Tango GeneBLAzer, Thermo Fischer) based on a beta-arrestin-linked transcription factor driving transgenic betalactamase transcription. HEK293M-cells over-expressing one of the GPCRs, adrenergic alpha-1 (ADRA1), adrenergic beta-2 (ADRB2), muscarinic type-2 (CHRM2) and opioid-receptor-like 1 (OPRL1) receptor were plated in 96-well optical plates and allowed to re-attach during 48 hrs. The cells were treated with 10% sera diluted in RPMI for 5 hours, followed by addition of the FRET-substrate, incubation for 60 min and quantification analysis in a CLARIOStar multi-purpose plate reader. Quantification of the activation of the GPCRs was log-transformed and related to the OHQ composite and individual scores in age-adjusted linear regression models. OHQ scores were compared according to the median of sera activation, using independent samples t-test. Results Serum ADRA1 activation associated with the OHQ composite score (beta 0.77 OHQ points per SD of activity; p=0.009), whereas there were no significant associations among controls (p=0.953). The association between ADRA1 and total OHQ was significant also after adjusting for heart rate and systolic blood pressure at 3 min (p=0.024). The OHQ composite score was higher in with above median serum ADRA1 activation (Figure 1). ADRA1 activation associated with a higher score for vision problems (p<0.001) and symptoms during prolonged standing (p=0.037) and walking for short (p=0.042) or long periods (p=0.001). The activity of ARB2, CHRM2 and OPRL1 did not associate with OHQ composite score in neither POTS patients nor controls. Figure 1. OHQ according to ADRA1 activation Conclusions Activating serum proteins for the adrenergic alpha-1 receptor are associated with the severity of orthostatic symptoms in POTS patients, partly independent of the orthostatic hemodynamic response. Serum activity against alpha1 receptors is related particularly to symptoms of disturbed vision and symptoms during walking or prolonged standing. Serum activity against the adrenergic alpha 1 receptor may be a one factor underlying the orthostatic symptoms in POTS. Acknowledgement/Funding The Swedish Heart-Lung Foundation, The Swedish Heart and Lung Association, Solidex, ALF funds, Crafoord Foundation, Ernhold Lundströms Foundation.

Roles of K+ and cation channels in ORL-1 receptor-mediated depression of neuronal excitability and epileptic activities in the medial entorhinal cortex

Nociceptin (NOP) is an endogenous opioid-like peptide that selectively activates the opioid receptor-like (ORL-1) receptors. The entorhinal cortex (EC) is closely related to temporal lobe epilepsy and expresses high densities of ORL-1 receptors. However, the functions of NOP in the EC, especially in modulating the epileptiform activity in the EC, have not been determined. We demonstrated that activation of ORL-1 receptors remarkably inhibited the epileptiform activity in entorhinal slices induced by application of picrotoxin or by deprivation of extracellular Mg2+. NOP-mediated depression of epileptiform activity was independent of synaptic transmission in the EC, but mediated by inhibition of neuronal excitability in the EC. NOP hyperpolarized entorhinal neurons via activation of K+ channels and inhibition of cation channels. Whereas application of Ba2+ at 300 μM which is effective for the inward rectifier K+ (Kir) channels slightly inhibited NOP-induced hyperpolarization, the current-voltage (I-V) curve of the net currents induced by NOP was linear without showing inward rectification. However, a role of NOP-induced inhibition of cation channels was revealed after inhibition of Kir channels by Ba2+. Furthermore, NOP-mediated augmentation of membrane currents was differently affected by application of the blockers selective for distinct subfamilies of Kir channels. Whereas SCH23390 or ML133 blocked NOP-induced augmentation of membrane currents at negative potentials, application of tertiapin-Q exerted no actions on NOP-induced alteration of membrane currents. Our results demonstrated a novel cellular and molecular mechanism whereby activation of ORL-1 receptors depresses epilepsy.

Nociceptin/Orphanin-FQ Inhibits Gonadotropin-Releasing Hormone Neurons via G-Protein-Gated Inwardly Rectifying Potassium Channels.

