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Background and Purpose: Fentanyl analogues have been implicated in many cases of intoxication and death with overdose worldwide. The aim of this study is to investigate the pharmaco‐toxicology of two fentanyl analogues: butyrylfentanyl (BUF) and 4‐fluorobutyrylfentanyl (4F‐BUF). Experimental Approach: In vitro, we measured agonist opioid receptor efficacy, potency, and selectivity and ability to promote interaction of the μ receptor with G protein and β‐arrestin 2. In vivo, we evaluated thermal antinociception, stimulated motor activity and cardiorespiratory changes in female and male CD‐1 mice injected with BUF or 4F‐BUF (0.1–6 mg·kg−1). Opioid receptor specificity was investigated using naloxone (6 mg·kg−1). We investigated the possible role of stress in increasing cardiorespiratory toxicity using the corticotropin‐releasing factor 1 (CRF1) antagonist antalarmin (10 mg·kg−1). Key Results: Agonists displayed the following rank of potency at μ receptors: fentanyl > 4F‐BUF > BUF. Fentanyl and BUF behaved as partial agonists for the β‐arrestin 2 pathway, whereas 4F‐BUF did not promote β‐arrestin 2 recruitment. In vivo, we revealed sex differences in motor and cardiorespiratory impairments but not antinociception induced by BUF and 4F‐BUF. Antalarmin alone was effective in blocking respiratory impairment induced by BUF in both sexes but not 4F‐BUF. The combination of naloxone and antalarmin significantly enhanced naloxone reversal of the cardiorespiratory impairments induced by BUF and 4F‐BUF in mice. Conclusion and Implications: In this study, we have uncovered a novel mechanism by which synthetic opioids induce respiratory depression, shedding new light on the role of CRF1 receptors in cardiorespiratory impairments by μ agonists. [ABSTRACT FROM AUTHOR]

Rationale: Recently, we demonstrated that the activation of the nociceptin/orphanin FQ (N/OFQ) receptor (NOP) signaling facilitates depressive-like behaviors. Additionally, literature findings support the ability of the N/OFQ-NOP system to modulate the hypothalamic-pituitary-adrenal (HPA) axis. Objectives: Considering that dysfunctional HPA axis is strictly related to stress-induced psychopathologies, we aimed to study the role of the HPA axis in the pro-depressant effects of NOP agonists. Methods: Mice were treated prior to stress with the NOP agonist Ro 65-6570, and immobility time in the forced swimming task and corticosterone levels were measured. Additionally, the role of endogenous glucocorticoids and CRF was investigated using the glucocorticoid receptor antagonist mifepristone and the CRF1 antagonist antalarmin in the mediation of the effects of Ro 65-6570. Results: The NOP agonist in a dose-dependent manner further increased the immobility of mice in the second swimming session compared to vehicle. By contrast, under the same conditions, the administration of the NOP antagonist SB-612111 before stress reduced immobility, while the antidepressant nortriptyline was inactive. Concerning in-serum corticosterone in mice treated with vehicle, nortriptyline, or SB-612111, a significant decrease was observed after re-exposition to stress, but no differences were detected in Ro 65-6570-treated mice. Administration of mifepristone or antalarmin blocked the Ro 65-6570-induced increase in the immobility time in the second swimming session. Conclusions: Present findings suggest that NOP agonists increase vulnerability to depression by hyperactivating the HPA axis and then increasing stress circulating hormones and CRF1 receptor signaling. [ABSTRACT FROM AUTHOR]

Background and Purpose: In animal models of sepsis, increased activation of the Nociceptin/Orphanin FQ (N/OFQ) receptor NOP is associated with mortality and NOP antagonists improved survival. We have explored the role of the N/OFQ‐NOP system in freshly isolated volunteer human B‐ and T‐cells incubated with lipopolysaccharide (LPS) and peptidoglycan G (PepG) as a model of in vitro sepsis. Experimental Approach: B‐ and T‐cell NOP expression was measured using the NOP fluorescent probe N/OFQATTO594, N/OFQ content was measured using immunofluorescence, N/OFQ release was tracked using a CHOhNOPGαiq5 biosensor assay and NOP function was measured using transwell migration and cytokine/chemokine release using a 25‐plex assay format. Cells were challenged with LPS/PepG. Key Results: CD19‐positive B‐cells bound N/OFQATTO594; they also contain N/OFQ. Stimulation with CXCL13/IL‐4 increased N/OFQ release. N/OFQ trended to reduced migration to CXCL13/IL‐4. Surface NOP expression was unaffected by LPS/PepG, but this treatment increased GM‐CSF release in an N/OFQ sensitive manner. CD3‐positive T‐cells did not bind N/OFQATTO594; they did contain N/OFQ. Stimulation with CXCL12/IL‐6 increased N/OFQ release. When incubated with LPS/PepG, NOP surface expression was induced leading to N/OFQATTO594 binding. In LPS/PepG‐treated cells, N/OFQ reduced migration to CXCL12/IL‐6. LPS/PepG increased GM‐CSF release in an N/OFQ sensitive manner. Conclusions and Implications: We suggest both a constitutive and sepsis‐inducible N/OFQ‐NOP receptor autocrine regulation of B‐ and T‐cell function, respectively. These NOP receptors variably inhibit migration and reduce GM‐CSF release. These data provide mechanistic insights to the detrimental role for increased N/OFQ signalling in sepsis and suggest a potential role for NOP antagonists as treatments. [ABSTRACT FROM AUTHOR]

The mu opioid receptor agonists are the most efficacious pain controlling agents but their use is accompanied by severe side effects. More recent developments indicate that some ligands can differentially activate receptor downstream pathways, possibly allowing for dissociation of analgesia mediated through the G protein from the opioid-related side effects mediated by ß-arrestin pathway. In an effort to identify such biased ligands, here we present a series of thirteen endomorphin-2 (EM-2) analogs with modifications in positions 1, 2, and/or 3. All obtained analogs behaved as mu receptor selective agonists in calcium mobilization assay carried out on cells expressing opioid receptors and chimeric G proteins. A Bioluminescence Resonance Energy Transfer (BRET) approach was employed to determine the ability of analogs to promote the interaction of the mu opioid receptor with G protein or ß-arrestin 2. Nearly half of the developed analogs showed strong bias towards G protein, in addition four compounds were nearly inactive towards ß-arrestin 2 recruitment while blocking the propensity of EM-2 to evoke mu-ß-arrestin 2 interaction. The data presented here contribute to our understanding of EM-2 interaction with the mu opioid receptor and of the transductional propagation of the signal. In addition, the generation of potent and selective mu receptor agonists strongly biased towards G protein provides the scientific community with novel tools to investigate the in vivo consequences of biased agonism at this receptor. [ABSTRACT FROM AUTHOR]

Sepsis is a dysregulated host response to infection that can cause widespread effects on other organs including cardiovascular depression, hypotension and organ failure. The receptor for Nociceptin/Orphanin FQ (N/OFQ), NOP is expressed on immune cells and these cells can release the peptide. Exogenous N/OFQ can dilate blood vessels and this peptide is increased in animal and human sepsis. We hypothesise that NOP receptors are present on vascular endothelial cells and therefore provide the target for released N/OFQ to cause vasodilation and hence hypotension. Using human umbilical vein endothelial cells (HUVEC) and human vascular smooth muscle cells (HVSMC) freshly prepared from umbilical cords and up to passage 4, we assessed NOP mRNA expression by Polymerase Chain Reaction (PCR), NOP surface receptor expression using a fluorescent NOP selective probe (N/OFQATTO594) and NOP receptor function with N/OFQ stimulated ERK1/2 phosphorylation. As an in vitro sepsis mimic we variably incubated cells with 100ng/ml Lipopolysaccharide and Peptidoglycan G (LPS/PepG). HUVECs express NOP mRNA and this was reduced by ~80% (n = 49) after 24–48 hours treatment with LPS/PepG. Untreated cells do not express surface NOP receptors but when treated with LPS/PepG the reduced mRNA was translated into protein visualised by N/OFQATTO594 binding (n = 49). These NOP receptors in treated cells produced an N/OFQ (1μM) driven increase in ERK1/2 phosphorylation (n = 20). One (of 50) HUVEC lines expressed NOP mRNA and receptor protein in the absence of LPS/PepG treatment. In contrast, HVSMC expressed NOP mRNA and surface receptor protein (n = 10) independently of LPS/PepG treatment. These receptors were also coupled to ERK1/2 where N/OFQ (1μM) increased phosphorylation. Collectively these data show that an in vitro sepsis mimic (LPS/PepG) upregulates functional NOP expression in the vascular endothelium. Activation of these endothelial receptors as suggested from in vivo whole animal work may contribute to the hypotensive response seen in sepsis. Moreover, blockade of these receptors might be a useful adjunct in the treatment of sepsis. [ABSTRACT FROM AUTHOR]

