The aim of the present study was to describe the activity of a set of opioid drugs, including partial agonists, in a human embryonic kidney cell system stably expressing only the mouse kappa-opioid receptors. Receptor activation was assessed by measuring the inhibition of cyclic adenosine mono phosphate (cAMP) production stimulated by 5 microM forskolin. Intrinsic activities and potencies of these ligands were determined relative to the endogenous ligand dynorphin and the kappa agonist with the highest intrinsic activity that was identified in this study, fentanyl.

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.

To directly compare the regulation of the cloned kappa and mu opioid receptor, we expressed them in the same cells, the mouse anterior pituitary cell line AtT-20. The coupling of an endogenous somatostatin receptor to adenylyl cyclase and an inward rectifier K+ current has been well characterized in these cells, enabling us to do parallel studies comparing the regulation of both the kappa and the mu receptor to this somatostatin receptor. We show that the kappa receptor readily uncoupled from the K+ current and from adenylyl cyclase after a 1 h pretreatment with agonist, as indicated by the loss in the ability of the agonist to induce a functional response. The desensitization of the kappa receptor was homologous, as the ability of somatostatin to mediate inhibition of adenylyl cyclase or potentiation of the K+ current was not altered by kappa receptor desensitization. The mu receptor uncoupled from the K+ current but not adenylyl cyclase after a 1 h pretreatment with agonist. Somatostatin was no longer able to potentiate the K+ current after mu receptor desensitization, thus this desensitization was heterologous. Interestingly, pretreatment with a somatostatin agonist caused uncoupling of the mu receptor but not the kappa receptor from the K+ current. These results show that in the same cell line, after a 1 h pretreatment with agonist, the kappa receptor displays homologous regulation, whereas the mu receptor undergoes only a heterologous form of desensitization. mu receptor desensitization may lead to the alterations of diverse downstream events, whereas kappa receptor regulation apparently occurs at the level of the receptor itself. Broad alterations of non-opioid systems by the mu receptor could be relevant to the addictive properties of mu agonists. Comparison of kappa and mu receptor regulation may help define the properties of the mu receptor which are important in the development of addiction, tolerance, and withdrawal to opioid drugs. These are the first studies to directly compare the coupling of the kappa and mu receptors to two different effectors in the same mammalian expression system.

An immunofluorescent technique that is more sensitive than radioligand binding was developed in order to detect opioid receptors expressed on leukocytes. The current study was designed to optimize the method for fluorescently labeling kappa opioid receptors. For these experiments, the opioid antagonist naltrexamine was conjugated to either fluorescein (FITC-NTXamine) or biotin (biotin-NTXamine). One-step, two-step, and three-step protocols were compared to determine which procedure resulted in optimal staining of the kappa opioid receptor expressed on intact, unfixed R1E/TL8x.1.OUAr.1(R1EGO) cells, a thymoma known to express kappa opioid receptors. The one-step method involved incubating cells with FITC-NTXamine, and the fluorescein intensity was measured by flow cytometry. In the two-step method, cells were incubated with biotin-NTXamine, followed by extravidin-conjugated phycoerythrin, and the phycoerythrin fluorescence was measured. Finally, in the three-step protocol, cells were incubated with FITC-NTXamine, followed by biotin-conjugated anti-fluorescein IgG, then extravidin-phycoerythrin. The one-step protocol stained the cells, but the signal was not diminished in the presence of opioid competitors. The two-step approach did not stain cells significantly above background levels. Only the three-step approach yielded staining that was displaced by the kappa-selective antagonist nor-binaltorphimine. Thus, the addition of a secondary biotinylated antibody, resulting in the amplification of binding, which was detected using phycoerythrin as a fluorophore, was required to detect low levels of opioid receptor expression on leukocytes.

