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Sample records for opioid receptor ligands

  1. Positron Emission Tomography (PET) Imaging of Opioid Receptors

    van Waarde, Aren; Absalom, Anthony; Visser, Anniek; Dierckx, Rudi; Dierckx, Rudi AJO; Otte, Andreas; De Vries, Erik FJ; Van Waarde, Aren; Luiten, Paul GM

    2014-01-01

    The opioid system consists of opioid receptors (which mediate the actions of opium), their endogenous ligands (the enkephalins, endorphins, endomorphins, dynorphin, and nociceptin), and the proteins involved in opioid production, transport, and degradation. PET tracers for the various opioid

  2. Synthesis of triated N1'-alkyl derivatives of the delta opioid receptor ligand naltrindole

    Lever, J.R.; Johnson, S.M.

    1997-01-01

    Tritiated N1'-methyl and N1'-ethyl analogues of naltrindole (NTI) have been synthesized for evaluation as radioligands for studies of delta opioid receptors. The two N1'-alkyl-5',7'-dibromoNTI precursors for radiolabeling were prepared by base-promoted alkylation of 2,4-dibromophenylhydrazine with either iodomethane or iodoethane followed by condensation with naltrexone using the Fischer indole synthesis. Catalytic debromotritiation followed by HPLC purification afforded [ 3 H]MeNTI (17.3 Ci/mmol) and [ 3 H]EtNTI (22.5 Ci/mmol) with high chemical and radiochemical purities (≥ 99.8%). (author)

  3. Ligand- and cell-dependent determinants of internalization and cAMP modulation by delta opioid receptor (DOR) agonists

    Charfi, Iness; Nagi, Karim; Mnie-Filali, Ouissame; Thibault, Dominic; Balboni, Gianfranco; Schiller, Peter W.; Trudeau, Louis-Eric

    2014-01-01

    Signaling bias refers to G protein-coupled receptor ligand ability to preferentially activate one type of signal over another. Bias to evoke signaling as opposed to sequestration has been proposed as a predictor of opioid ligand potential for generating tolerance. Here we measured whether delta opioid receptor agonists preferentially inhibited cyclase activity over internalization in HEK cells. Efficacy (τ) and affinity (KA) values were estimated from functional data and bias was calculated from efficiency coefficients (log τ/KA). This approach better represented the data as compared to alternative methods that estimate bias exclusively from τ values. Log (τ/KA) coefficients indicated that SNC-80 and UFP-512 promoted cyclase inhibition more efficiently than DOR internalization as compared to DPDPE (bias factor for SNC-80: 50 and for UFP-512: 132). Molecular determinants of internalization were different in HEK293 cells and neurons with βarrs contributing to internalization in both cell types, while PKC and GRK2 activities were only involved in neurons. Rank orders of ligand ability to engage different internalization mechanisms in neurons were compared to rank order of Emax values for cyclase assays in HEK cells. Comparison revealed a significant reversal in rank order for cyclase Emax values and βarr-dependent internalization in neurons, indicating that these responses were ligand-specific. Despite this evidence, and because kinases involved in internalization were not the same across cellular backgrounds, it is not possible to assert if the magnitude and nature of bias revealed by rank orders of maximal responses is the same as the one measured in HEK cells. PMID:24022593

  4. Anti-analgesic effect of the mu/delta opioid receptor heteromer revealed by ligand-biased antagonism.

    Laura Milan-Lobo

    Full Text Available Delta (DOR and mu opioid receptors (MOR can complex as heteromers, conferring functional properties in agonist binding, signaling and trafficking that can differ markedly from their homomeric counterparts. Because of these differences, DOR/MOR heteromers may be a novel therapeutic target in the treatment of pain. However, there are currently no ligands selective for DOR/MOR heteromers, and, consequently, their role in nociception remains unknown. In this study, we used a pharmacological opioid cocktail that selectively activates and stabilizes the DOR/MOR heteromer at the cell surface by blocking its endocytosis to assess its role in antinociception. We found that mice treated chronically with this drug cocktail showed a significant right shift in the ED50 for opioid-mediated analgesia, while mice treated with a drug that promotes degradation of the heteromer did not. Furthermore, promoting degradation of the DOR/MOR heteromer after the right shift in the ED50 had occurred, or blocking signal transduction from the stabilized DOR/MOR heteromer, shifted the ED50 for analgesia back to the left. Taken together, these data suggest an anti-analgesic role for the DOR/MOR heteromer in pain. In conclusion, antagonists selective for DOR/MOR heteromer could provide an avenue for alleviating reduced analgesic response during chronic pain treatment.