The pulsatile release of gonadotropin-releasing hormone (GnRH) is a key feature of the hypothalamic–pituitary–gonadal axis. Kisspeptin neurons in the arcuate nucleus (ARC) trigger GnRH neuronal activity, but how GnRH neurons return to baseline electrical activity is unknown. Nociceptin/orphanin-FQ (OFQ) is an inhibitory neuromodulator. ARC proopiomelanocortin (POMC) neurons, known to receive inputs from ARC kisspeptin neurons, contact GnRH neurons and coexpress OFQ in the rat. In the present study, the effect of OFQ(1-13) on GnRH neurons was determined in the mouse. We identified transcripts for the OFQ receptor [opioid receptor like 1 (ORL1)] in GnRH neurons, and, using two-model systems (explants and slices), we found that OFQ exerted a potent inhibition on GnRH neurons, with or without excitatory inputs. We confirmed that the inhibition was mediated by ORL1 via Gi/o-protein coupling. The inhibition, occurring independently of levels of intracellular cyclic adenosine monophosphate, was sensitive to inwardly rectifying potassium channels. The only specific blocker of Gi/o-protein-coupled inwardly rectifying potassium (GIRK) channels, tertiapin-Q (TPNQ), was ineffective in the inhibition of OFQ. Two GIRK activators, one sharing the binding site of TPNQ and one active only on GIRK1-containing GIRK channels, failed to trigger an inhibition. In contrast, protein kinase C phosphorylation activation, known to inhibit GIRK2-mediated currents, prevented the OFQ inhibition. These results indicate a specific combination of GIRK subunits, GIRK2/3 in GnRH neurons. In vivo, double-labeled OFQ/POMC fibers were found in the vicinity of GnRH neurons, and OFQ fibers apposed GnRH neurons. Together, this study brings to light a potent neuromodulator of GnRH neurons.

Activation of spinal nociceptin receptors induces cardiovascular depression and antinociception in an independent manner in mice

PurposeThe nociceptin receptor (NOP) was discovered in 1994 and was designated opioid-like receptor; activation of NOP leads to reduced neuronal excitability. Although suggested by the anatomical localization of NOP in brain or spinal cord, the cardiovascular or nociceptive effects of its endogenous ligand, nociceptin, are equivocal. Taking advantage from intrathecal application of nociceptin to simultaneously activate NOP on sympathetic preganglionic neurons in the intermediolateral column (IML) and superficial laminae of dorsal horn, we investigated whether the nociceptin-induced cardiovascular effects engage the participation of baroreflex, and whether the concurrently elicited changes in blood pressure and pain responses are interrelated.MethodsNOPs in the thoracic spinal cord of ICR or C57BL/6 mice were identified with immunofluorescence staining and were activated through intrathecal administration of nocicetpin. The elicited changes in cardiovascular parameters and tail-flick nociceptive responses were measured.ResultsPositive immunoreactivity against NOP colocalized with neurons in the IML and superficial dorsal horn layers of thoracic spinal cord. Intrathecal administration of nociceptin (1, 2, or 5 nmol) elicited a significant and dose-dependent decrease in blood pressure or heart rate that was paralleled by reduced baroreflex-mediated sympathetic vasomotor tone and mirrored by augmented cardiac vagal baroreflex, alongside prolonged tail-flick latency with an efficacy of hypotension <<< antinociception. Coadministration of the specific NOP antagonist, UFP101 (10 nmol), blunted all nociceptin-elicited responses. However, restoring blood pressure to baseline level failed to affect the antinociceptive actions of nociceptin.ConclusionActivation of thoracic spinal NOP in ICR and C57BL/6 mice induces blood pressure and heart rate by decreasing the sympathetic outflow of both arms of the baroreflex arc to the blood vessels and the heart, and the antinociceptive responses to nociceptin are independent of and disproportional to its cardiovascular actions.

ORL1 Activation Mediates a Novel ORL1 Receptor Agonist SCH221510 Analgesia in Neuropathic Pain in Rats.

