The opioid receptor family comprises classical; MOP (mu/µ), KOP (kappa/k), DOP(delta/δ) receptors along with non-classical Nociceptin/Orphanin FQ (N/OFQ) peptide receptor (NOP). Expression on glial cells is controversial where there is a role for glia and opioids in pain processing and immunomodulation. Here we detail expression and function of opioid receptors in a wide range of established and primary glia. Namely, 1321N1 human astrocytoma, C6 rat astrocytoma, mouse primary astrocytes, human MO3.13 oligodendrocyte like cells, human HOG oligodendrocytoma, mouse EOC-20 microglia and mouse primary microglia. We used (i)-PCR for mRNA, (ii)-radioligand binding and (iii)-fluorescent probe binding for expression, (iv)-MAPK for activation and (v)-scratch assay for migration. MOP mRNA was detected in C6 and mouse primary astrocytes only. NOP mRNA was found in 1321N1, C6 and mouse primary astrocytes along with MO3.13 and HOG cells. There was variable expression of DOP and KOP; this was not probed further. Surprisingly, microglia (EOC-20 or primary mouse) did not express mRNA for opioid receptors unless treated with media from astrocytes. mRNA expression profile was generally matched by [3H]-DPN binding to classical and [3H]-N/OFQ binding to non-classical opioid receptors. In addition, C6, mouse primary and 1321N1 astrocytes along with MO3.13 and HOG cells bound the fluorescent NOP probe N/OFQATTO594. C6 and primary mouse astrocytes also bound the MOP fluorescent probe DermorphinATTO488. Where expressed both MOP and NOP supported Endomorphin-1 and N/OFQ-induced phosphorylation of ERK1/2 and reduced scratch migration. In microglia, astrocyte modulated opioid receptor expression could influence central opioid-immunomodulation and pain processing; further studies are required.

Background and PurposeIn 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 ApproachB- and T-cell NOP expression was measured using the NOP fluorescent probe N/OFQ(ATTO594), N/OFQ content was measured using immunofluorescence, N/OFQ release was tracked using a CHOhNOPG alpha iq5 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 ResultsCD19-positive B-cells bound N/OFQ(ATTO594); 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/OFQ(ATTO594); 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/OFQ(ATTO594) 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 ImplicationsWe 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.

Nociceptin/Orphanin FQ (N/OFQ) is the endogenous opioid agonist for the N/OFQ receptor or NOP. This receptor system is involved in pain processing but also has a role in immune regulation. Indeed, polymorphonuclear cells (PMNs) express mRNA for N/OFQ precursor and are a potential source for circulating N/OFQ. Current measurements are based on ELISA and RIA techniques. In this study we have designed a bioassay to measure N/OFQ release from single PMNs. Chinese Hamster Ovary (CHO) cells transfected with the human (h) NOP receptor and Gαiq5 chimera force receptor coupling in biosensor cells to increase intracellular Ca2+; this can be measured with FLUO-4 dye. If isolated PMNs from healthy human volunteers are layered next to CHOhNOPGαiq5 biosensor cells then stimulated with the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMLP) we hypothesise that released N/OFQ will activate the biosensor. PMNs also release ATP and CHO cells express purinergic receptors coupled to elevated Ca2+. In a system where these receptors (P2Y1, P2Y2 and P2X7) are blocked with high concentrations of PPADS and oATP, PMN stimulation with fMLP increases Ca2+ in PMNs then shortly afterwards the biosensor cells. Our data therfore reports detection of single cell N/OFQ release from immune cells. This was absent when cells were preincubated with the selective NOP antagonist; SB-612111. Collectively this is the first description of single cell N/OFQ release. We will deploy this assay with further purified individual cell types and use this to further study the role of the N/OFQ-NOP system in disease; in particular sepsis where there is strong evidence for increased levels of N/OFQ worsening outcome.