The present study investigated the in vitro pharmacology of the human kappa opioid receptor using multiple assays, including calcium mobilization in cells expressing chimeric G proteins, the dynamic mass redistribution (DMR) label-free assay, and a bioluminescence resonance energy transfer (BRET) assay that allows measurement of receptor interaction with G protein and β-arrestin 2. In all assays, dynorphin A, U-69,593, and [D-Pro10]dyn(1-11)-NH2 behaved as full agonists with the following rank order of potency [D-Pro10]dyn(1-11)-NH2 > dynorphin A ≥ U-69,593. [Dmt1,Tic2]dyn(1-11)-NH2 behaved as a moderate potency pure antagonist in the kappa-β-arrestin 2 interaction assay and as low efficacy partial agonist in the other assays. Norbinaltorphimine acted as a highly potent and pure antagonist in all assays except kappa-G protein interaction, where it displayed efficacy as an inverse agonist. The pharmacological actions of novel kappa ligands, namely the dynorphin A tetrameric derivative PWT2-Dyn A and the palmitoylated derivative Dyn A-palmitic, were also investigated. PWT2-Dyn A and Dyn A-palmitic mimicked dynorphin A effects in all assays showing similar maximal effects but 3–10 fold lower potency. In conclusion, in the present study, multiple in vitro assays for the kappa receptor have been set up and pharmacologically validated. In addition, PWT2-Dyn A and Dyn A-palmitic were characterized as potent full agonists; these compounds are worthy of further investigation in vivo for those conditions in which the activation of the kappa opioid receptor elicits beneficial effects e.g. pain and pruritus. [ABSTRACT FROM AUTHOR]

Neurons containing neuropeptide S (NPS) and orexins are activated during stress. Previously, we reported that orexins released during stress, via orexin OX1 receptors (OX1 Rs), contribute to the reinstatement of cocaine seeking through endocannabinoid/CB1 receptor (CB1 R)-mediated dopaminergic disinhibition in the ventral tegmental area (VTA). Here, we further demonstrated that NPS released during stress is an up-stream activator of this orexin-endocannabinoid cascade in the VTA, leading to the reinstatement of cocaine seeking. Mice were trained to acquire cocaine conditioned place preference (CPP) by context-pairing cocaine injections followed by the extinction training with context-pairing saline injections. Interestingly, the extinguished cocaine CPP in mice was significantly reinstated by intracerebroventricular injection (i.c.v.) of NPS (1 nmol) in a manner prevented by intraperitoneal injection (i.p.) of SHA68 (50 mg/kg), an NPS receptor antagonist. This NPS-induced cocaine reinstatement was prevented by either i.p. or intra-VTA microinjection (i.vta.) of SB-334867 (15 mg/kg, i.p. or 15 nmol, i.vta.) and AM 251 (1.1 mg/kg, i.p. or 30 nmol, i.vta.), antagonists of OX1 Rs and CB1 Rs, respectively. Besides, NPS (1 nmol, i.c.v.) increased the number of c-Fos-containing orexin neurons in the lateral hypothalamus (LH) and increased orexin-A level in the VTA. The latter effect was blocked by SHA68. Furthermore, a 30-min restraint stress in mice reinstated extinguished cocaine CPP and was prevented by SHA68. These results suggest that NPS is released upon stress and subsequently activates LH orexin neurons to release orexins in the VTA. The released orexins then reinstate extinguished cocaine CPP via an OX1 R- and endocannabinoid-CB1 R-mediated signaling in the VTA. [ABSTRACT FROM AUTHOR]

The μ-opioid peptide (MOP) receptor is a member of the opioid receptor family and an important clinical target for analgesia. Measuring MOP receptor location and tracking its turnover traditionally used radiolabels or antibodies with attendant problems of utility of radiolabels in whole cells and poor antibody selectivity. To address these issues we have synthesized and characterised a novel ATTO488 based fluorescent Dermorphin analogue; [Cys(ATTO 488)8]Dermorphin-NH2 (DermATTO488). We initially assessed the binding profile of DermATTO488 in HEK cells expressing human MOP and CHO cells expressing human MOP, δ-opioid peptide (DOP), κ-opioid peptide (KOP) and Nociceptin/Orphanin FQ peptide (NOP) receptors using radioligand binding. Functional activity of the conjugated peptide was assessed by measuring (i) the ability of the ligand to engage G-protein by measuring the ability to stimulate GTPγ[35S] binding and (ii) the ability to stimulate phosphorylation of ERK1/2. Receptor location was visualised using confocal scanning laser microscopy. Dermorphin and DermATTO488 bound to HEKMOP (pKi: 8.29 and 7.00; p<0.05), CHOMOP (pKi: 9.26 and 8.12; p<0.05) and CHODOP (pKi: 7.03 and 7.16; p>0.05). Both ligands were inactive at KOP and NOP. Dermorphin and DermATTO488 stimulated the binding of GTPγ[35S] with similar pEC50 (7.84 and 7.62; p>0.05) and Emax (1.52 and 1.34fold p>0.05) values. Moreover, Dermorphin and DermATTO488 produced a monophasic stimulation of ERK1/2 phosphorylation peaking at 5mins (6.98 and 7.64-fold; p>0.05). Finally, in confocal microscopy DermATTO488 bound to recombinant MOP receptors on CHO and HEK cells in a concentration dependent manner that could be blocked by pre-incubation with unlabelled Dermorphin or Naloxone. Collectively, addition to ATTO488 to Dermorphin produced a ligand not dissimilar to Dermorphin; with ~10fold selectivity over DOP. This new ligand DermATTO488 retained functional activity and could be used to visualise MOP receptor location. [ABSTRACT FROM AUTHOR]

Rationale: Depression and anxiety frequently co-occur, and this has important clinical implications. Previous studies showed that activation of the nociceptin/orphanin FQ receptor (NOP) elicits anxiolytic effects, while its blockade promotes consistent antidepressant actions. NOP antagonists are effective in reversing footshock-induced depressive-like behaviors, but their effects on stress-induced anxiety are still unclear. Objective: This study aimed to investigate the effects of the NOP antagonist SB-612111 on footshock stress-induced anxiety behaviors. Methods: Male Swiss mice were exposed to inescapable electric footshock stress, and behavioral phenotype was screened based on the ability to escape from footshock (i.e., helpless or non-helpless). Animals were then treated with diazepam (1 mg/kg) and SB-612111 (0.1–10 mg/kg), and their behavior was assessed in the elevated plus-maze (EPM) and open field test. Results: When compared with non-stressed mice, helpless, but not non-helpless, animals displayed significant reductions in the time spent in and entries into open arms in the EPM. Diazepam significantly increased open arms exploration in helpless, non-helpless, and non-stressed mice. However, treatment with the NOP antagonist SB-612111 was inactive in naive mice, while it reversed anxiogenic-related behaviors in helpless mice and increased anxiety states in non-helpless mice. No effects on locomotion were observed. Conclusion: Helpless mice displayed increased anxiety compared to non-stressed and non-helpless animals, thus supporting use of this approach as an animal model to investigate anxiety/depression comorbidity. Additionally, SB-612111 modulated anxiety-like behaviors in male mice depending on individual stress susceptibility. Ultimately, NOP antagonists could be useful for treating anxiety in depressed patients. [ABSTRACT FROM AUTHOR]

Background: Stress-induced analgesia (SIA) is an evolutionarily conserved phenomenon during stress. Neuropeptide S (NPS), orexins, substance P, glutamate and endocannabinoids are known to be involved in stress and/or SIA, however their causal links remain unclear. Here, we reveal an unprecedented sequential cascade involving these mediators in the lateral hypothalamus (LH) and ventrolateral periaqueductal gray (vlPAG) using a restraint stress-induced SIA model. Methods: Male C57BL/6 mice of 8–12 week-old were subjected to intra-cerebroventricular (i.c.v.) and/or intra-vlPAG (i.pag.) microinjection of NPS, orexin-A or substance P alone or in combination with selective antagonists of NPS receptors (NPSRs), OX1 receptors (OX1Rs), NK1 receptors (NK1Rs), mGlu5 receptors (mGlu5Rs) and CB1 receptors (CB1Rs), respectively. Antinociceptive effects of these mediators were evaluated via the hot-plate test. SIA in mice was induced by a 30-min restraint stress. NPS levels in the LH and substance P levels in vlPAG homogenates were compared in restrained and unrestrained mice. Results: NPS (i.c.v., but not i.pag.) induced antinociception. This effect was prevented by i.c.v. blockade of NPSRs. Substance P (i.pag.) and orexin-A (i.pag.) also induced antinociception. Substance P (i.pag.)-induced antinociception was prevented by i.pag. Blockade of NK1Rs, mGlu5Rs or CB1Rs. Orexin-A (i.pag.)-induced antinociception has been shown previously to be prevented by i.pag. blockade of OX1Rs or CB1Rs, and here was prevented by NK1R or mGlu5R antagonist (i.pag.). NPS (i.c.v.)-induced antinociception was prevented by i.pag. blockade of OX1Rs, NK1Rs, mGlu5Rs or CB1Rs. SIA has been previously shown to be prevented by i.pag. blockade of OX1Rs or CB1Rs. Here, we found that SIA was also prevented by i.c.v. blockade of NPSRs or i.pag. blockade of NK1Rs or mGlu5Rs. Restrained mice had higher levels of NPS in the LH and substance P in the vlPAG than unrestrained mice. Conclusions: These results suggest that, during stress, NPS is released and activates LH orexin neurons via NPSRs, releasing orexins in the vlPAG. Orexins then activate OX1Rs on substance P-containing neurons in the vlPAG to release substance P that subsequently. Activates NK1Rs on glutamatergic neurons to release glutamate. Glutamate then activates perisynaptic mGlu5Rs to initiate the endocannabinoid retrograde inhibition of GABAergic transmission in the vlPAG, leading to analgesia. [ABSTRACT FROM AUTHOR]