  5. N1'-fluoroethyl-naltrindole (BU97001) and N1'-fluoroethyl-(14-formylamino)-naltrindole (BU97018) potential δ-opioid receptor PET ligands

    Tyacke, Robin J.; Robinson, Emma S.J.; Schnabel, Rebecca; Lewis, John W.; Husbands, Stephen M.; Nutt, David J.; Hudson, Alan L.

    2002-01-01

    The properties of two prospective positron emission tomography (PET) ligands for the δ-opioid receptor, N1'-fluoroethyl-naltrindole (BU97001) and N1'-fluoroethyl-(14-formylamino)-naltrindole (BU97018) were investigated. Both were antagonists in the mouse vas deferens, and showed high affinity and selectivity, 1.81 nM and 3.09 nM respectively. [ 3 H]BU97001 binding to rat whole brain was also of high affinity, K D of 0.42 nM of and B MAX of 59.95 fmol mg of protein -1 . In autoradiographic studies, it was found to bind to brain areas previously shown to be associated with the δ-opioid receptor and good correlations were found to exist with naltrindole and DPDPE. BU97018 and especially BU97001 appear to show good potential as δ-opioid receptor PET ligands with the incorporation of 18 F

  6. Molecular characterization of opioid receptors

    Howard, A.D.

    1986-01-01

    The aim of this research was to purify and characterize active opioid receptors and elucidate molecular aspects of opioid receptor heterogeneity. Purification to apparent homogeneity of an opioid binding protein from bovine caudate was achieved by solubilization in the non-ionic detergent, digitonin, followed by sequential chromatography on the opiate affinity matrix, ..beta..-naltrexylethylenediamine-CH-Sepharose 4B, and on the lectine affinity matrix, wheat germ agglutinin-agarose. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) followed by autoradiography revealed that radioiodinated purified receptor gave a single band. Purified receptor preparations showed a specific activity of 12,000-15,000 fmol of opiate bound per mg of protein. Radioiodinated human beta-endorphin (/sup 125/I-beta-end/sub H/) was used as a probe to investigate the ligand binding subunits of mu and delta opioid receptors. /sup 125/I-beta-end/sub H/ was shown to bind to a variety of opioid receptor-containing tissues with high affinity and specificity with preference for mu and delta sites, and with little, if any, binding to kappa sites. Affinity crosslinking techniques were employed to covalently link /sup 125/I-beta-end/sub H/ to opioid receptors, utilizing derivatives of bis-succinimidyl esters that are bifunctional crosslinkers with specificities for amino and sulfhydryl groups. This, and competition experiments with high type-selective ligands, permitted the assignment of two labeled peptides to their receptor types, namely a peptide of M/sub r/ = 65,000 for mu receptors and one of M/sub r/ = 53,000 for delta receptors.

  7. Gender dependent rate of metabolism of the opioid receptor-PET ligand [18F]fluoroethyl-diprenorphine

    Henriksen, G.; Spilker, M.E.; Hauser, A.I.; Boecker, H.; Schwaiger, M.; Wester, H.J.; Sprenger, T.; Platzer, S.; Toelle, T.R.