Opioid receptor like 1 (ORL1) receptor activation displayed an anti-nociceptive effect at spinal level for acute and neuropathic pain. SCH221510, an orally active non-peptide ORL1 agonist, was reported to be effective in treating neuropathic pain. The present study used ORL1 antagonist and siRNA to investigate that ORL1 activation mediates intrathecal SCH221510 analgesia in neuropathic pain induced by chronic constrictive injury (CCI) to rat sciatic nerve. Paw withdrawal latency and 50% mechanical threshold were measured for thermal and mechanical hypersensitivity in rats. CCI significantly decreased paw withdrawal latency and mechanical threshold. SCH221510 (3, 10, 30μg) or ORL1 antagonist ([Nphe1]nociceptin(1-13)NH2, 10μg) was intrathecally injected to test the behavioral effects on neuropathic pain. Intrathecal siRNA was started on 1day before CCI surgery and maintained for 7days. L4-L5 spinal cord ORL1 mRNA and protein were measured by real-time PCR and Western blot. The effect of intrathecal siRNA on SCH2210510 was tested in CCI rats on day 7. Intrathecal SCH221510 dose-dependently reduced thermal and mechanical hypersensitivity induced by CCI. [Nphe1]nociceptin(1-13)NH2 blocked SCH221510 analgesia in CCI rats. Intrathecal siRNA blocked ORL1 mRNA and protein increase induced by CCI. Intrathecal ORL1 siRNA did not change thermal and mechanical hypersensitivity induced by nerve injury. Intrathecal siRNA blocked SCH221510 analgesia in neuropathic pain at spinal level. Conclusively, ORL1 activation mediates SCH221510 analgesia in neuropathic pain at spinal level. The results warrant a potential clinically applicable drug in treating neuropathic pain.

JTC-801 inhibits the proliferation and metastasis of ovarian cancer cell SKOV3 through inhibition of the PI3K - AKT signaling pathway.

Ovarian cancer (OC) is the commonest cause of gynaecological cancer-associated death because of the wide metastasis and frequent recidivation. JTC-801 is a new synthetic compound with the function of reversing pain and anxiety symptoms as a selective opioid receptor-like1 receptor (belonging to the G-protein-coupled receptor) antagonist. We investigated the role and possible mechanisms of JTC-801 in the cell growth and metastasis of OC. It was observed that JTC-801 inhibited the proliferation, invasion and migration of cancer in SKOV3 cells. The apoptosis rate of SKOV3 cells treated with JTC-801 was significantly increased (P<0.05), and the expression results of relevant apoptosis proteins (BCL2, BAX, Active Caspase-3) indicated the JTC-801 could induce the apoptosis of SKOV3. Further, the expression levels of phosphorylated AKT, phosphorylated mTOR, P70 and CyclinD1 in the PI3K/AKT signaling pathway were obviously reduced in the JTC-801 treated SKOV3 group. This suggests that JTC-801 exerts its anticancer effect through the PI3K/AKT signaling pathway. Our data also highlights the possibility of using JTC-801 as a novel therapeutic drug for OC treatment mean while it plays the analgesic effect.

A Novel and Selective Nociceptin Receptor (NOP) Agonist (1-(1-((cis)-4-isopropylcyclohexyl)piperidin-4-yl)-1H-indol-2-yl)methanol (AT-312) Decreases Acquisition of Ethanol-Induced Conditioned Place Preference in Mice.