Background: The peptide nociceptin/orphanin FQ (N/OFQ) and its receptor (NOP) are implicated in the modulation of emotional states. Previous human and rodent findings support NOP antagonists as antidepressants. However, the role played by the N/OFQ-NOP receptor system in resilience to stress is unclear.Aims: The present study investigated the effects of activation or blockade of NOP receptor signaling before exposure to acute stress.Methods: The behavioral effects of the administration before stress of the NOP agonists Ro 65-6570 (0.01-1 mg/kg) and MCOPPB (0.1-10 mg/kg), and the NOP antagonist SB-612111 (1-10 mg/kg) were assessed in mice exposed to inescapable electric footshock and forced swim as stressors. The behavioral phenotype of mice lacking the NOP receptor (NOP(-/-)) exposed to inescapable electric footshock was also investigated.Results: The activation of NOP receptor signaling with the agonists increased the percentage of mice developing helpless behavior and facilitated immobile posture. In contrast, the blockade of NOP receptor reduced the acquisition of depressive-like phenotypes, and similar resistance to develop helpless behaviors was observed in NOP(-/-) mice. Under the same stressful conditions, the antidepressant nortriptyline (20 mg/kg) did not change the acquisition of helpless behavior and immobile posture.Conclusions: These findings support the view that NOP activation during acute stress facilitates the development of depressive-related behaviors, whereas NOP blockade has a protective outcome. This study showed for first time that NOP antagonists are worthy of investigation as preemptive treatments in patients with severe risk factors for depression. [ABSTRACT FROM AUTHOR]

Neuropeptide S (NPS) is the endogenous ligand of the neuropeptide S receptor (NPSR). NPS modulates several biological functions including anxiety, wakefulness, pain, and drug abuse. The aim of this study was the investigation of the pharmacological profile of NPSR using the dynamic mass redistribution (DMR) assay. DMR is a label-free assay that offers a holistic view of cellular responses after receptor activation. HEK293 cells stably transfected with the murine NPSR (HEK293mNPSR) have been used. To investigate the nature of the NPS-evoked DMR signaling, FR900359 (Gq inhibitor), pertussis toxin (Gi inhibitor), and rolipram (phosphodiesterase inhibitor) were used. To determine the pharmacology of NPSR, several selective ligands (agonists, partial agonists, antagonists) have been tested. NPS, through selective NPSR activation, evoked a robust DMR signal with potency in the nanomolar range. This signal was predominantly, but not completely, blocked by FR900359, suggesting the involvement of the Gq-dependent signaling cascade. NPSR ligands (agonists and antagonists) displayed potency values in DMR experiments similar, but not identical, to those reported in the literature. Furthermore, partial agonists produced a higher efficacy in DMR than in calcium experiments. DMR can be successfully used to study the pharmacology and signaling properties of novel NPSR ligands. This innovative approach will likely increase the translational value of in vitro pharmacological studies. [ABSTRACT FROM AUTHOR]

Rationale: Nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand of a Gi protein-coupled receptor named NOP. Both N/OFQ and NOP receptor are widely expressed in brain areas involved in the control of emotional processes. Clinical and preclinical studies support antidepressant effects due to the blockade of NOP receptor signaling. By contrast, NOP receptor activation did not evoke any change in behavioral despair tests.Objectives: The present study aimed to investigate the effects of the co-administration of NOP agonists and classic antidepressant drugs in the forced swimming test (FST) and learned helplessness model (LH) in mice.Methods: Male Swiss mice were co-administered with NOP agonists (N/OFQ and Ro 65-6570) and antidepressants (nortriptyline, fluoxetine, and R-ketamine) or SB-612111 (NOP antagonist) and the behavioral effects were assessed in the FST and LH tests.Results: Fluoxetine, nortriptyline, R-ketamine and the NOP antagonist SB-612111 displayed antidepressant-like effects in the FST. The administration of the NOP agonists N/OFQ and Ro 65-6570 did not induce any behavioral change. However, co-administration of NOP agonists blocked the antidepressant effects of SB-612111, fluoxetine, and nortriptyline, but not R-ketamine in the FST. Similarly, in the LH, the systemic injection of SB-612111, nortriptyline, and R-ketamine reversed helplessness. The co-administration of Ro 65-6570 blocked the antidepressant-like effects of SB-612111 and nortriptyline, but not R-ketamine.Conclusions: NOP receptor activation inhibits the acute antidepressant effects of nortriptyline and fluoxetine, but not R-ketamine. The present findings contribute to further understand the role played by the N/OFQ-NOP receptor system in regulating mood states. [ABSTRACT FROM AUTHOR]

The Nociceptin/Orphanin FQ (N/OFQ) peptide NOP receptor is coupled to pertussis toxin (PTX)-sensitive G proteins (Gi/o) whose activation leads to the inhibition of both cAMP production and calcium channel activity, and to the stimulation of potassium currents. The label free dynamic mass redistribution (DMR) approach has been demonstrated useful for investigating the pharmacological profile of G protein-coupled receptors. Herein, we employ DMR technology to systematically characterize the pharmacology of a large panel of NOP receptor ligands. These are of peptide and non-peptide nature and display varying degrees of receptor efficacy, ranging from full agonism to pure antagonism. Using Chinese hamster ovary (CHO) cells expressing the human NOP receptor we provide rank orders of potency for full and partial agonists as well as apparent affinities for selective antagonists. We find the pharmacological profile of NOP receptor ligands to be similar but not identical to values reported in the literature using canonical assays for Gi/o-coupled receptors. Our data demonstrate that holistic label-free DMR detection can be successfully used to investigate the pharmacology of the NOP receptor and to characterize the cellular effects of novel NOP receptor ligands. [ABSTRACT FROM AUTHOR]

Background: Neuropeptide S (NPS) is an endogenous neuropeptide controlling anxiolysis, wakefulness, and analgesia. NPS containing neurons exist near to the locus coeruleus (LC) involved in the descending anti-nociceptive system. NPS interacts with central noradrenergic neurons; thus brain noradrenergic signaling may be involved in NPS-induced analgesia. We tested NPS analgesia in noradrenergic neuron-lesioned rats using a selective LC noradrenergic neurotoxin, N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4).Methods: A total 66 male Sprague–Dawley rats weighing 350–450 g were used. Analgesic effects of NPS were evaluated using hot-plate and tail-flick test with or without DSP-4. The animal allocated into 3 groups; hot-plate with NPS alone intracerebroventricular (icv) (0.0, 1.0, 3.3, and 10.0 nmol), tail-flick NPS alone icv (0.0 and 10.0 nmol), and hot-plate with NPS and DSP-4 (0 or 50 mg/kg ip). In hot-plate with NPS and DSP-4 group, noradrenaline content in the cerebral cortex, pons, hypothalamus, were measured.Results: NPS 10 nmol icv prolonged hot plate (%MPE) but not tail flick latency at 30 and 40 min after administration. DSP-4 50 mg/kg decreased noradrenaline content in the all 3 regions. The NA depletion inhibited NPS analgesic effect in the hot plate test but not tail flick test. There was a significant correlation between hot plate latency (percentage of maximum possible effect: %MPE) with NPS 10 nmol and NA content in the cerebral cortex (p = 0.017, r2 = 0.346) which noradrenergic innervation arisen mainly from the LC. No other regions had the correlation.Conclusions: NPS analgesia interacts with LC noradrenergic neuronal activity. [ABSTRACT FROM AUTHOR]

The aim of the study was to investigate the in vitro and in vivo pharmacological profile of cebranopadol, a novel agonist for opioid and nociceptin/orphanin FQ (N/OFQ) receptors (NOP). In vitro cebranopadol was assayed in calcium mobilization studies in cells coexpressing NOP or opioid receptors and chimeric G-proteins and in a bioluminescence resonance energy transfer (BRET) assay for studying receptor interaction with G-protein and b-arrestin 2. The mouse tail withdrawal and formalin tests were used for investigating cebranopadol antinociceptive properties. In calcium mobilization studies cebranopadol showed the following rank order of potency NOP = mu > kappa ≥ delta. In BRET studies, cebranopadol promoted NOP and mu receptors interaction with G-protein with similar high potency and efficacy. However, cebranopadol did not stimulated NOP–b-arrestin 2 interactions and displayed reduced potency at mu/b-arrestin 2. In vivo, cebranopadol exhibits highly potent and extremely long-lasting antinociceptive effects. The effects of cebranopadol in the tail withdrawal assay were sensitive to both SB-612111 and naloxone. Collectively the present results confirm and extend previous finding demonstrating that cebranopadol, by acting as mixed NOP/opioid receptor agonist, elicits robust analgesic effects in different pain models. [ABSTRACT FROM AUTHOR]