    2006-01-01

    Aim: The morphinane-derivate 6-O-(2-[ 18 F]fluoroethyl)-6-O-desmethyldiprenorphine ([ 18 F]FDPN) is a non-selective opioid receptor ligand currently used in positron emission tomography (PET). Correction for plasma metabolites of the arterial input function is necessary for quantitative measurements of [ 18 ]FDPN binding. A study was undertaken to investigate if there are gender dependent differences in the rate of metabolism of [ 18 F]FDPN. Methods: The rate of metabolism of [ 18 F]FDPN was mathematically quantified by fitting a bi-exponential function to each individual's dynamic metabolite data. Results: No statistically significant gender differences were found for age, weight, body mass index or dose. However, significant differences (p 18 F]FDPN faster than men. These differences were found in the contribution of the fast and slow kinetic components of the model describing the distribution of radioactive species in plasma, indicating a higher rate of enzyme-dependent degradation of [ 18 F]FDPN in women than in men. Conclusion: The findings reinforce the need for individualized metabolite correction during [ 18 F]FDPN-PET scans and also indicate that in certain cases, grouping according to gender could be performed in order to minimize methodological errors of the input function prior to kinetic analyses. (orig.)

  8. Identification in the mu-opioid receptor of cysteine residues responsible for inactivation of ligand binding by thiol alkylating and reducing agents.

    Gaibelet, G; Capeyrou, R; Dietrich, G; Emorine, L J

    1997-05-19

    Inactivation by thiol reducing and alkylating agents of ligand binding to the human mu-opioid receptor was examined. Dithiothreitol reduced the number of [3H]diprenorphine binding sites. Replacement by seryl residues of either C142 or C219 in extracellular loops 1 and 2 of the mu receptor resulted in a complete loss of opioid binding. A disulfide bound linking C142 to C219 may thus be essential to maintain a functional conformation of the receptor. We also demonstrated that inactivation of ligand binding upon alkylation by N-ethylmaleimide occurred at two sites. Alteration of the more sensitive (IC50 = 20 microM) did not modify antagonists binding but decreased agonist affinity almost 10-fold. Modification of the less reactive site (IC50 = 2 mM) decreased the number of both agonist and antagonist binding sites. The alkylation site of higher sensitivity to N-ethylmaleimide was shown by mutagenesis experiments to be constituted of both C81 and C332 in transmembrane domains 1 and 7 of the mu-opioid receptor.

  9. The opioid receptor pharmacology of GSK1521498 compared to other ligands with differential effects on compulsive reward-related behaviours.

    Kelly, Eamonn; Mundell, Stuart J; Sava, Anna; Roth, Adelheid L; Felici, Antonio; Maltby, Kay; Nathan, Pradeep J; Bullmore, Edward T; Henderson, Graeme

    2015-01-01

    The novel opioid receptor antagonist, GSK1421498, has been shown to attenuate reward-driven compulsive behaviours, such as stimulant drug seeking or binge eating, in animals and humans. Here, we report new data on the receptor pharmacology of GSK121498, in comparison to naltrexone, naloxone, 6-β-naltrexol and nalmefene. To determine whether the novel opioid antagonist, GSK1521498, is an orthosteric or allosteric antagonist at the μ opioid receptor (MOPr) and whether it has neutral antagonist or inverse agonist properties. A combination of radioligand binding assays and [(35)S]GTPγS binding assays was employed. GSK1521498 completely displaced [(3)H]naloxone binding to MOPr and did not alter the rate of [(3)H]naloxone dissociation from MOPr observations compatible with it binding to the orthosteric site on MOPr. GSK1521498 exhibited inverse agonism when MOPr was overexpressed but not when the level of MOPr expression was low. In parallel studies under conditions of high receptor expression density, naloxone, naltrexone, 6-β-naltrexol and nalmefene exhibited partial agonism, not inverse agonism as has been reported previously for naloxone and naltrexone. In brain tissue from mice receiving a prolonged morphine pre-treatment, GSK1521498 exhibited slight inverse agonism. Differences between GSK1521498 and naltrexone in their effects on compulsive reward seeking are arguably linked to the more selective and complete MOPr antagonism of GSK1521498 versus the partial MOPr agonism of naltrexone. GSK1521498 is also pharmacologically differentiated by its inverse agonist efficacy at high levels of MOPr expression, but this may be less likely to contribute to behavioural differentiation at patho-physiological levels of expression.