Nociceptin/orphanin FQ, the endogenous peptide agonist for the opioid receptor-like receptor (also known as NOP or the nociceptin receptor), has been shown to block the acquisition and expression of ethanol (EtOH)-induced conditioned place preference (CPP). Here, we report the characterization of a novel small-molecule NOP ligand AT-312 (1-(1-((cis)-4-isopropylcyclohexyl)piperidin-4-yl)-1H-indol-2-yl)methanol) in receptor binding and GTPγS functional assays invitro. We then investigated the effect of AT-312 on the rewarding action of EtOH in mice using the CPP paradigm. Further, using mice lacking the NOP receptor and their wild-type controls, we also examined the involvement of NOP in the effect of AT-312. Motivational effects of AT-312 alone were also assessed in the CPP paradigm. Female mice lacking NOP and/or their wild-type controls received conditioning in the presence or absence of the NOP agonist [AT-312 (1, 3, and 10mg/kg) or the control NOP agonist SCH221510 (10mg/kg)] followed by saline/EtOH for 3 consecutive days (twice daily) and tested for CPP in a drug-free state on the next day. Our invitro data showed that AT-312 is a high-affinity, selective NOP full agonist with 17-fold selectivity over the mu opioid receptor and >200-fold selectivity over the kappa opioid receptor. The results of our invivo studies showed that AT-312 reduced EtOH CPP at the lowest dose (1mg/kg) tested but completely abolished EtOH CPP at higher doses (3 or 10mg/kg) compared to their vehicle-treated control group. AT-312 (3mg/kg) did not alter EtOH-induced CPP in mice lacking NOP, confirming that AT-312 reduced EtOH CPP through its action at the NOP receptor. AT-312 (3mg/kg) did not induce reward or aversion when administered alone, showing that the novel small-molecule NOP agonist shows efficacy in blocking EtOH-induced CPP via the NOP receptor. Together, these data suggest that small-molecule NOP agonists have the potential to reduce alcohol reward and may be promising as medications to treat alcohol addiction.

In silico design of novel probes for the atypical opioid receptor MRGPRX2.

The primate-exclusive MRGPRX2 G protein-coupled receptor (GPCR) has been suggested to modulate pain and itch. Despite putative peptide and small molecule MRGPRX2 agonists, selective nanomolar potency probes have not yet been reported. To identify a MRGPRX2 probe, we first screened 5,695 small molecules and found many opioid compounds activated MRGPRX2, including (−)- and (+)-morphine, hydrocodone, sinomenine, dextromethorphan and the prodynorphin-derived peptides, dynorphin A, dynorphin B, and α- and β-neoendorphin. We used these to select for mutagenesis-validated homology models and docked almost 4 million small molecules. From this docking, we predicted ZINC-3573, which represents a potent MRGPRX2-selective agonist, showing little activity against 315 other GPCRs and 97 representative kinases, and an essentially inactive enantiomer. ZINC-3573 activates endogenous MRGPRX2 in a human mast cell line inducing degranulation and calcium release. MRGPRX2 is a unique atypical opioid-like receptor important for modulating mast cell degranulation, which can now be specifically modulated with ZINC-3573.

Characteristics of receptor- and transducer-coupled activation of the intracellular signalling in sensory neuron revealed by atomic force microscopy

The mechanical properties of sensory neurons upon activation of intracellular cascade processes by comenic acid binding to a membrane opioid-like receptor (receptor-coupled), as well as a very low (endogenous) concentration of ouabain (transducer-coupled), have been investigated. Using atomic force microscopy, it is established that exposure to ouabain, in contrast to the impact of comenic acid, leads to a hardening of the neuron soma. This suggests that the receptor-coupled signal transmission to the cell genome is carried out through mechanisms that are different from the transducer-coupled signal pathways.

Opiates Modulate Noxious Chemical Nociception through a Complex Monoaminergic/Peptidergic Cascade.