Rationale: Pharmacological and genetic evidence support antidepressant-like effects elicited by the blockade of the NOP receptor. The learned helplessness (LH) model employs uncontrollable and unpredictable electric footshocks as a stressor stimulus to induce a depressive-like phenotype that can be reversed by classical antidepressants. Objectives: The present study aimed to evaluate the action of NOP receptor antagonists in helpless mice. Methods: Male Swiss mice were subjected to the three steps of the LH paradigm (i.e., (1) induction, (2) screening, and (3) test). Only helpless animals were subjected to the test session. During the test session, animals were placed in the electrified chamber and the latency to escape after the footshock and the frequency of escape failures were recorded. The effect of the following treatments administered before the test session were evaluated: nortriptyline (30 mg/kg, ip, 60 min), fluoxetine (30 mg/kg, ip, four consecutive days of treatment), and NOP antagonists SB-612111 (1-10 mg/kg, ip, 30 min) and UFP-101 (1-10 nmol, icv, 5 min). To rule out possible biases, the effects of treatments on controllable stressful and non stressful situations were assessed. Results: In helpless mice, nortriptyline, fluoxetine, UFP-101 (3-10 nmol), and SB-612111 (3-10 mg/kg) significantly reduced escape latencies and escape failures. No effects of drug treatments were observed in mice subjected to the controllable electric footshocks and non stressful situations. Conclusions: Acute treatment with NOP antagonists reversed helplessness similarly to the classical antidepressants. These findings support the proposal that NOP receptor antagonists are worthy of development as innovative antidepressant drugs. [ABSTRACT FROM AUTHOR]

ABSTRACT The study reports the synthesis and biological evaluation of two opioid analogs, a monomer and a dimer, obtained as products of the solid-phase, side-chain to side-chain cyclization of the pentapeptide Tyr-d-Lys-Phe-Phe-AspNH2. The binding affinities to the mu, delta, and kappa opioid receptors, as well as results obtained in a calcium mobilization functional assay are reported. Tyr-[d-Lys-Phe-Phe-Asp]2-NH2 1 was a potent and selective full agonist of mu with sub-nanomolar affinity, while the dimer (Tyr-[d-Lys-Phe-Phe-Asp]2-NH2)2 2 showed a significant mixed mu/kappa affinity, acting as an agonist at the mu. Molecular docking computations were utilized to explain the ability of the dimeric cyclopeptide 2 to interact with the receptor. Interestingly, in spite of the increased ring size, the higher flexibility allowed 2 to fold and fit into the mu receptor binding pocket. Both cyclopeptides were shown to elicit strong antinociceptive activity after intraventricular injection but only cyclomonomer 1 was able to cross the blood-brain barrier. However, the cyclodimer 2 displayed a potent peripheral antinociceptive activity in a mouse model of visceral inflammatory pain. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 309-317, 2016. [ABSTRACT FROM AUTHOR]

Neuropeptide S (NPS) has generated substantial interest due to its anxiolytic and fear-attenuating effects in rodents, while a corresponding receptor polymorphism associated with increased NPS receptor (NPSR1) surface expression and efficacy has been implicated in an increased risk of panic disorder in humans. To gain insight into this paradox, we examined the NPS system in rats and mice bred for high anxiety-related behavior (HAB) versus low anxiety-related behavior, and, thereafter, determined the effect of central NPS administration on anxiety- and fear-related behavior. The HAB phenotype was accompanied by lower basal NPS receptor (Npsr1) expression, which we could confirm via in vitro dual luciferase promoter assays. Assessment of shorter Npsr1 promoter constructs containing a sequence mutation that introduces a glucocorticoid receptor transcription factor binding site, confirmed via oligonucleotide pull-down assays, revealed increased HAB promoter activity--an effect that was prevented by dexamethasone. Analogous to the human NPSR1 risk isoform, functional analysis of a synonymous single nucleotide polymorphism in the coding region of HAB rodents revealed that it caused a higher cAMP response to NPS stimulation. Assessment of the behavioral consequence of these differences revealed that intracerebroventricular NPS reversed the hyperanxiety of HAB rodents as well as the impaired cued-fear extinction in HAB rats and the enhanced fear expression in HAB mice, respectively. These results suggest that alterations in the NPS system, conserved across rodents and humans, contribute to innate anxiety and fear, and that HAB rodents are particularly suited to resolve the apparent discrepancy between the preclinical and clinical findings to date. [ABSTRACT FROM AUTHOR]

The peptide welding technology (PWT) is a novel chemical strategy that allows the synthesis of multibranched peptides with high yield, purity, and reproducibility. With this approach, a tetrabranched derivative of neuropeptide S (NPS) has been synthesized and pharmacologically characterized. The in vitro activity of PWT1-NPS has been studied in a calcium mobilization assay. In vivo, PWT1-NPS has been investigated in the locomotor activity (LA) and recovery of the righting reflex (RR) tests. In calcium mobilization studies, PWT1-NPS behaved as full agonist at the mouse NPS receptor (NPSR) being threefold more potent than NPS. The selective NPSR antagonists [ tBu-D-Gly5]NPS and SHA 68 displayed similar potency values against NPS and PWT1-NPS. In vivo, both NPS (1-100 pmol, i.c.v.) and PWT1-NPS (0.1-100 pmol, i.c.v.) stimulated mouse LA, with PWT1-NPS showing higher potency than NPS. In the RR assay, NPS (100 pmol, i.c.v.) was able to reduce the percentage of mice losing the RR after diazepam administration and their sleep time 5 min after the i.c.v. injection, but it was totally inactive 2 h after the injection. On the contrary, PWT1-NPS (30 pmol, i.c.v.), injected 2 h before diazepam, displayed wake-promoting effects. This PWT1-NPS stimulant effect was no longer evident in mice lacking the NPSR receptor. The PWT1 technology can be successfully applied to the NPS sequence. PWT1- NPS displayed in vitro a pharmacological profile similar to NPS. In vivo PWT1- NPS mimicked NPS effects showing higher potency and long-lasting action. [ABSTRACT FROM AUTHOR]

Studies have shown a close relationship between anxiety and aversive memory processing, but few animal models are suitable for investigating the effects of a given compound on anxiety and memory simultaneously. A growing body of evidence suggests anxiolytic and amnesic effects of nociceptin/orphanin FQ (N/OFQ). The mouse elevated T-maze (ETM) has been shown to detect the effects of drugs on anxiety and memory at the same time. In this study, the effects of intracerebroventricular N/OFQ injected before or immediately after training session were assessed in the ETM task. When pretraining injected, N/OFQ 0.1 nmol significantly decreased the latency to enter an open arm in the training session compared to control, which is suggestive of anxiolysis. In addition, N/OFQ (0.1 and 1 nmol) significantly reduced the latency to enter an open arm during the test session compared to control, thus suggesting memory impairments. However, when N/OFQ was administered posttraining, it did not affect memory retrieval. No alterations in locomotion were detected in N/OFQ-treated mice in the open field test. In conclusion, these findings are discussed considering the simultaneous anxiolytic and amnesic effects of N/OFQ. [ABSTRACT FROM AUTHOR]

Background and Purpose: Peptide welding technology (PWT) is a novel chemical strategy that allows the synthesis of multibranched peptides with high yield, purity and reproducibility. Using this technique, we have synthesized and pharmacologically characterized the tetrabranched derivatives of the tachykinins, substance P (SP), neurokinin A (NKA) and B (NKB).Experimental Approach: The following in vitro assays were used: calcium mobilization in cells expressing human recombinant NK receptors, BRET studies of G-protein - NK1 receptor interaction, guinea pig ileum and rat urinary bladder bioassays. Nociceptive behavioural response experiments were performed in mice following intrathecal injection of PWT2-SP.Key Results: In calcium mobilization studies, PWT tachykinin derivatives behaved as full agonists at NK receptors with a selectivity profile similar to that of the natural peptides. NK receptor antagonists display similar potency values when tested against PWT2 derivatives and natural peptides. In BRET and bioassay experiments PWT2-SP mimicked the effects of SP with similar potency, maximal effects and sensitivity to aprepitant. After intrathecal administration in mice, PWT2-SP mimicked the nociceptive effects of SP, but with higher potency and a longer-lasting action. Aprepitant counteracted the effects of PWT2-SP in vivo.Conclusions and Implications: The present study has shown that the PWT technology can be successfully applied to the peptide sequence of tachykinins to generate tetrabranched derivatives characterized with a pharmacological profile similar to the native peptides. In vivo, PWT2-SP displayed higher potency and a marked prolongation of action, compared with SP. [ABSTRACT FROM AUTHOR]

Background and Purpose Peptide welding technology ( PWT) is a novel chemical strategy that allows the synthesis of multibranched peptides with high yield, purity and reproducibility. Using this technique, we have synthesized and pharmacologically characterized the tetrabranched derivatives of the tachykinins, substance P ( SP), neurokinin A ( NKA) and B ( NKB). Experimental Approach The following in vitro assays were used: calcium mobilization in cells expressing human recombinant NK receptors, BRET studies of G-protein - NK1 receptor interaction, guinea pig ileum and rat urinary bladder bioassays. Nociceptive behavioural response experiments were performed in mice following intrathecal injection of PWT2- SP. Key Results In calcium mobilization studies, PWT tachykinin derivatives behaved as full agonists at NK receptors with a selectivity profile similar to that of the natural peptides. NK receptor antagonists display similar potency values when tested against PWT2 derivatives and natural peptides. In BRET and bioassay experiments PWT2- SP mimicked the effects of SP with similar potency, maximal effects and sensitivity to aprepitant. After intrathecal administration in mice, PWT2- SP mimicked the nociceptive effects of SP, but with higher potency and a longer-lasting action. Aprepitant counteracted the effects of PWT2- SP in vivo. Conclusions and Implications The present study has shown that the PWT technology can be successfully applied to the peptide sequence of tachykinins to generate tetrabranched derivatives characterized with a pharmacological profile similar to the native peptides. In vivo, PWT2- SP displayed higher potency and a marked prolongation of action, compared with SP. [ABSTRACT FROM AUTHOR]