  10. The development of [18F]cyclofoxy as a ligand for imaging opioid receptors in the CNS of conscious humans

    Rice, K.C.; Newman, A.H.; Ostrowski, N.L.; Cohen, R.M.; Pert, A.; Pert, C.B.; Burke, T.R. Jr.; McLellan, C.A.; Channing, M.A.; Finn, R.D.; Dunn, B.; Simpson, N.; Carson, R.W.; Larson, S.M.; Eckelman, W.C.; Bennett, J.M.; Kawai, R.; Sawada, Y.; Herscovitch, P.; Yolles, P.S.; Nordhal, T.; Gross, M.; Blasberg, R.

    1989-01-01

    Positron emission tomography (PET) is a unique, noninvasive technique applicable to real time visualization and quantitation of drug receptor occupancy in the brain of conscious humans. Such studies with the normal and abnormal human CNS can potentially provide insight into the biochemical basis of disease states and the effects of drug therapy. The (-)-enantiomer of cyclofoxy, a fluorinated, potent narcotic antagonist derived from naltrexone, has been developed at NIH as an agent for study of the opioid receptor-endorphin system using PET. The development and current status of this program is described, including application of the NIH Opiate Total Synthesis for production the pharmacologically inert (+)-[ 18 F]cyclofoxy required for quantitation of receptor occupancy

  11. Kappa-receptor selective binding of opioid ligands with a heterocyclic bicyclo[3.3.1]nonan-9-one structure.

    Benyhe, S; Márki, A; Nachtsheim, Corina; Holzgrabe, Ulrike; Borsodi, Anna

    2003-01-01

    Previous pharmacological results have suggested that members of the heterocyclic bicyclo[3.3.1]nonan-9-one-like compounds are potent kappa-opioid receptor specific agonists. One lead molecule of this series. called compound 1 (dimethyl 7-methyl-2,4-di-2-pyridyl-3.7-diazabicyclo[3.3.1]nonan-9-one-1,5-dicarboxylate) exhibited high affinity for [3H]ethylketocyclazocine and [3H]U-69.593 binding sites in guinea pig cerebellar membranes which known to be a good source for kappa1 receptors. It was shown by molecular modelling that heterocyclic bicyclo[3.3.1]nonan-9-ones fit very well with the structure of ketazocine, a prototypic kappa-selective benzomorphan compound; when compared to the arylacetamide structure of U-69.593, a specific kappa1-receptor agonist, a similar geometry was found with a slightly different distribution of the charges. It is postulated, that the essential structural skeleton involved in the opioid activity is an aryl-propyl-amine element distributed along the N7-C6-C5-C4-aryl bonds.

  12. Part I. Naltrexone-derived conjugate addition ligands for opioid receptors. Part II. Chemical and enantioselective aspects of the metabolism of verapamil

    Olsen, L.D.

    1987-01-01

    Selective chemoaffinity ligands to aid in identification and purification of opioid receptor subtypes were prepared from 6α- and 6β-naltrexol, obtained stereoselectively from the μ-receptor antagonist naltrexone. The targets were the 6α- and 6β-methacrylate ethers and 6α- and 6β-methacrylate esters prepared from reaction of 6α- and 6β-naltrexol with methyl α-(bromomethyl)acrylate or methacryloyl chloride. Of three methacrylate derivatives, the 6α-ether was the most potent in an opioid receptor binding assay with [ 3 H]-naltrexone. In the presence of sodium ion, preincubation of the 6α-ether resulted in recovery of about 60% of original [ 3 H]-naltrexone binding suggesting some irreversible effects. The methacrylate esters precipitated withdrawal in morphine dependent monkeys. The enantiomers of verapamil, a calcium channel antagonist, have different pharmacological and pharmacokinetic properties. The oxidative metabolism of verapamil was studied in rat and human liver microsomes and in man after a single oral dose

  13. Part I. Naltrexone-derived conjugate addition ligands for opioid receptors. Part II. Chemical and enantioselective aspects of the metabolism of verapamil

    Olsen, L.D.