There is a controversial narrative in the anaesthetic literature suggesting that anaesthetic technique (including opioids) may be detrimental to survival after tumour resection. The initial observations were retrospective. Several prospective studies are ongoing; one in breast cancer has reported no adverse outcome. The evidence for an effect of opioids stems from three pieces of information: (1) opioids depress the immune system, (2) opioids potentially promote angiogenesis, and (3) opioids potentially support tumour growth. Although the evidence for (2)/(3) is unclear, combinations of these effects are beneficial to tumours and potentially promote metastatic reseeding. Accepted wisdom suggests that opioid effects are driven by opioid receptor activation but the presence of opioid receptors on immune cells for example is unlikely. Immune cells, vascular endothelium and a range of tumour cells express Toll-like receptor 4 (TLR4) receptors (for Gram-negative bacterial wall components), and there is growing evidence for opioids interacting with this alternative receptor; and for some there is paradoxical naloxone sensitivity. Is the focus on opioid receptors and cancer the wrong target? TLR4 receptor activation produces immune activation, stimulates angiogenesis, and supports tumour survival. We know that some opioids are more immune suppressive than others (there is no such comparative information for angiogenesis and tumour survival); this may correlate with TLR4 activation. If there are clusters of opioids that have more opioid than TLR4 profiles and vice versa, this may influence outcome. If this is the case, then evidence-based advice could be given for perioperative use in the oncology–anaesthesia setting.

Opioids targeting mu;μ (MOP) receptors produce analgesia in the peri-operative period and palliative care. They also produce side effects including respiratory depression, tolerance/dependence and addiction. The N/OFQ opioid receptor (NOP) also produces analgesia but is devoid of the major MOP side effects. Evidence exists for MOP-NOP interaction and mixed MOP-NOP ligands produce analgesia with reduced side effects. We have generated a HEK MOP/NOP human expression system and used bivalent MOP-NOP and fluorescent ligands to (i) probe for receptor interaction and (ii) consequences of that interaction. We used HEK MOP/NOP cells and two bivalent ligands; Dermorphin-N/OFQ (MOP agonist-NOP agonist; DeNO) and Dermorphin-UFP101 (MOP agonist-NOP antagonist; De101). We have determined receptor binding profiles, GTPγ[ 35 S] binding, cAMP formation and ERK1/2 activation. We have also probed MOP and NOP receptor interactions in HEK cells and hippocampal neurones using the novel MOP fluorescent ligand, Dermorphin ATTO488 and the NOP fluorescent ligand N/OFQ ATTO594 . In HEK MOP/NOP MOP ligands displaced NOP binding and NOP ligands displaced MOP binding. Using fluorescent probes in HEK MOP/NOP cells we demonstrated MOP-NOP probe overlap and a FRET signal indicating co-localisation. MOP-NOP were also co-localised in hippocampal tissue. In GTPγ[ 35 S] and cAMP assays NOP stimulation shifted the response to MOP rightwards. At ERK1/2 the response to bivalent ligands generally peaked later. We provide evidence for MOP-NOP interaction in recombinant and native tissue. NOP activation reduces responsiveness of MOP activation; this was shown with conventional and bivalent ligands.

Opioid receptors are divided into the three classical types: MOP(μ:mu), DOP(δ:delta) and KOP(κ:kappa) that are naloxone-sensitive and an additional naloxone-insensitive nociceptin/orphanin FQ(N/OFQ) peptide receptor(NOP). Studies to determine opioid receptor location and turnover variably rely on; (i) measuring receptor mRNA, (ii) genetically tagging receptors, (iii) labelling receptors with radioligands, (iv) use of antibodies in immunohistochemistry/Western Blotting or (v) measuring receptor function coupled with the use of selective antagonists. All have their drawbacks with significant issues relating to mRNA not necessarily predicting protein, poor antibody selectivity and utility of radiolabels in low expression systems. In this minireview we discuss use of fluorescently labelled opioid receptor ligands. To maintain the pharmacological properties of the corresponding parent ligand fluorescently labelled ligands must take into account fluorophore (brightness and propensity to bleach), linker length and chemistry, and site to which the linker (and hence probe) will be attached. Use of donor and acceptor fluorophores with spectral overlap facilitates use in FRET type assays to determine proximity of ligand or tagged receptor pairs. There is a wide range of probes of agonist and antagonist nature for all four opioid receptor types; caution is needed with agonist probes due to the possibility for internalization. We have produced two novel ATTO based probes; DermorphinATTO488 (MOP) and N/OFQATTO594 (NOP). These probes label MOP and NOP in a range of preparations and using N/OFQATTO594 we demonstrate internalization and ligand-receptor interaction by FRET. Fluorescent opioid probes offer potential methodological advantages over more traditional use of antibodies and radiolabels.