Background: Opioid analgesics are the most effective drugs for the treatment of moderate to severe pain. However, they also produce several adverse effects that can complicate pain management. The μ opioid (MOP) receptor, a G protein-coupled receptor, is recognized as the opioid receptor type which primarily mediates the pharmacological actions of clinically used opioid agonists. The morphinan class of analgesics including morphine and oxycodone are of main importance as therapeutically valuable drugs. Though the natural alkaloid morphine contains a C-6-hydroxyl group and the semisynthetic derivative oxycodone has a 6-carbonyl function, chemical approaches have uncovered that functionalizing position 6 gives rise to a range of diverse activities. Hence, position 6 of N-methylmorphinans is one of the most manipulated sites, and is established to play a key role in ligand binding at the MOP receptor, efficacy, signaling, and analgesic potency. We have earlier reported on a chemically innovative modification in oxycodone resulting in novel morphinans with 6-acrylonitrile incorporated substructures. Results: This study describes in vitro and in vivo pharmacological activities and signaling of new morphinans substituted in position 6 with acrylonitrile and amido functions as potent agonists and antinociceptive agents interacting with MOP receptors. We show that the presence of a 6-cyano group in N-methylmorphinans has a strong influence on the binding to the opioid receptors and post-receptor signaling. One 6-cyano-N-methylmorphinan of the series was identified as the highest affinity and most selective MOP agonist, and very potent in stimulating G protein coupling and intracellular calcium release through the MOP receptor. In vivo, this MOP agonist showed to be greatly effective against thermal and chemical nociception in mice with marked increased antinociceptive potency than the lead molecule oxycodone. Conclusion: Development of such novel chemotypes by targeting position 6 provides valuable insights on ligand-receptor interaction and molecular mode of action, and may aid in identification of opioid therapeutics with enhanced analgesic properties and fewer undesirable effects. [ABSTRACT FROM AUTHOR]

Morphine and structurally related derivatives are highly effective analgesics, and the mainstay in the medical management of moderate to severe pain. Pharmacological actions of opioid analgesics are primarily mediated through agonism at the µ opioid peptide (MOP) receptor, a G protein-coupled receptor. Position 17 in morphine has been one of the most manipulated sites on the scaffold and intensive research has focused on replacements of the 17-methyl group with other substituents. Structural variations at the N-17 of the morphinan skeleton led to a diversity of molecules appraised as valuable and potential therapeutics and important research probes. Discovery of therapeutically useful morphine-like drugs has also targeted the C-6 hydroxyl group, with oxymorphone as one of the clinically relevant opioid analgesics, where a carbonyl instead of a hydroxyl group is present at position 6. Herein, we describe the effect of N-substituent variation in morphine and oxymorphone on in vitro and in vivo biological properties and the emerging structure-activity relationships. We show that the presence of a N-phenethyl group in position 17 is highly favorable in terms of improved affinity and selectivity at the MOP receptor, potent agonism and antinociceptive efficacy. The N-phenethyl derivatives of morphine and oxymorphone were very potent in stimulating G protein coupling and intracellular calcium release through the MOP receptor. In vivo, they were highly effective against acute thermal nociception in mice with marked increased antinociceptive potency compared to the lead molecules. It was also demonstrated that a carbonyl group at position 6 is preferable to a hydroxyl function in these N-phenethyl derivatives, enhancing MOP receptor affinity and agonist potency in vitro and in vivo. These results expand the understanding of the impact of different moieties at the morphinan nitrogen on ligand-receptor interaction, molecular mode of action and signaling, and may be instrumental to the development of new opioid therapeutics. [ABSTRACT FROM AUTHOR]

Amygdala plasticity is an important contributor to the emotionalaffective dimension of pain. Recently discovered neuropeptide S (NPS) has anxiolytic properties through actions in the amygdala. Behavioral data also suggest antinociceptive effects of centrally acting NPS, but site and mechanism of action remain to be determined. This is the first electrophysiological analysis of pain-related NPS effects in the brain. We combined whole cell patch-clamp recordings in brain slices and behavioral assays to test the hypothesis that NPS activates synaptic inhibition of amygdala output to suppress pain behavior in an arthritis pain model. Recordings of neurons in the laterocapsular division of the central nucleus (CeLC), which serves pain-related amygdala output functions, show that NPS inhibited the enhanced excitatory drive [monosynaptic excitatory postsynaptic currents (EPSCs)] from the basolateral amygdala (BLA) in the pain state. As shown by miniature EPSC analysis, the inhibitory effect of NPS did not involve direct postsynaptic action on CeLC neurons but rather a presynaptic, action potential-dependent network mechanism. Indeed, NPS increased external capsule (EC)-driven synaptic inhibition of CeLC neurons through PKA-dependent facilitatory postsynaptic action on a cluster of inhibitory intercalated (ITC) cells. NPS had no effect on BLA neurons. High-frequency stimulation (HFS) of excitatory EC inputs to ITC cells also inhibited synaptic activation of CeLC neurons, providing further evidence that ITC activation can control amygdala output. The cellular mechanisms by which ECdriven synaptic inhibition controls CeLC output remain to be determined. Administration of NPS into ITC, but not CeLC, also inhibited vocalizations and anxiety-like behavior in arthritic rats. A selective NPS receptor antagonist ([D-Cys(tBu)5]NPS) blocked electrophysiological and behavioral effects of NPS. Thus NPS is a novel tool to control amygdala output and pain-related affective behaviors through a direct action on inhibitory ITC cells. [ABSTRACT FROM AUTHOR]

Neuropeptide S (NPS) regulates various biological functions by selectively activating the NPS receptor (NPSR). Recently, epidemiological studies revealed an association between NPSR single nucleotide polymorphisms and susceptibility to panic disorders. Here we investigated the effects of NPS in mice subjected to the elevated T maze (ETM), an assay which has been proposed to model anxiety and panic. Diazepam [1 mg/kg, intraperitoneally (i.p.)] elicited clear anxiolytic effects reducing the latency to emerge from the closed to the open (CO) arm without modifying the latencies from the open to the closed (OC) arm. By contrast, chronic fluoxetine (10 mg/kg i.p., once a day for 21 days) selectively increased OC latency, suggesting a panicolytic-like effect. NPS given intracerebroventricularly at 0.001-1 nmol elicited both anxiolytic- and panicolytic-like effects. However, although the NPS anxiolytic dose-response curve displayed the classical sigmoidal shape, the dose-response curve of the putative panicolytic-like effect was bell shaped with peak effect at 0.01 nmol. The behaviour of wild-type [NPSR(+/+)] and receptor knock out [NPSR(−/−)] mice in the ETM task was superimposable. NPS at 0.01 nmol elicited anxiolytic- and panicolytic-like effects in NPSR(+/+) but not in NPSR(−/−) mice. In conclusion, this study demonstrated that NPS, via selective activation of the NPSR, promotes both anxiolytic- and panicolytic-like actions in the mouse ETM. [ABSTRACT FROM AUTHOR]

Members of the opioid receptor family of G-protein-coupled receptors (GPCRs) are found throughout the peripheral and central nervous system, where they have key roles in nociception and analgesia. Unlike the 'classical' opioid receptors, ?, ? and ? (?-OR, ?-OR and ?-OR), which were delineated by pharmacological criteria in the 1970s and 1980s, the nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP, also known as ORL-1) was discovered relatively recently by molecular cloning and characterization of an orphan GPCR. Although it shares high sequence similarity with classical opioid GPCR subtypes (?60%), NOP has a markedly distinct pharmacology, featuring activation by the endogenous peptide N/OFQ, and unique selectivity for exogenous ligands. Here we report the crystal structure of human NOP, solved in complex with the peptide mimetic antagonist compound-24 (C-24) (ref. 4), revealing atomic details of ligand-receptor recognition and selectivity. Compound-24 mimics the first four amino-terminal residues of the NOP-selective peptide antagonist UFP-101, a close derivative of N/OFQ, and provides important clues to the binding of these peptides. The X-ray structure also shows substantial conformational differences in the pocket regions between NOP and the classical opioid receptors ? (ref. 5) and ? (ref. 6), and these are probably due to a small number of residues that vary between these receptors. The NOP-compound-24 structure explains the divergent selectivity profile of NOP and provides a new structural template for the design of NOP ligands. [ABSTRACT FROM AUTHOR]

Nociceptin/orphanin FQ (N/OFQ) controls several biological functions via selective activation of the N/OFQ peptide receptor (NOP). [(pF)PheAibArgLys]N/OFQ-NH (UFP-112) is an NOP receptor ligand designed using a combination of several chemical modifications in the same peptide sequence that increase NOP receptor affinity/potency and/or reduce susceptibility to enzymatic degradation. In the present review article, we summarize data from the literature and present original findings on the in vitro and in vivo pharmacological features of UFP-112. Moreover, important biological actions and possible therapeutic indications of NOP receptor agonists are discussed based on the results obtained with UFP-112 and compared with other peptide and nonpeptide NOP receptor ligands. [ABSTRACT FROM AUTHOR]