Activation of kappa opioid receptors (KORs) in the spinal cord can diminish nociception. Humans and rodents show sex differences in the analgesia produced by KOR agonists, and female rats show fluctuations in KOR density and sensitivity across the estrous cycle. However, it is unclear whether there are sex differences in the amount and/or distribution of spinal KORs. In the present study, immunocytochemically labeled KORs were examined in laminae I and II of the lumbosacral spinal dorsal horn of male and normally cycling female Sprague-Dawley rats. The basic pattern of KOR labeling was determined in both sexes using qualitative electron microscopy (EM), and sex-linked differences in the density and subcellular distribution of KOR immunoreactivity were determined with quantitative EM and light microscopy. KOR labeling was visualized with immunoperoxidase for optimally sensitive detection, or with immunogold for precise subcellular localization. By EM, the general pattern of KOR immunoreactivity was similar in males and females. KOR immunoreactivity was common in dendrites, axons, and axon terminals, and was in a few glia and neuronal somata. Most KOR-immunoreactive (-ir) axons were fine-diameter and unmyelinated. Most KOR-ir terminals were small or medium-sized, and a minority formed asymmetric or symmetric synapses with unlabeled dendrites. KOR immunoreactivity was associated both with the plasma membrane and with cytoplasmic organelles, notably including dense core vesicles in terminals. Light microscopic densitometry revealed that KOR immunoreactivity was significantly denser in estrus and proestrus females than in males. By EM, the distribution of KOR-immunogold labeling within axon terminals differed, with a greater proportion of cytoplasmic KOR labeling in estrus females compared with males. In contrast, the abundance and types of KOR-immunoperoxidase-labeled profiles did not show sex-linked differences. We conclude that in both sexes, KORs are positioned to influence both pre- and postsynaptic neurotransmission and are present in morphologically heterogeneous neuron populations. These findings are consistent with complex consequences of KOR activation in the spinal cord. In addition, the presence of increased KOR density and proportionally elevated intracellular KORs in proestrus/estrus females suggests a basis for sex-linked differences in KOR-mediated antinociception.

Increases in drug consumption over time, also known as escalation, is a key behavioral component of substance use disorder (SUD) that is related to potential harm to users, such as overdose. Studying escalation also allows researchers to investigate the transition from casual drug use to more SUD-like drug use. Understanding the neurobiological systems that drive this transition will inform therapeutic treatments in the aim to prevent increases in drug use and the development of SUD. The kappa opioid receptor (KOR) system is typically known for its role in negative affect, which is commonly found in SUD as well. Furthermore, the KOR system has also been implicated in drug use and importantly, modulating the negative effects of drug use. However, the specific neuronal subpopulation expressing KOR involved has not been identified. Here, we first demonstrated that pharmacologically inhibiting KOR in the nucleus accumbens core (NAcC), as a whole, blocks cocaine escalation under long-access self-administration conditions. We then demonstrated that KOR expressed on ventral tegmental area (VTA) neurons but not NAcC neurons is sufficient for blocking cocaine escalation by utilizing a novel virally-mediated CRISPR-SaCas9 knock-out of the oprk1 gene. Together, this suggests that activation of KOR on VTA terminals in the NAcC drives the transition to the SUD-like phenotype of escalation of cocaine consumption.

The recent mu-opioid receptor (MOPr) and kappa-opioid receptor (KOPr) crystal structures have inspired hypotheses of physiologically relevant dimerization contacts, specifically: a closely packed interface involving transmembrane (TM) helices TM5 and TM6, and a less compact interface, involving TM1, TM2, and helix 8 (H8). While the former was only found in MOPr crystals, similar arrangements of the latter were identified for both KOPr and MOPr. The relevance of these interfaces outside of a crystal lattice is called into question by the possibility that they might be influenced by the specific crystallization conditions. In this study, we have employed umbrella sampling molecular dynamics simulations of coarse-grained representations of the interacting MOPr or KOPr crystallographic structures, in the absence of the T4 lysozyme, and in an explicit lipid-water environment, to determine the strength of receptor dimerization at the different crystallographic interfaces. We note that the shape of the interface plays a dominant role in the strength of the interaction, and the pattern of contacting residues defines the shape of the potential of mean force. This information can be used to guide experiments aimed at exploring the role of dimerization in opioid receptor function.

Bone resorption associated to chronic diseases, such as arthritis and periodontitis, results from exacerbated immuno-inflammatory host response that leads to tissue breakdown. The significance of opioid pathways as endogenous modulators of inflammatory events has already been described. Thus, the aim of this work is to determine whether some of the main three opioid receptors are endogenously activated to prevent bone loss during experimentally-induced alveolar bone resorption.