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.

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.

Sepsis results from dysregulation of the immune response to infection that can lead to life-threatening organ dysfunction. Impaired cardiovascular function and hypotension are critical features of sepsis, and estimated mortality from sepsis is 44 000 deaths per year in the UK. Previous work by our laboratory has identified increased immune release of the non-classical opioid peptide nociceptin/orphanin FQ (N/OFQ) in sepsis.1 Moreover, this peptide is capable of producing hypotension in laboratory animals.2 The release of N/OFQ from immune cells may interact with its receptor (NOP) on the vasculature to cause or exacerbate hypotension. To study the role of NOP-N/OFQ in sepsis, we have used freshly isolated human umbilical vascular endothelial cells (HUVECs) challenged with lipopolysaccharide (LPS) and peptidoglycan (PepG). We determined NOP receptor expression and function before and after challenge. Freshly isolated HUVECs were cultured in the presence or absence of 100 ng ml–1 LPS and PepG. After incubation for 24 or 48 h, NOP mRNA and NOP functional activity was measured using quantitative polymerase chain reaction (QPCR),1 confocal microscopy, and western blot.3 mRNA encoding for human NOP was present in all conditions (both native tissue and LPS/PepG-treated samples). Twenty-four hour treatment with LPS/PepG led to a reduction in NOP mRNA expression in five out of six samples, with 48 h LPS/PepG treatment producing a decrease in all samples. The mean fold-decrease at 48 h was 4 (n=6). In confocal microscopy experiments using the NOP-selective fluorescent ligand N/OFQATTO594, freshly isolated HUVECs did not express NOP receptor protein on the cell surface. Treatment with LPS/PepG lead to an increase in NOP receptor expression (n=24). In the absence of LPS/PepG, incubation of HUVECs with N/OFQ (100 nM) failed to activate extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) in western blot time course assays (time scale of 1, 2.5, 5, 10, 15, and 30 min intervals). In LPS/PepG-treated (24 h) cells, N/OFQ (100 nM) activated ERK1/2 peaking 15 min after addition (n=9). In conclusion, the addition of the inflammatory agents LPS and PepG leads to significant changes in NOP-N/OFQ expression and function. This increase might explain, in part, the sepsis induced vasodilation and hypotension. British Heart Foundation PG/18/54/33896.