Neuropeptide S (NPS) is the last neuropeptide identified via reverse pharmacology techniques. NPS selectively binds and activates a previous orphan GPCR, now named NPSR, producing intracellular calcium mobilization and increases in cAMP levels. Biological functions modulated by the NPS/NPSR system include anxiety, arousal, locomotion, food intake, memory, and drug addiction. The primary sequence of NPS (in humans SFRNGVGTGMKKTSFQRAKS) is highly conserved among vertebrates especially at the N-terminus. Ala- and D-scan studies demonstrated that this part of the molecule is crucial for biological activity. Focused structure-activity studies performed on Phe2, Arg3, and Asn4 confirmed this indication and revealed the chemical requirements of these positions for NPSR binding and activation. The sequence Gly5-Val6-Gly7 seems to be important for shaping the bioactive conformation of the peptide. Structure-activity studies on Gly5 enabled identification of the first generation of peptidergic NPSR pure antagonists including [ D-Cys(tBu)5]NPS and [ D-Val5]NPS whose antagonist properties were confirmed in vivo. Finally, the pharmacological features of substituted bicyclic piperazine molecules (e.g. SHA 68 (3-oxo-1,1-diphenyl-tetrahydro-oxazolo[3,4-a]pyrazine-7-carboxylic acid 4-fluoro-benzylamide) were recently published making available the first generation of nonpeptide NPSR antagonists. The use in future studies of NPSR antagonists will be of paramount importance for understanding which biological functions are controlled by the NPS/NPSR system and for defining the therapeutic potential of selective NPSR ligands. © 2009 Wiley Periodicals, Inc. Med Res Rev, 30, No. 5, 751-777, 2010 [ABSTRACT FROM AUTHOR]

Neuropeptide S (NPS) is the endogenous ligand of a G-protein-coupled receptor named as NPSR. Behavioral effects have been recently attributed to NPS, i.e. hyperlocomotion, anxiolysis, and wakefulness. However, little is known about the mechanisms by which NPS evokes such biological actions. The present study aimed to investigate the role played by the adenosine A2A and A1 receptors in hyperlocomotion induced by NPS. Spontaneous locomotion was assessed in an activity cage for 30 min in mice acutely treated with caffeine (a nonselective adenosine receptor antagonist), ZM241385 (a selective A2A receptor antagonist), or CPT (a selective A1 receptor antagonist) before NPS challenge (0.1 nmol, i.c.v.), which induce hyperlocomotion in mice. The pretreatment with caffeine (3 mg/kg, i.p.), in an inactive dose per se, prevented the increase in locomotion evoked by NPS. The co-administration of NPS (0.1 nmol, i.c.v.) and ZM241385 (0.1 pmol, i.c.v.) counteracted hyperlocomotion evoked by NPS. The co-administration of NPS and CPT (0.1 pmol, i.c.v.) slightly facilitated the increase in locomotion evoked by NPS alone. In summary, the pharmacological blockade of A2A receptors significantly attenuated the stimulatory effects of NPS. By contrast, the antagonism of A1 receptors facilitated NPS-induced hyperlocomotion in mice, but we cannot rule out a merely additive effect of two stimulatory systems in the brain. Altogether, this is the first evidence of a putative role played by A2A and A1 receptors in modulating hyperlocomotion induced by NPS. [ABSTRACT FROM AUTHOR]

Human urotensin-II (U-II) is an 11-amino-acid cyclic peptide that activates a Gq-coupled receptor named UT. Little is known about the desensitisation profile of this receptor as peptide binding is essentially irreversible. In the present study, we have examined the effects of U-II and carbachol on Ca2+ signalling in SJCRH30 rhabdomyosarcoma (receptor density, Bmax ~0.1 pmol/mg protein) and human embroynic kidney (HEK)hUT ( Bmax ~1.4 pmol/mg protein) cells expressing native and recombinant UT, respectively. In SJCRH30, HEKhUT and human peripheral blood mononuclear cells induced to express native UT by treatment with 2 μg/ml lipopolysaccharide (LPS), we have measured the effects of U-II treatment on UT mRNA. In SJCRH30 cells, primary stimulation with carbachol (250 μM) did not affect a secondary challenge with U-II (1 μM) and primary challenge with U-II did not affect a secondary challenge with carbachol. In contrast, in HEKhUT cells, primary stimulation with carbachol (250 μM) reduced a secondary challenge to U-II (1 μM) by 84% and primary challenge with U-II reduced a secondary challenge to carbachol by 76%. Pre-treatment of SJCRH30 cells with U-II reduced UT mRNA after 6 h and this returned to basal after 24 h. In recombinant HEKhUT cells, UT mRNA expression increased following a 6 h treatment with 1 μM U-II. U-II treatment of naïve un-stimulated peripheral blood mononuclear cells was without effect. However, when UT expression is up-regulated following 15 h of LPS treatment, a 6 h U-II challenge reduced UT mRNA by 66%. In summary, we report differences in the desensitisation profiles of native and recombinant U-II receptors. Design and interpretation of functional experiments are hampered by irreversibility of U-II binding. [ABSTRACT FROM AUTHOR]

In this study, the Gαqi5 protein was used to force the human nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor to signal through the Ca2+ pathway in CHO cells. [Ca2+]i levels were monitored using the fluorometer FlexStation II and the Ca2+ dye Fluo 4 AM. Concentration response curves were generated with a panel of full and partial agonists, while NOP antagonists were assessed in inhibition-response curves. The following rank order of potency of antagonists was measured: $${\text{SB}} - {\text{612111}} >{\text{J}} - 113397 = {\text{Trap}} - 101 \geqslant {\text{UFP}} - 101 >\left[ {{\text{Nphe}}^{\text{1}} } \right]{{\text{N}} \mathord{\left/ {\vphantom {{\text{N}} {{\text{OFQ}}}}} \right. \kern-\nulldelimiterspace} {{\text{OFQ}}}}\left( {1 - 13} \right){\text{NH}}_{\text{2}} >>$$ naloxone, which is superimposable to literature findings. The rank order of potency of full and partial agonists is also similar to that obtained in previous studies with the exception of a panel of ligands (UFP-112, Ro 64-6198, ZP120, UFP-113) whose potency was relatively low in the Gαqi5–NOP receptor calcium assay. Interestingly, these NOP ligands are characterized by slow kinetic of interaction with the NOP receptor, as demonstrated by bioassay experiments. These results demonstrated that the FlexStation II–Gαqi5–NOP receptor calcium assay represents an adequate and useful screening for NOP receptor ligands, particularly for antagonists. [ABSTRACT FROM AUTHOR]

Nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand for the N/OFQ peptide receptor (NOP). Binding studies have relied on [leucyl-3H]N/OFQ, but as this is an agonist G-protein coupling will affect affinity. In this paper, we describe a new [3H]labeled NOP antagonist, [Nphe1,4’-3H-Phe4,Arg14,Lys15]N/OFQ-NH2 ([3H]UFP-101). We have characterized [3H]UFP-101 at recombinant human NOP expressed in Chinese hamster ovary cells (CHOhNOP) and native rat NOP in cerebrocortex. Radioligand saturation and competition studies were performed on membranes, and [3H]UFP-101 (antagonist) was compared with [3H]N/OFQ (agonist). The effects of GTPγS (10 µM) and Na+ were investigated alone and in combination in competition experiments with both radioligands. In CHOhNOP, B max, and p K D, values were 561 and 580 fmol mg protein−1 and 9.97 and 10.19 for [3H]UFP-101 and [leucyl-3H]N/OFQ, respectively. In rat cerebrocortex B max and p K D, values were 65 and 88 fmol mg protein−1 and 10.12 and 10.34 for [3H]UFP-101 and [leucyl-3H]N/OFQ. The binding of both radioligands was displaced by a range of peptide and non-peptide NOP ligands at both isoforms with good correlation ( r 2 0.92 in Rat and 0.97 in CHOhNOP). Naloxone was inactive. The binding of both radioligands was Na+-dependent with [3H]UFP-101 being more sensitive (IC50 ~20 mM). Unlike the agonist [leucyl-3H]N/OFQ, the antagonist [3H]UFP-101 was unaffected by GTPγS. [3H]UFP-101 binds to human and rat NOP with high affinity and good agreement with standard [leucyl-3H]N/OFQ in competition experiments. In addition, the binding of [3H]UFP-101 is unaffected by GTPγS. This radioligand will be useful to further characterize NOP in a range of binding paradigms. [ABSTRACT FROM AUTHOR]

The aim of this study was investigate the effects of nociceptin/orphanin FQ (N/OFQ) and ([Nphe1,Arg14,Lys15]N/OFQ–NH2) (UFP-101), the endogenous N/OFQ peptide receptor (NOP) ligand and a selective NOP antagonist, respectively, in the inflammatory response after cecal ligation and puncture (CLP) model of sepsis in rats. Prospective, controlled experiment. Animal basic science laboratory. Male Wistar rats, weighing 300–350 g. Rats subjected to CLP were treated with N/OFQ (0.001, 0.01 or 0.1 mg/kg) or UFP-101 (0.03, 0.03 or 0.3 mg/kg) subcutaneously administered immediately after surgery. Twelve hours after surgery, blood was collected by cardiac puncture and bronchoalveolar (BAL) and peritoneal lavage were performed. In a separate set of experiments mortality was evaluated monitoring CLP rats for 10 days. Our findings showed that UFP-101 (0.03 mg/kg, sc, but not 0.003 mg/kg) modified parameters related to the systemic inflammatory response by effectively preventing cells migration, bacterial dissemination, and by modulating the release of pro-inflammatory cytokines and chemokines, and reducing animal mortality in a clinically relevant model of sepsis. By contrast, N/OFQ (0.1 mg/kg, sc) increased mortality in the CLP model. Our findings point to a functional relationship between the N/OFQ-NOP receptor system and inflammatory response in the CLP model of sepsis and suggest that NOP receptor antagonists are worthy of testing as innovative drugs for the treatment of sepsis. [ABSTRACT FROM AUTHOR]

Nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand for the N/OFQ peptide receptor (NOP). N/OFQ causes hypotension and vasodilation, and we aimed to determine the role of histamine in inflammatory microvascular responses to N/OFQ. Male Wistar rats (220-300 g, n = 72) were anesthetized with thiopental (30 mg/kg bolus, 40-90 mg·kg-1· h-1iv), and the mesentery was prepared for fluorescent intravital microscopy using fluorescein isothiocyanate-conjugated BSA (FITC-BSA, 0.25 ml/100 g iv) or 1 μm fluorescently labeled microspheres. N/OFQ (0.6-60 nmol/kg iv) caused hypotension (SAP, baseline: 154 ± 11 mmHg, 15 nmol/kg N/OFQ: 112 ± 10 mmHg, P = 0.009), vasodilation (venules: 23.9 ± 1.2 μm, 26.7 ± 1.2 μm, P = 0.006), macromolecular leak (interstitial gray level FITC-BSA: 103.7 ± 3.4, 123.5 ± 11.8, P = 0.009), and leukocyte adhesion (2.0 ± 0.9, 15.2 ± 0.9/100 μm, P = 0.036). Microsphere velocity also decreased (venules: 1,230 ± 370 μm/s, P = 0.037), but there were no significant changes in blood flow. Flow cytometry measured a concurrent increase in neutrophil expression of cd11b with N/OFQ vs. controls (Geo mean fluorescence: 4.19 ± 0.13 vs. 2.06 ± 0.38, P < 0.05). The NOP antagonist [Nphe1,Arg14,Lys15]N/OFQ-NH2 (UFP-101; 60 and 150 nmol/kg iv), H1 and H2 antagonists pyrilamine (mepyramine, 1 mg/kg iv) and ranitidine (1 mg/kg iv), and mast cell stabilizer cromolyn (1 mg·kg-1·m-1) also abolished vasodilation and macromolecular leak to N/OFQ in vivo (P < 0.05), but did not affect hypotension. Isolated mesenteric arteries (~200 μm, n = 25) preconstricted with U-46619 were also mounted on a pressure myograph (60 mmHg), and both intraluminally arid extraluminally administered N/OFQ (10-5 M) caused dilation, inhibited by pyrilamine in the extraluminal but not the intraluminal (control: -6.9 ± 3.8%; N/OFQ: 32.6 ± 8.4%; pyrilamine: 31.5 ± 6.8%, n = 18, P < 0.05) experiments. We conclude that, in vivo, mesenteric microvascular dilation and macromolecular leak occur via N/OFQ-NOP-mediated release of histamine from mast cells. Therefore, N/OFQ-NOP has an important role in microvascular inflammation, and this may be targeted during disease, particularly as we have proven that UFP-101 is an effective antagonist of microvascular responses in vivo. [ABSTRACT FROM AUTHOR]

Several peptidic urotensin-II (UT) receptor antagonists exert ‘paradoxical’ agonist activity in recombinant cell- and tissue-based bioassay systems, likely the result of differential urotensin-II receptor (UT receptor) signal transduction/coupling efficiency between assays. The present study has examined this phenomenon in mammalian arteries and recombinant UT-HEK (human embryonic kidney) cells.BacMam-mediated recombinant UT receptor upregulation in HEK cells augmented agonist activity for all four peptidic UT ligands studied. The nominal rank order of relative intrinsic efficacy was U-II>urantide ([Pen5-DTrp7-Orn8]hU-II4–11)>SB-710411 (Cpa-c[DCys-Pal-DTrp-Lys-Val-Cys]-Cpa-amide)≫GSK248451 (Cin-c[DCys-Pal-DTrp-Orn-Val-Cys]-His-amide) (the relative coupling efficiency of recombinant HEK cells was cat>human≫rat UT receptor).The present study further demonstrated that the use of high signal transduction/coupling efficiency isolated blood vessel assays (primate>cat arteries) is required in order to characterize UT receptor antagonism thoroughly. This cannot be attained simply by using the rat isolated aorta, an artery with low signal transduction/coupling efficiency in which low-efficacy agonists appear to function as antagonists.In contrast to the ‘low-efficacy agonists’ urantide and SB-710411, GSK248451 functioned as a potent UT receptor antagonist in all native isolated tissues studied (UT receptor selectivity was confirmed in the rat aorta). Further, GSK248451 exhibited an extremely low level of relative intrinsic activity in recombinant HEK cells (4–5-fold less than seen with urantide). Since GSK248451 (1 mg kg−1, i.v.) blocked the systemic pressor actions of exogenous U-II in the anaesthetized cat, it represents a suitable peptidic tool antagonist for delineating the role of U-II in the aetiology of mammalian cardiometabolic diseases.British Journal of Pharmacology (2006) 148, 173–190. doi:10.1038/sj.bjp.0706716; published online 20 March 2006 [ABSTRACT FROM AUTHOR]

Urotensin II (U-II) is the peptide ligand for the G-protein-coupled U-II receptor (UT). U-II has been dubbed “the most potent vasoconstrictor identified to date”. However, in vivo studies with this system are hampered by the paucity of available ligands. Here, we characterise Chinese hamster ovary (CHO) cells expressing the human UT receptor in the following assays; (1) [125I]U-II binding, (2) GTPγ[35S] binding, (3) cAMP formation, and (4) intracellular Ca2+. We assess activity of 9 U-II analogues using these paradigms and examine their ability to contract isolated rat aorta. CHOhUT cells bound [125I]U-II with a B max and K d of 1,110±70 fmol/mg protein and 742 pM, respectively. hU-II stimulated GTPγ[35S] binding (pEC50 8.38), optimal at low (0.1 μM) GDP concentrations. The hU-II GTPγ[35S] response was partially PTx sensitive and there was a potent (pEC50 9.23) low efficacy (∼20% inhibition) coupling to adenylyl cyclase. In CHOhUT cells hU-II stimulates calcium release from intracellular stores (pEC50 8.80) and calcium influx in a PTx-insensitive manner. In our structure-activity relationship study most ligands acted as full agonists. However, urantide behaved as a partial agonist (pEC50 7.67/pKB 7.55) in GTPγ[35S] binding, a full agonist (pEC50 8.11) for increases in intracellular Ca2+ and a competitive antagonist in the rat aorta bioassay (pKB 8.59). Collectively, these data show promiscuity at high expression and indicate the need for careful multi-assay evaluation of novel U-II analogues. Further modification of urantide, in order to eliminate residual agonist activity and to identify novel ligands for in vivo cardiovascular studies are clearly warranted. [ABSTRACT FROM AUTHOR]

Many studies point toward the nociceptin/orphanin FQ (N/OFQ) and the N/OFQ peptide receptor (NOP) as targets for the development of innovative drugs for treating affective disorders. It has been reported that the activation of NOP receptors produces anxiolytic-like effects in rodents in a large series of behavioral assays, i.e., elevated plus maze, light-dark aversion, operant conflict, fear-potentiated startle, pup ultrasonic vocalizations, and hole board tests. In contrast, the blockade of N/OFQ signaling obtained with NOP-selective antagonists promotes antidepressant-like effects in the forced swimming and tail suspension tests. In these assays, N/OFQ is inactive per se, but reverses the antidepressant-like effects of NOP antagonists. NOP receptor knockout mice show an antidepressant-like phenotype, and NOP antagonists are inactive in these animals. Thus, the activation of the NOP receptor seems to evoke anxiolytic-like effects while its blockade antidepressant-like effects. This appears to be a rather unique behavioral profile since the activation or the blockade of a given neuropeptide receptor produces, in most of the cases, both antidepressant- and anxiolytic-like effects. This particular behavioral profile, the possible mechanisms of action, and the therapeutic potential of NOP receptor ligands for the treatment of depression and anxiety disorders are discussed in this review article. [ABSTRACT FROM AUTHOR]

ABSTRACT Nociceptin/orphanin FQ modulates various biological functions at central and peripheral levels by selectively activating a G-protein coupled receptor named N/OFQ peptide (NOP) receptor. For extending our knowledge on the biological roles of the N/OFQ - NOP receptor system the identification of selective NOP ligands, especially antagonists, is mandatory. [Nphe1, Arg14, Lys15] N/OFQ-NH2 (UFP-101) is a novel NOP ligand that was designed by combining, in the same molecule, the [Nphe1] chemical modification which eliminates efficacy and the [Arg14, Lys15] substitution which increases ligand potency and duration of action in vivo. In the present article, we summarize the pharmacological features of UFP-101 as determined in a series of in vitro and in vivo assays. Moreover, some biological actions and possible therapeutic indications of NOP ligands are discussed on the basis of results obtained with UFP-101. Data obtained with this compound were compared with those generated using other NOP antagonists, especially J-113397 and [Nphe1]N/OFQ(1-13)-NH2, receptor or peptide knockout mice and other pharmacological tools useful for blocking N/OFQ - NOP receptor signaling. The analysis of the available data demonstrates that UFP-101 is a useful pharmacological tool for the investigation of the central and peripheral biological functions regulated by the N/OFQ - NOP receptor system and for defining the therapeutic potential of NOP receptor ligands [ABSTRACT FROM AUTHOR]