Apelin receptor (APJ) and kappa opioid receptor (KOR) are members of the family A of G protein-coupled receptors (GPCRs). These two receptors are involved in the central nervous system regulation of the cardiovascular system. Here, we explore the possibility of heterodimerization between APJ and KOR and investigate their novel signal transduction characteristics. Co-immunoprecipitation (Co-IP), co-localization and bioluminescence resonance energy transfer (BRET) assays confirmed the heterodimerization of APJ and KOR. In APJ and KOR stably transfected HEK293 cells, treatment with APJ ligand apelin-13 or KOR ligand dynorphinA (1-13) resulted in higher phosphorylation levels of extracellular-regulated kinases 1/2 (ERK1/2) compared to HEK293 cells transfected with either APJ or KOR alone. The siRNA knockdown of either APJ or KOR receptor in human umbilical vein endothelial cells (HUVECs) resulted in significant reduction of the apelin-13 induced ERK activation. Additionally both forskolin (FSK)-induced cAMP levels and cAMP response element reporter activities were significantly reduced, whereas the serum response element luciferase (SRE-luc) reporter activity was significantly upregulated. Moreover, the ERK phosphorylation and SRE-luc activity were abrogated by the protein kinase C (PKC) inhibitor. These results demonstrate for the first time that human APJ forms a heterodimer with KOR and leads to increased PKC and decreased protein kinase A activity leading to a significant increase in cell proliferation, which may translate to the regulation of diverse biological actions and offers the potential for the development of more selective and tissue specific drug therapies.

The dynorphin (DYN)-kappa opioid receptor (kappaOR) system has been implicated in stress modulation, depression, and relapse to drug-seeking behaviors. Previous anatomical and physiological data have indicated that the noradrenergic nucleus locus coeruleus (LC) is one site at which DYN may contribute to these effects. Using light microscopy, immunofluorescence, and electron microscopy, the present study investigated the cellular substrates for pre- and postsynaptic interactions of kappaOR in the LC. Dual immunocytochemical labeling for kappaOR and tyrosine hydroxylase (TH) or kappaOR and preprodynorphin (ppDYN) was examined in the same section of tissue. Light microscopic analysis revealed prominent kappaOR immunoreactivity in the nuclear core of the LC and in the peri-coerulear region where noradrenergic dendrites extend. Fluorescence and electron microscopy revealed kappaOR immunoreactivity within TH-immunoreactive somata and dendrites in the LC as well as localized to ppDYN-immunoreactive processes. In sections processed for kappaOR and TH, approximately 29% (200/688) of the kappaOR-containing axon terminals identified targeted TH-containing profiles. Approximately 49% (98/200) of the kappaOR-labeled axon terminals formed asymmetric synapses with TH-labeled dendrites. Sections processed for kappaOR and ppDYN showed that, of the axon terminals exhibiting kappaOR, 47% (223/477) also exhibited ppDYN. These findings indicate that kappaORs are poised to modulate LC activity by their localization to somata and dendrites. Furthermore, kappaORs are strategically localized to presynaptically modulate DYN afferent input to catecholamine-containing neurons in the LC. These data add to the growing literature showing that kappaORs can modulate diverse afferent signaling to the LC.

Kappa opioid receptor (KOR) ligands alter nociceptive responses when applied to the rostral ventromedial medulla (RVM). However, the effects of kappa opioid receptor ligands are distinct in males and females. The present study examined the distribution of kappa opioid receptor immunoreactivity in the RVM of male and female rats. KOR immunoreactivity was found at pre- and postsynaptic sites within the RVM of both sexes. The most common KOR-immunoreactive (KOR-ir) neuronal structures were unmyelinated axons, followed by axon terminals, dendrites, and somata. Different proportions of KOR-ir axon terminals and dendrites were found in females at different estrous stages. Specifically, dendrites containing KOR immunoreactivity were less abundant in proestrus females compared with estrus females and showed a trend toward being less abundant in males, suggesting that KOR ligands applied to the RVM may be less potent in proestrus females. These findings suggest that the distribution of KORs in the RVM may be influenced by reproductive hormone levels. We also found KOR immunoreactivity in many spinally projecting neurons within the RVM of female rats. These findings are consistent with the hypothesis that KOR ligands influence nociceptive behaviors by altering the activity of specific populations of neurons within the RVM. The abundance of KOR in axons and axon terminals in RVM indicates a substantial role for presynaptic effects of KOR ligands through pathways that have not been clearly delineated. Altering the balance between pre- and postsynaptic receptive sites may underlie differences in the effects of KOR agonists on nociceptive responses in males and females.