Opioid receptors are classified as classical or naloxone sensitive MOP (mu), DOP (delta) and KOP (kappa) and the non-classical or naloxone insensitive nociceptin/orphanin FQ (N/OFQ) receptor (NOP).1 Designing and evaluating potential novel opioid analgesics for translation into the clinic requires sensitive and selective receptor tool kits that can be used to identify, characterise, and track receptor populations. Typically, receptor expression and tracking requires use of radiolabels or antibodies and opioid receptor antibodies display very poor selectivity. In this study we have conjugated a fluorescent probe, ATTO488 (green) to the opioid peptide Dermorphin and assessed its binding affinity and ability to visualise MOP opioid receptors. Dermorphin displays moderate MOP selectivity, 34-fold over DOP.2 Experiments were performed in cultured human embryonic kidney (HEK) cells expressing recombinant MOP receptors (HEKhMOP). Binding affinity (expressed as Ki) was determined using displacement of the non-selective opioid radiolabel [3H]-diprenorphine ([3H]-DPN).2 Visualisation of binding was determined using confocal scanning laser microscopy.3 Dermorphin and dermorphinATTO488 displaced the binding of [3H]-DPN to HEKhMOP in a concentration dependent manner with a pKi+SEM (Ki) of 7.86+0.24 (14nM) and 6.92+0.14 (120nM, n=5) respectively. Adding ATTO488 reduced binding affinity 9-fold (p<0.05). We are currently using confocal experiments with 100nM and 1μM dermorphinATTO488 to visualise cell surface receptor binding. These data suggest that dermorphinATTO488 might be a useful fluorescent probe to study MOP receptors but the reduction in binding affinity may affect its selectivity to MOP. Further studies to determine opioid receptor selectivity profile of dermorphinATTO488 and to compare with dermorphin are underway. BJA/RCoA.

Classical opioids (μ: mu, MOP; δ: delta, DOP and κ: kappa, KOP) variably affect immune function; they are immune depressants and there is good clinical evidence in the periphery. In addition, there is evidence for a central role in the control of a number of neuropathologies, e.g., neuropathic pain. Nociceptin/Orphanin FQ (N/OFQ) is the endogenous ligand for the N/OFQ peptide receptor, NOP; peripheral and central activation can modulate immune function. In the periphery, NOP activation generally depresses immune function, but unlike classical opioids this is in part driven by NOP located on circulating immune cells. Peripheral activation has important implications in pathologies like asthma and sepsis. NOP is expressed on central neurones and glia where activation can modulate glial function. Microglia, as resident central ‘macrophages’, increase/infiltrate in pain and following trauma; these changes can be reduced by N/OFQ. Moreover, the interaction with other glial cell types such as the ubiquitous astrocytes and their known cross talk with microglia open a wealth of possibilities for central immunomodulation. At the whole animal level, clinical ligands with wide central and peripheral distribution have the potential to modulate immune function, and defining the precise nature of that interaction is important in mitigating or even harnessing the adverse effect profile of these important drugs.

Background and Purpose Experimental Approach The nociceptin/orphanin FQ (N/OFQ) receptor (NOP) is a member of the opioid receptor family and is involved in a number of physiological responses, pain and immune regulation as examples. In this study, we conjugated a red fluorophore-ATTO594 to the peptide ligand N/OFQ (N/OFQ(ATTO594)) for the NOP receptor and explored NOP receptor function at high (in recombinant systems) and low (on immune cells) expression. We assessed N/OFQ(ATTO594) receptor binding, selectivity and functional activity in recombinant (CHO) cell lines. Live cell N/OFQ(ATTO594) binding was measured in (i) HEK cells expressing NOP and NOPGFP receptors, (ii) CHO cells expressing the hNOPG alpha qi5 chimera (to force coupling to measurable Ca2+ responses) and (iii) freshly isolated human polymorphonuclear cells (PMN). Key Results Conclusions and Implications N/OFQ(ATTO594) bound to NOP receptor with nM affinity and high selectivity. N/OFQ(ATTO594) activated NOP receptor by reducing cAMP formation and increasing Ca2+ levels in CHOhNOPG alpha qi5 cells. N/OFQ(ATTO594) was also able to visualize NOP receptors at low expression levels on PMN cells. In NOP-GFP-tagged receptors, N/OFQ(ATTO594) was used in a FRET protocol where GFP emission activated ATTO, visualizing ligand-receptor interaction. When the NOPGFP receptor is activated by N/OFQ(ATTO594), movement of ligand and receptor from the cell surface to the cytosol can be measured. In the absence of validated NOP receptor antibodies and issues surrounding the use of radiolabels (especially in low expression systems), these data indicate the utility of N/OFQ(ATTO594) to study a wide range of N/OFQ-driven cellular responses.