The ability to detect noxious stimuli, process the nociceptive signal, and elicit an appropriate behavioral response is essential for survival. In Caenorhabditis elegans, opioid receptor agonists, such as morphine, mimic serotonin, and suppress the overall withdrawal from noxious stimuli through a pathway requiring the opioid-like receptor, NPR-17. This serotonin- or morphine-dependent modulation can be rescued in npr-17-null animals by the expression of npr-17 or a human κ opioid receptor in the two ASI sensory neurons, with ASI opioid signaling selectively inhibiting ASI neuropeptide release. Serotonergic modulation requires peptides encoded by both nlp-3 and nlp-24, and either nlp-3 or nlp-24 overexpression mimics morphine and suppresses withdrawal. Peptides encoded by nlp-3 act differentially, with only NLP-3.3 mimicking morphine, whereas other nlp-3 peptides antagonize NLP-3.3 modulation. Together, these results demonstrate that opiates modulate nociception in Caenorhabditis elegans through a complex monoaminergic/peptidergic cascade, and suggest that this model may be useful for dissecting opiate signaling in mammals. Opiates are used extensively to treat chronic pain. In Caenorhabditis elegans, opioid receptor agonists suppress the overall withdrawal from noxious chemical stimuli through a pathway requiring an opioid-like receptor and two distinct neuropeptide-encoding genes, with individual peptides from the same gene functioning antagonistically to modulate nociception. Endogenous opioid signaling functions as part of a complex, monoaminergic/peptidergic signaling cascade and appears to selectively inhibit neuropeptide release, mediated by a α-adrenergic-like receptor, from two sensory neurons. Importantly, receptor null animals can be rescued by the expression of the human κ opioid receptor, and injection of human opioid receptor ligands mimics exogenous opiates, highlighting the utility of this model for dissecting opiate signaling in mammals.

Intrathecal administration of low-dose nociceptin/orphanin FQ induces allodynia via c-Jun N-terminal kinase and monocyte chemoattractant protein-1.

Pathological chronic pain, which is frequently associated with prolonged tissue damage, inflammation, tumour invasion, and neurodegenerative diseases, gives rise to hyperalgesia and allodynia. We previously reported that intrathecal administration of nociceptin/orphanin FQ (N/OFQ), an endogenous ligand for the orphan opioid receptor-like receptor, in the femtomole range induces touch-evoked allodynia. N/OFQ has been implicated in multiple signalling pathways, such as inhibition of cAMP production and Ca(2+) channels, or activation of K(+) channels and mitogen-activated protein kinase, although the signalling pathways of N/OFQ-induced allodynia remain unclear. To address these issues, we developed an exvivo mitogen-activated protein kinase assay by using intact slices of mouse spinal cord. N/OFQ markedly increased the phosphorylation of c-Jun N-terminal kinase (JNK) in the superficial dorsal horn of the spinal cord. The N/OFQ-stimulated JNK phosphorylation was significantly inhibited by pertussis toxin, the phospholipase C inhibitor U73122, and the inositol trisphosphate receptor antagonist Xestospongin C. Intrathecal administration of the JNK inhibitor SP600125 inhibited N/OFQ-evoked allodynia. The N/OFQ-induced increase in JNK phosphorylation was observed in astrocytes that expressed glial fibrillary acidic protein. N/OFQ also induced monocyte chemoattractant protein-1 (MCP-1) release via the JNK pathway, and N/OFQ-induced JNK phosphorylation was observed in MCP-1-immunoreactive astrocytes. Intrathecal administration of the MCP-1 receptor antagonist RS504393 inhibited N/OFQ-evoked allodynia. These results suggest that, in the spinal dorsal horn, N/OFQ induces allodynia through activation of JNK via the phospholipase C-inositol trisphosphate pathway, which is coupled to pertussis toxin-sensitive G-protein, and following the release of MCP-1 from astrocytes.

Antidepressant-like Effects of Buprenorphine are Mediated by Kappa Opioid Receptors.