    1987-01-01

    Selective chemoaffinity ligands to aid in identification and purification of opioid receptor subtypes were prepared from 6..cap alpha..- and 6..beta..-naltrexol, obtained stereoselectively from the ..mu..-receptor antagonist naltrexone. The targets were the 6..cap alpha..- and 6..beta..-methacrylate ethers and 6..cap alpha..- and 6..beta..-methacrylate esters prepared from reaction of 6..cap alpha..- and 6..beta..-naltrexol with methyl ..cap alpha..-(bromomethyl)acrylate or methacryloyl chloride. Of three methacrylate derivatives, the 6..cap alpha..-ether was the most potent in an opioid receptor binding assay with (/sup 3/H)-naltrexone. In the presence of sodium ion, preincubation of the 6..cap alpha..-ether resulted in recovery of about 60% of original (/sup 3/H)-naltrexone binding suggesting some irreversible effects. The methacrylate esters precipitated withdrawal in morphine dependent monkeys. The enantiomers of verapamil, a calcium channel antagonist, have different pharmacological and pharmacokinetic properties. The oxidative metabolism of verapamil was studied in rat and human liver microsomes and in man after a single oral dose.

  14. Quantitative analysis of multiple kappa-opioid receptors by selective and nonselective ligand binding in guinea pig spinal cord: Resolution of high and low affinity states of the kappa 2 receptors by a computerized model-fitting technique

    Tiberi, M.; Magnan, J.

    1990-01-01

    The binding characteristics of selective and nonselective opioids have been studied in whole guinea pig spinal cord, using a computer fitting method to analyze the data obtained from saturation and competition studies. The delineation of specific binding sites labeled by the mu-selective opioid [3H]D-Ala2,MePhe4,Gly-ol5-enkephalin (Kd = 2.58 nM, R = 4.52 pmol/g of tissue) and by the delta-selective opioid [3H]D-Pen2, D-Pen5-enkephalin (Kd = 2.02 nM, R = 1.47 pmol/g of tissue) suggests the presence of mu and delta-receptors in the spinal cord tissue. The presence of kappa receptors was probed by the kappa-selective opioid [3H]U69593 (Kd = 3.31 nM, R = 2.00 pmol/g of tissue). The pharmacological characterization of the sites labeled by [3H]U69593 confirms the assumption that this ligand discriminates kappa receptors in guinea pig spinal cord. The benzomorphan [3H]ethylketazocine labels a population of receptors with one homogeneous affinity state (Kd = 0.65 nM, R = 7.39 pmol/g of tissue). The total binding capacity of this ligand was not different from the sum of the binding capacities of mu, delta-, and kappa-selective ligands. Under mu- and delta-suppressed conditions, [3H]ethylketazocine still binds to receptors with one homogeneous affinity state (Kd = 0.45 nM, R = 1.69 pmol/g of tissue). Competition studies performed against the binding of [3H]ethylketazocine under these experimental conditions reveal that the pharmacological profile of the radiolabeled receptors is similar to the profile of the kappa receptors labeled with [3H]U69593. Saturation studies using the nonselective opioid [3H]bremazocine demonstrate that this ligand binds to spinal cord membranes with heterogeneous affinities (Kd1 = 0.28 nM, R1 = 7.91 pmol/g of tissue; Kd2 = 3.24 nM, R2 = 11.2 pmol/g of tissue)

  15. Quantitative analysis of multiple kappa-opioid receptors by selective and nonselective ligand binding in guinea pig spinal cord: Resolution of high and low affinity states of the kappa 2 receptors by a computerized model-fitting technique

    Tiberi, M.; Magnan, J. (Universite de Montreal, Quebec (Canada))