The opioid receptor family is comprised of four members, the naloxone-sensitive Mu (μ), Delta (δ) and Kappa (κ) receptors, as well as the naloxone-insensitive NOP receptor. Activation of the NOP receptor has been shown to influence, amongst others, pain pathways, anxiety and depression. In the periphery, NOP activity has been implicated in immune cell function, heart failure and sepsis.1 While it is possible to measure NOP receptor protein levels in certain tissue, the sparsity of available protein in peripheral cells often means NOP expression is implied through functional studies. In this current study, we demonstrate the capability of a red fluorescent peptide based on the endogenous ligand for NOP (Nociceptin/Orphanin FQ; N/OFQ), N/OFQATTO to identify NOP receptors in various expression systems. Confocal microscopy and radioligand binding ([3H]-N/OFQ) was used to characterise binding of N/OFQATTO in Human embryonic kidney (HEK) cells expressing the NOP receptor or green fluorescent tagged (GFP) NOP receptors.2 For assessment of N/OFQATTO binding in low expression systems, human polymorphonuclear cells were extracted from whole blood using density gradient centrifugation and binding of N/OFQATTO measured using confocal microscopy. All data are mean±SEM of at least 5 experiments. In [3H]-N/OFQ binding assays N/OFQATTO bound with high affinity (pKi: 8.98±0.09) and selectivity for the NOP receptor, behaving in a similar manner to unconjugated N/OFQ (9.03±0.05). Using confocal microscopy, it is possible to measure increases in fluorescence (binding) in a concentration-dependent manner, allowing the affinity of N/OFQATTO to be determined (pKd: 8.53±0.34). N/OFQATTO binds to the surface of polymorphonuclear cells, an event that can be blocked by the high affinity NOP antagonist SB 612,111 or unlabelled N/OFQ, indicating selective binding to NOP receptors on the cell surface. In high expression systems, tracking of N/OFQATTO allows for determination of NOP receptor internalisation post-activation. Furthermore, coupling of N/OFQATTO to NOP receptors tagged with green fluorescent protein allows for visualisation of both ligand and receptor interaction. Conjugation of the N/OFQ peptide with the red fluorescent ATTO dye has not altered the functional properties of this peptide. The detectable range of N/OFQATTO in confocal microscopy is such that concentration-dependent binding; binding affinities can be measured. The sensitivity of N/OFQATTO in low expression system experiments is such that binding of this ligand can be seen on polymorphonuclear cells, demonstrating the presence of NOP receptor protein on these cells for the first time. N/OFQATTO is a versatile and robust ligand that will act to further understand localisation and function of the NOP receptor. Funded by Biotechnology and Biological Sciences Research Council (BB/N000188/1). 1.Lambert DG. Nature Reviews Drug Discovery. 2008; 7: 694–710.2.Bird MF, Cerlesi MC, Brown M, et al. Plos One. 2016; 11: e0156897.