Previous studies have identified potential antidepressant effects of buprenorphine (BPN), a drug with high affinity for mu opioid receptor (MORs) and kappa opioid receptors (KORs) and some affinity at delta opioid receptor (DOR) and opioid receptor-like 1 (ORL-1) receptors. Therefore, these studies examined which opioid receptors were involved in BPN's effects on animal behavior tests sensitive to antidepressant drugs. The acute effects of BPN were tested in the forced swim test (FST) using mice with genetic deletion of individual opioid receptors or after pharmacological blockade of receptors. For evaluating the effects of BPN on chronic stress, separate groups of mice were exposed to unpredictable chronic mild stress (UCMS) for 3 weeks and treated with BPN for at least 7 days before behavioral assessment and subsequent measurement of Oprk1, Oprm1, and Pdyn mRNA expression in multiple brain regions. BPN did not reduce immobility in mice with KOR deletion or after pretreatment with norbinaltorphimine, even though desipramine remained effective. In contrast, BPN reduced immobility in MOR and DOR knockout mice and in mice pretreated with the ORL-1 antagonist JTC-801. UCMS reduced sucrose preference, decreased time in the light side of the light/dark box, increased immobility in the FST and induced region-specific alterations in Oprk1, Oprm1, and PDYN mRNA expression in the frontal cortex and striatum. All of these changes were normalized following BPN treatment. The KOR was identified as a key player mediating the effects of BPN in tests sensitive to antidepressant drugs in mice. These studies support further development of BPN as a novel antidepressant.

Estradiol Rapidly Attenuates ORL-1 Receptor-Mediated Inhibition of Proopiomelanocortin Neurons via Gq-Coupled, Membrane-Initiated Signaling

Estradiol rapidly regulates the activity of arcuate nucleus (ARH) proopiomelanocortin (POMC) neurons that project to the medial preoptic nucleus (MPN) to regulate lordosis. Orphanin FQ/nociceptin (OFQ/N) acts via opioid receptor-like (ORL)-1 receptors to inhibit these POMC neurons. Therefore, we tested the hypothesis that estradiol excites POMC neurons by rapidly attenuating inhibitory ORL-1 signaling in these cells. Hypothalamic slices through the ARH were prepared from ovariectomized rats injected with Fluorogold into the MPN. Electrophysiological recordings were generated in ARH neurons held at or near -60 mV, and neuronal phenotype was determined post hoc by immunohistofluorescence. OFQ/N application induced robust outward currents and hyperpolarizations via G protein-gated, inwardly rectifying K⁺ (GIRK) channels that were attenuated by pretreatment with either 17-β estradiol (E₂) or E₂ conjugated to bovine serum albumin. This was blocked by the estrogen receptor (ER) antagonist ICI 182,780 and mimicked by the G_q-coupled membrane ER (G_q-mER) ligand STX and the ERα agonist PPT. Inhibiting phosphatidylinositol-3-kinase (PI3K) blocked the estrogenic attenuation of ORL-1/GIRK currents. Antagonizing either phospholipase C (PLC), protein kinase C (PKC), protein kinase A (PKA) or neuronal nitric oxide synthase (nNOS) also abrogated E₂ inhibition of ORL-1/GIRK currents, whereas activation of PKC, PKA, protein kinase B (Akt) and nNOS substrate <smlcap>L</smlcap>-arginine all attenuated the OFQ/N response. This was observed in 92 MPN-projecting, POMC-positive ARH neurons. Thus, ORL-1 receptor-mediated inhibition of POMC neurons is rapidly and negatively modulated by E₂, an effect which is stereoselective and membrane initiated via G_q-mER and ERα activation that signals through PLC, PKC, PKA, PI3K and nNOS.

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.

A Visible‐Light‐Mediated Radical Smiles Rearrangement and its Application to the Synthesis of a Difluoro‐Substituted Spirocyclic ORL‐1 Antagonist

AbstractA visible‐light‐mediated radical Smiles rearrangement has been developed to address the challenging synthesis of the gem‐difluoro group present in an opioid receptor‐like 1 (ORL‐1) antagonist that is currently in development for the treatment of depression and/or obesity. This method enables the direct and efficient introduction of the difluoroethanol motif into a range of aryl and heteroaryl systems, representing a new disconnection for the synthesis of this versatile moiety. When applied to the target compound, the photochemical step could be conducted on 15 g scale using industrially relevant [Ru(bpy)3Cl2] catalyst loadings of 0.01 mol %. This transformation is part of an overall five‐step route to the antagonist that compares favorably to the current synthetic sequence and demonstrates, in this specific case, a clear strategic benefit of photocatalysis.