Opioid receptors (OR) are widely expressed in the central nervous system (CNS). Opioid antinociception might be initiated by activation of OR outside the CNS, indicating targeting of peripheral OR could be useful in the treatment of chronic pain. This study was designed to detect OR in skin tissues of healthy volunteers at both mRNA and protein levels. Skin samples from 10 healthy individuals were investigated. Total isolated RNAs were reverse transcribed, amplified and quantified by real-time PCR. Tissue and skin fibroblast OR protein was detected by immunohistochemistry, Western blot, and immunofluorescence. All skin tissue samples expressed delta- (DOR) and kappa-OR (KOR) mRNAs. Using immunohistochemistry, DOR and KOR were localized in skin fibroblast-like and mononuclear cells. Skin fibroblasts in culture expressed DOR and KOR mRNA. Using immunofluorescence, both DOR and KOR proteins were expressed predominantly on the cell membrane with minor staining in the cytoplasm. We suggest that enhanced expression of DOR and KOR in skin justifies the exploration of selective novel delta and kappa agonists for local pain treatment.

Mice lacking the mu-opioid receptor (MOR) gene have been successfully developed by homologous recombination and these animals show complete loss of analgesic responses to morphine as well as loss of place-preference activity and physical dependence on this opioid. We report here quantitative autoradiographic mapping of opioid receptor subtypes in the brains of wild-type, heterozygous and homozygous mutant mice to demonstrate the deletion of the MOR gene, to investigate the possible existence of any mu-receptor subtypes derived from a different gene and to determine any modification in the expression of other opioid receptors. Mu-, delta-, kappa1- and total kappa-receptors, in adjacent coronal sections in fore- and midbrain and in sagittal sections, were labelled with [3H]DAMGO (D-Ala2-MePhe4-Gly-ol5 enkephalin), [3H]DELTI (D-Ala2 deltorphinI), [3H]CI-977 and [3H]bremazocine (in the presence of DAMGO and DPDPE) respectively. In heterozygous mice, deficient in one copy of the MOR gene, mu-receptors were detectable throughout the brain at about 50% compared to wild-type. In brains from mu-knockout mice there were no detectable mu-receptors in any brain regions and no evidence for mu-receptors derived from another gene. Delta-, kappa1- and total kappa-receptor binding was present in all brain regions in mutant mice where binding was detected in wild-type animals. There were no major quantitative differences in kappa- or delta-binding in mutant mice although there were some small regional decreases. The results indicate only subtle changes in delta- and kappa-receptors throughout the brains of animals deficient in mu-receptors.

Opioid receptors (ORs) have been observed as homo- and heterodimers, but it is unclear if the dimers are stable under physiological conditions, and whether monomers or dimers comprise the predominant fraction in a cell. Here, we use three live-cell imaging approaches to assess dimerization of ORs at expression levels that are 10-100 × smaller than in classical biochemical assays. At membrane densities around 25/µm2, a split-GFP assay reveals that κOR dimerizes, while µOR and δOR stay monomeric. At receptor densities < 5/µm2, single-molecule imaging showed no κOR dimers, supporting the concept that dimer formation depends on receptor membrane density. To directly observe the transition from monomers to dimers, we used a single-molecule assay to assess membrane protein interactions at densities up to 100 × higher than conventional single-molecule imaging. We observe that κOR is monomeric at densities < 10/µm2 and forms dimers at densities that are considered physiological. In contrast, µOR and δOR stay monomeric even at the highest densities covered by our approach. The observation of long-lasting co-localization of red and green κOR spots suggests that it is a specific effect based on OR dimerization and not an artefact of coincidental encounters.

Endothelin (ET) type A receptor antagonists enhance morphine-induced antinociception and restore morphine analgesia in morphine tolerant rats [Peptides 23 (2002) 1837; Peptides 24 (2003) 553]. These studies suggest that the central ET and opioid systems functionally interact. To explore this idea further, we determined the effect of i.c.v. administration of anti-ET-1 IgG (rabbit) on brain opioid receptor and ET receptor expression. Three days after implanting cannula into the lateral ventricle, male Sprague-Dawley rats were administered 10 microl (i.c.v.) of either control rabbit IgG (2.5 microg/microl) or anti-ET IgG (2.5 microg/microl) on day 1, day 3, and day 5. On day 6, animals were killed and the caudate and hippocampus collected. Anti-ET IgG had no significant effect on expression, measured by Western blots, of mu, delta or ET-B receptors, but increased kappa opioid (59%) and ET-A (33%) receptor protein expression in the caudate. [35S]-GTP-gamma-S binding assays demonstrated that anti-ET IgG decreased [D-Ala2-MePhe4, Gly-ol5]enkephalin efficacy, but not potency in the caudate. Control experiments showed that there was no detectable rabbit IgG in caudate and hippocampal samples. These results suggest that ET in the CSF negatively regulates kappa opioid and ET-A receptors in certain brain regions. These findings support the hypothesis that CSF neuropeptides have regulatory effects and further demonstrate a link between ET and the opioid receptor system.