The nociceptin opioid peptide (NOP) receptor and its endogenous ligand, nociceptin/orphanin FQ (N/OFQ), are members of the opioid receptor family. The NOP–N/OFQ system is most commonly associated with pain and analgesia; however, there is mounting evidence to support the involvement of this receptor–ligand complex in immune cell function.1 For instance, activation of T cells pre-incubated with staphylococcal enterotoxin B (SEB) is modulated by N/OFQ.2 In this current study, we evaluate the live-cell release of N/OFQ from both B and T cells, taken from healthy volunteers, through a novel bioassay and determine the NOP receptor expression using a novel probe on these cells. B and T cells were obtained from healthy volunteers using Miltenyi MACSExpress (UK) separation kits. Confocal microscopy was used to measure live-cell N/OFQ release and N/OFQATTO binding to immune cells to determine the NOP receptor expression. In live-cell-release assays, B and T cells labelled with identifier antibodies (CD19 B cells and CD3 T cells) were injected onto a coverslip containing a CHOhNOPαi/q feeder layer loaded with Fluo-4 AM calcium dye (Sigma, UK); these cells were then forced to respond to N/OFQ via an increase in Ca2+ that can be measured in the confocal microscope.3 After stimulation of the B/T cells with FMLP (N-formylmethionine-leucyl-phenylalanine), increases in calcium release were measured as a surrogate for the release of N/OFQ. The NOP antagonist SB 612,111 was used to determine the specificity of the NOP activity. For detection of the NOP receptor on immune cells, N/OFQATTO, the endogenous peptide conjugated to a fluorescent fluorophore, was added to B or T cells, and binding was measured through increases in red light wavelengths. The NOP antagonist, SB612,111, was used to determine the binding specificity of N/OFQATTO. All experiments were repeated five times. In live-cell-assay results, the addition of FMLP led to the activation of B and T cells, and, in turn, the activation of CHOhNOPαi/q cells; N/OFQ was being released. The activation produced by both B and T cells was inhibited in the presence of the NOP antagonist, SB612,111. In assays to determine NOP receptor expression, the fluorescent peptide N/OFQATTO bound to the surface of B cells. This binding was inhibited in the presence of the NOP receptor antagonist, SB612,111. Conversely, no binding of N/OFQATTO was detected on the surface of T cells. In this study, we demonstrated that both B and T lymphocytes release N/OFQ when stimulated by FMLP. Furthermore, B cells exhibit NOP receptor expression on the cell surface. Interestingly, T cells taken from healthy volunteers do not express NOP receptors. Further study into the environments leading to NOP receptor expression in these T cells will expand our understanding of the role the NOP-N/OFQ system plays in health and inflammatory disease. Biotechnology and Biological Sciences Research Council (BB/N000188/1).

Deorphanization of ORL-1/LC132 in 1995 by reverse pharmacology in two simultaneously published landmark studies added a new member to the opioid family of G-protein coupled receptors. Meunier and Reinscheid used cells expressing recombinant ORL-1 (human) or LC132 (rat) and the presumed intracellular inhibition of cyclic AMP formation to ‘fish’ for endogenous peptide ligands in rat whole-brain and pig hypothalamic extracts. Both studies reported the isolation of a 17-amino-acid peptide, which was named nociceptin and orphanin FQ by the two authors, respectively. The behaviour of the isolated peptide was a complete surprise, as a general hyperalgesia was observed when the peptide was administered at supraspinal sites. We now know that this peptide has, in fact, anti-opioid action, particularly in the medulla. The endogenous peptide exerts a multitude of effects both in the nervous system and, unlike classical opioids, has efficacy in neuropathic pain.

Nociceptin/orphanin FQ (N/ OFQ ) regulates several biological functions via selective activation of the N/ OFQ receptor ( NOP ), a member of the opioid receptor family. We recently identified a new high affinity and highly selective NOP agonist AT ‐403. In this study, we characterized the functional profile of AT ‐403 and compared it to other known nonpeptide NOP agonists Ro 65‐6570, Ro 2q, SCH ‐221510, MCOPPB , AT ‐202 and SCH ‐486757, using the following assays: GTP γ [ 35 S] stimulated binding, calcium mobilization assay in cells‐expressing human NOP or classical opioid receptors and chimeric G proteins, bioluminescence resonance energy transfer (BRET) based assay for studying NOP receptor interaction with G protein and arrestin, and the electrically stimulated mouse vas deferens bioassay. All compounds behaved as NOP full agonists consistently showing the following rank order of potency MCOPPB > AT ‐403 > Ro 65‐6570 = Ro 2q > SCH ‐221510 > AT ‐202 > SCH ‐486757. AT ‐403 and MCOPPB displayed the highest NOP selectivity both at human and murine receptors. Interestingly, while all the other nonpeptide NOP agonists displayed bias toward G protein‐mediated signaling in the BRET assay, AT ‐403, similar to the natural ligand N/ OFQ , behaved as an unbiased agonist, activating G‐protein‐mediated function as well as arrestin recruitment. AT ‐403 may be a useful nonpeptide tool compound to study the pharmacology of NOP activation in disease states.