The vasoactive cyclic undecapeptide urotensin-II (U-II) has been identified as an endogenous ligand for the G-protein coupled receptor now referred to as the UT receptor. The U-II/UT receptor system might be relevant for cardiovascular functions. A structure-activity study of human U-II investigating 31 peptides in the rat aorta bioassay is reported. Ala- and D-scan investigations indicated that the sequence Phe6-Trp7-Lys8-Tyr9 is essential for biological activity and that Lys8 and Tyr9 are particularly important. These two residues were substituted with a series of coded and non-coded amino acids. These studies demonstrated that the positive charge of the primary aliphatic amine at position 8 and its relative spatial orientation is crucial for both receptor occupation and activation, while the only chemical requirement at position 9 is the presence of an aromatic moiety. Moreover, this study led to the identification of UT receptor partial agonists (compounds 23 and 24) which can be used as chemical templates for further investigations aimed at the identification of selective antagonists. Copyright © 2004 European Peptide Society and John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Abstract The peptide urotensin II (U-Il) is the cognate ligand of the 6-protein coupled receptor UT (formerly GPRI4). A role in the regulation of cardiovascular functions has been proposed for this novel peptide/ receptor system. In the present study, we evaluated the ability of U-Il to induce plasma extravasation in mice and attempted to characterize the receptor involved using the novel UT receptor ligand, [Orn8] U-Il. The Evans blue technique was used to quantify plasma extravasation. U-Il (0.01, 0.1, 1 and 10 nmol/kg) dose-dependently stimulated plasma extravasation in airways, gastrointestinal and urogenital tract tissues from mice, but not in the skin. In most tissues, the dose/response curves to U-Il were bell shaped with the maximal effect induced by I nmol/kg. [Orn8]U-II at 30 nmol/kg was per se either inactive or produced a non-significant increase in plasma extravasation; in the presence of 30 nmol/kg [Orn8]U-II, the effects of I nmol/kg U-Il were always reduced and, in some tissues, abolished. The present findings demonstrate that U-Il promotes plasma extravasation in various mouse vascular regions via activation of UT receptors. The mouse plasma extravasation assay will be a useful tool in future studies aimed at characterizing the pharmacological features of novel UT receptor ligands in vivo. [ABSTRACT FROM AUTHOR]

Nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand for the N/OFQ peptide receptor (NOP) and has been shown previously to produce bradycardia and hypotension in rodents. In this study we have measured the effects of intravenous N/OFQ, and the NOP antagonists [Nphe1]N/OFQ(1–13)-NH2 ([Nphe1]) and [Nphe1,Arg14,Lys15]N/OFQ-NH2 (UFP-101) on cardiovascular parameters and plasma catecholamine concentrations. Female Hartley guinea pigs were anesthetized with pentobarbital and ventilated artificially. MAP and HR were measured via a femoral arterial catheter and ECG, respectively. Plasma catecholamine concentrations were measured by HPLC. Animals received saline, N/OFQ (0.25, 1.25, 6.25 and 25 nmol cumulatively at 10-min intervals), [Nphe1] (600 nmol) and UFP-101 (60 nmol) i.v. in various combinations. After establishing a stable baseline, MAP and HR measurements and blood sampling were performed at the beginning and 3 min following each drug administration. N/OFQ significantly decreased MAP, HR and the plasma noradrenaline concentrations in a dose dependent manner (maximally by 29.1±1.8%, 13.8±0.8% and 46.6±7.8%, respectively) To the contrary, N/OFQ tended to increase plasma adrenaline concentration but did not affect plasma dopamine concentrations. There was a significant correlation between percent change in MAP (0.69, P<0.01) or HR (0.84, P<0.01) and that in plasma noradrenaline. [Nphe1], but not UFP-101, alone significantly decreased MAP. [Nphe1] partially antagonized N/OFQ-induced hypotension, bradycardia and the decrease in plasma concentration of noradrenaline. UFP-101 fully prevented the effects of N/OFQ in this model. In conclusion, the present study shows that intravenous N/OFQ, via NOP receptors, elicits hypotension and bradycardia also in the anaesthetized guinea pig and that the decrease in MAP and HR are positively correlated with the decrease in the plasma noradrenaline level. [ABSTRACT FROM AUTHOR]

1 The pharmacological profiles of presynaptic nociceptin/orphanin FQ (N/OFQ) peptide receptors (NOP) modulating 5-hydroxytryptamine (5-HT) and noradrenaline (NE) release in the rat neocortex were characterized in a preparation of superfused synaptosomes challenged with 10 mM KCl. 2 N/OFQ concentration-dependently inhibited K(+)-evoked [(3)H]-5-HT and [(3)H]-NE overflow with similar potency (pEC(50) approximately 7.9 and approximately 7.7, respectively) and efficacy (maximal inhibition approximately 40%). 3 N/OFQ (0.1 micro M) inhibition of [(3)H]-5-HT and [(3)H]-NE overflow was antagonized by selective NOP receptor antagonists of peptide ([Nphe(1)]N/OFQ(1-13)NH(2) and UFP-101; 10 and 1 microM, respectively) and non-peptide (J-113397 and JTC-801; both 0.1 microM) nature. Antagonists were routinely applied 3 min before N/OFQ. However, a 21 min pre-application time was necessary for J-113397 and JTC-801 to prevent N/OFQ inhibition of [(3)H]-NE overflow. 4 The NOP receptor ligand [Phe(1)psi(CH(2)-NH)Gly(2)]N/OFQ(1-13)NH(2) ([F/G]N/OFQ(1-13)NH(2); 3 microM) did not affect K(+)-evoked [(3)H]-NE but inhibited K(+)-evoked [(3)H]-5-HT overflow in a UFP-101 sensitive manner. [F/G]N/OFQ(1-13)NH(2) antagonized N/OFQ actions on both neurotransmitters. 5 The time-dependency of JTC-801 action was studied in CHO cells expressing human NOP receptors. N/OFQ inhibited forskolin-stimulated cAMP accumulation and JTC-801, tested at different concentrations (0.1-10 microM) and pre-incubation times (0, 40 and 90 min), antagonized this effect in a time-dependent manner. The Schild-type analysis excluded a competitive type of antagonism. 6 We conclude that presynaptic NO receptors inhibiting 5-HT and NE release in the rat neocortex have similar pharmacological profiles. Nevertheless, they can be differentiated pharmacologically on the basis of responsiveness to [F/G]N/OFQ(1-13)NH(2) and time-dependent sensitivity towards non-peptide antagonists. [ABSTRACT FROM AUTHOR]

1 This study reports on the pharmacological characterization of ZP120, a novel ligand of the nociceptin/orphanin FQ (N/OFQ) peptide receptor, NOP. ZP120 is a structure inducing probes modified NOP ligand: Zealand Pharma proprietary SIP technology was used to increase the enzymatic stability and half-life of peptide. 2 In vitro, ZP120 mimicked the inhibitory effects of N/OFQ in the electrically stimulated mouse vas deferens, showing however higher potency (pEC(50) 8.88 vs 7.74), lower maximal effects (E(max) 69+/-5% vs 91+/-2%), and slower onset of action. Like N/OFQ, the effects of ZP120 were not modified by 1 micro M naloxone, but they were antagonized by the NOP receptor selective antagonist J-113397 (pA(2) 7.80 vs ZP120, 7.81 vs N/OFQ). 3 In vivo, ZP120 mimicked the effects of N/OFQ, producing pronociceptive effects in the tail withdrawal assay and decreased locomotor activity after i.c.v., but not after i.v. administration in mice. ZP120 elicited similar maximal effects as N/OFQ, but it was about 10 fold more potent and its effects lasted longer. 4 In conclusion, the novel NOP receptor ligand ZP120 is a highly potent and selective partial agonist of the NOP receptor with prolonged effects in vivo. [ABSTRACT FROM AUTHOR]

1 Nocistatin (NST) antagonizes several actions of nociceptin orphanin FQ (N/OFQ), but acts on distinct receptors. As N/OFQ exerts anxiolytic-like actions in various tests, its behavioural actions in the elevated plus-maze (EPM) test were compared with those of bovine NST. 2 Five minutes after i.c.v. treatment, mice were placed on the EPM for 5 min and entries into and lime spent on open and closed arms were recorded alongside other parameters. 3 NST (0.1 -3 pmol) reduced percentages of entries into (control 39.6±3.1%, peak effect at 1 pmol NST 8.5±2.9%) and time spent on open arms (control 30.8±2.3%. NST 2.7 ± 1.5%), The C-terminal hexapeptide of NST (NST-C6; 0.01 -10 pmol) closely mimicked these actions of NST, with peak effects at 0.1 pmol. 4 N/OFQ (1-100 pmol) increased percentages of entries into (control 38.5±3.4%: peak effect at 10 pmol N/OFQ 67.9 ± 4.9%) and time spent on open arms (control 32.0±3.8%; N/OFQ 74.9± 5.8%). Closed arm entries, an index of locomotor activity, were unchanged by all peptides. 5 Effects of NST or NST-C6, but not N/OFQ, were still detectable 15 min after injection. Behaviour of animals co-injected with NST (1 pmol) or NST-C6 (0.1 pmol) plus N/OFQ (10 pmol) was indistinguishable from that of controls. 6 These results reveal potent anxiogenic-like actions of NST and NST-C6, and confirm the anxiolytic-like properties of N/OFQ. As NST and N/OFQ both derive from preproN/OF, anxiety may be modulated in opposing directions depending on how this precursor is processed. [ABSTRACT FROM AUTHOR]