Studies in laboratory animals and humans indicate that endogenous opioids play an important role in regulating the rewarding value of various drugs, including ethanol (EtOH). Indeed, opioid antagonists are currently a front-line treatment for alcoholism in humans. Although roles for mu- and delta-opioid receptors have been characterized, the contribution of kappa-opioid receptors (KORs) is less clear. There is evidence that changes in KOR system function can decrease or increase EtOH drinking, depending on test conditions. For example, female mice lacking preprodynorphin - the precursor to the endogenous KOR ligand dynorphin - have reduced EtOH intake. Considering that KORs can regulate dopamine (DA) transmission, we hypothesized that KORs expressed on DA neurons would play a prominent role in EtOH intake in females.

If activation of recombinant G protein-coupled receptors in host cells (by drugs or other ligands) has predictive value, similar data must be obtained with native receptors naturally expressed in tissues. Using mouse and human recombinant κ opioid receptors transfected into a host cell, two selectively-acting compounds (ICI204448, asimadoline) equi-effectively activated both receptors, assessed by measuring two different cell signalling pathways which were equally affected without evidence of bias. In mouse intestine, naturally expressing κ receptors within its nervous system, both compounds also equi-effectively activated the receptor, inhibiting nerve-mediated muscle contraction. However, whereas ICI204448 acted similarly in human intestine, where κ receptors are again expressed within its nervous system, asimadoline was inhibitory only at very high concentrations; instead, low concentrations of asimadoline reduced the activity of ICI204448. This demonstration of species-dependence in activation of native, not recombinant κ receptors may be explained by different mouse/human receptor structures affecting receptor expression and/or interactions with intracellular signalling pathways in native environments, to reveal differences in intrinsic efficacy between receptor agonists. These results have profound implications in drug design for κ and perhaps other receptors, in terms of recombinant-to-native receptor translation, species-dependency and possibly, a need to use human, therapeutically-relevant, not surrogate tissues.

Stress is related to heavy alcohol use and relapse in alcoholics. Using the reinstatement model, we have shown that corticotropin-releasing factor (CRF) underlies stress-induced relapse to alcohol seeking in laboratory rodents. Little is known about how other neurotransmitters interact with CRF in these effects. Dynorphin and its receptor (kappa opioid receptor, KOR) are involved in stress responses and in alcohol seeking. KOR and CRF receptors (CRF R) may interact in the production of stress-related behaviors but it is not known whether this interaction is involved in reinstatement of alcohol seeking.

Stressful experiences potently activate kappa opioid receptors (κORs). κORs in the ventral tegmental area regulate multiple aspects of dopaminergic and non-dopaminergic cell function. Here we show that at GABAergic synapses on rat VTA dopamine neurons, a single exposure to a brief cold-water swim stress induces prolonged activation of κORs. This is mediated by activation of the receptor during the stressor followed by a persistent, ligand-independent constitutive activation of the κOR itself. This lasting change in function is not seen at κORs at neighboring excitatory synapses, suggesting distinct time courses and mechanisms of regulation of different subsets of κORs. We also provide evidence that constitutive activity of κORs governs the prolonged reinstatement to cocaine-seeking observed after cold water swim stress. Together, our studies indicate that stress-induced constitutive activation is a novel mechanism of κOR regulation that plays a critical role in reinstatement of drug seeking.

The hippocampus is part of a neural network which regulates the renewal of fear following extinction. Both the ventral (VH) and dorsal (DH) hippocampus have been shown to be necessary for renewal, however the critical receptors and neurotransmitters mediating these contributions are poorly understood. One candidate mechanism is the kappa opioid receptor (KOR) system, which has been implicated in fear learning and anxiety. Here we examined the effect of the KOR antagonist norbinaltorphamine hydrochloride (norBNI), infused into either the VH or DH, on the renewal of extinguished fear. We found that an infusion of norBNI into the VH significantly reduced the relapse of fear on test compared to that seen in saline controls (Experiment 1), while similar infusions of norBNI into the DH had no effect on renewal (Experiment 2). These findings show that hippocampal KORs are involved in fear renewal and also identify a dissociation in the contribution of VH and DH KORs to the expression of renewed fear.