An innovative chemical strategy named peptide welding technology (PWT) has been developed for the facile synthesis of tetrabranched peptides. [Dmt1]N/OFQ(1–13)-NH2 acts as a universal agonist for nociceptin/orphanin FQ (N/OFQ) and classical opioid receptors. The present study investigated the pharmacological profile of the PWT derivative of [Dmt1]N/OFQ(1–13)NH2 (PWT2-[Dmt1]) in several assays in vitro and in vivo after spinal administration in monkeys subjected to the tail withdrawal assay. PWT2-[Dmt1] mimicked the effects of [Dmt1]N/OFQ(1–13)-NH2 displaying full agonist activity, similar affinity/potency and selectivity at human recombinant N/OFQ (NOP) and opioid receptors in receptor binding, stimulation of [35S]GTPγS binding, calcium mobilization in cells expressing chimeric G proteins, and BRET studies for measuring receptor/G-protein and receptor/β-arrestin 2 interaction. In vivo in monkeys PWT2-[Dmt1] elicited dose-dependent and robust antinociceptive effects being more potent and longer lasting than [Dmt1]N/OFQ(1–13)-NH2. The analgesic action of PWT2-[Dmt1] was sensitive to the NOP receptor antagonist J-113397, but not naltrexone. Thus, the present study demonstrated that the tetrabranched derivative of [Dmt1]N/OFQ(1–13)-NH2 obtained with the PWT technology maintains the in vitro pharmacological profile of the parent peptide but displays higher potency and longer lasting action in vivo.

Introduction Opioid receptors are currently classified as Mu (μ), Delta (δ), Kappa (κ) plus the opioid related nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP). Despite compelling evidence for interactions and benefits of targeting more than one receptor type in producing analgesia, clinical ligands are Mu agonists. In this study we have designed a Mu-NOP agonist named DeNo. The Mu agonist component is provided by dermorphin, a peptide isolated from the skin of Phyllomedusa frogs and the NOP component by the endogenous agonist N/OFQ. Methods We have assessed receptor binding profile of DeNo and compared with dermorphin and N/OFQ. In a series of functional screens we have assessed the ability to (i) increase Ca2+ in cells coexpressing recombinant receptors and a the chimeric protein Gαqi5, (ii) stimulate the binding of GTPγ[35S], (iii) inhibit cAMP formation, (iv) activate MAPKinase, (v) stimulate receptor-G protein and arrestin interaction using BRET, (vi) electrically stimulated guinea pig ileum (gpI) assay and (vii) ability to produce analgesia via the intrathecal route in rats. Results DeNo bound to Mu (pKi; 9.55) and NOP (pKi; 10.22) and with reasonable selectivity. This translated to increased Ca2+ in Gαqi5 expressing cells (pEC50 Mu 7.17; NOP 9.69), increased binding of GTPγ[35S] (pEC50 Mu 7.70; NOP 9.50) and receptor-G protein interaction in BRET (pEC50 Mu 8.01; NOP 9.02). cAMP formation was inhibited and arrestin was activated (pEC50 Mu 6.36; NOP 8.19). For MAPK DeNo activated p38 and ERK1/2 at Mu but only ERK1/2 at NOP. In the gpI DeNO inhibited electrically-evoked contractions (pEC50 8.63) that was sensitive to both Mu and NOP antagonists. DeNo was antinociceptive in rats. Conclusion Collectively these data validate the strategy used to create a novel bivalent Mu-NOP peptide agonist by combining dermorphin (Mu) and N/OFQ (NOP). This molecule behaves essentially as the parent compounds in vitro. In the antonocicoeptive assays employed in this study DeNo displays only weak antinociceptive properties.