    1990-05-01

    The binding characteristics of selective and nonselective opioids have been studied in whole guinea pig spinal cord, using a computer fitting method to analyze the data obtained from saturation and competition studies. The delineation of specific binding sites labeled by the mu-selective opioid (3H)D-Ala2,MePhe4,Gly-ol5-enkephalin (Kd = 2.58 nM, R = 4.52 pmol/g of tissue) and by the delta-selective opioid (3H)D-Pen2, D-Pen5-enkephalin (Kd = 2.02 nM, R = 1.47 pmol/g of tissue) suggests the presence of mu and delta-receptors in the spinal cord tissue. The presence of kappa receptors was probed by the kappa-selective opioid (3H)U69593 (Kd = 3.31 nM, R = 2.00 pmol/g of tissue). The pharmacological characterization of the sites labeled by (3H)U69593 confirms the assumption that this ligand discriminates kappa receptors in guinea pig spinal cord. The benzomorphan (3H)ethylketazocine labels a population of receptors with one homogeneous affinity state (Kd = 0.65 nM, R = 7.39 pmol/g of tissue). The total binding capacity of this ligand was not different from the sum of the binding capacities of mu, delta-, and kappa-selective ligands. Under mu- and delta-suppressed conditions, (3H)ethylketazocine still binds to receptors with one homogeneous affinity state (Kd = 0.45 nM, R = 1.69 pmol/g of tissue). Competition studies performed against the binding of (3H)ethylketazocine under these experimental conditions reveal that the pharmacological profile of the radiolabeled receptors is similar to the profile of the kappa receptors labeled with (3H)U69593. Saturation studies using the nonselective opioid (3H)bremazocine demonstrate that this ligand binds to spinal cord membranes with heterogeneous affinities (Kd1 = 0.28 nM, R1 = 7.91 pmol/g of tissue; Kd2 = 3.24 nM, R2 = 11.2 pmol/g of tissue).

  16. Chronic nicotine-induced changes in gene expression of delta and kappa-opioid receptors and their endogenous ligands in the mesocorticolimbic system of the rat.

    Ugur, Muzeyyen; Kaya, Egemen; Gozen, Oguz; Koylu, Ersin O; Kanit, Lutfiye; Keser, Aysegul; Balkan, Burcu

    2017-09-01

    Delta and kappa opioid receptors (DOR and KOR, respectively) and their endogenous ligands, proenkephalin (PENK) and prodynorphin (PDYN)-derived opioid peptides are proposed as important mediators of nicotine reward. This study investigated the regulatory effect of chronic nicotine treatment on the gene expression of DOR, KOR, PENK and PDYN in the mesocorticolimbic system. Three groups of rats were injected subcutaneously with nicotine at doses of 0.2, 0.4, or 0.6 mg/kg/day for 6 days. Rats were decapitated 1 hr after the last dose on day six, as this timing coincides with increased dopamine release in the mesocorticolimbic system. mRNA levels in the ventral tegmental area (VTA), lateral hypothalamic area (LHA), amygdala (AMG), dorsal striatum (DST), nucleus accumbens, and medial prefrontal cortex were measured by quantitative real-time PCR. Our results showed that nicotine upregulated DOR mRNA in the VTA at all of the doses employed, in the AMG at the 0.4 and 0.6 mg/kg doses, and in the DST at the 0.4 mg/kg dose. Conversely, PDYN mRNA was reduced in the LHA with 0.6 mg/kg nicotine and in the AMG with 0.4 mg/kg nicotine. KOR mRNA was also decreased in the DST with 0.6 mg/kg nicotine. Nicotine did not regulate PENK mRNA in any brain region studied. © 2017 Wiley Periodicals, Inc.

  17. Methyl-orvinol-Dual activity opioid receptor ligand inhibits gastrointestinal transit and alleviates abdominal pain in the mouse models mimicking diarrhea-predominant irritable bowel syndrome.

    Zielińska, Marta; Jarmuż, Agata; Wasilewski, Andrzej; Cami-Kobeci, Gerta; Husbands, Stephen; Fichna, Jakub

    2017-04-01

    Diarrhea-predominant irritable bowel syndrome (IBS-D) is a functional disorder of the gastrointestinal (GI) tract. The major IBS-D symptoms include diarrhea, abdominal pain and discomfort. High density of opioid receptors (ORs) in the GI tract and their participation in the maintenance of GI homeostasis make ORs ligands an attractive option for developing new anti-IBS-D treatments. The aim of this study was to characterize the effect of methyl-orvinol on the GI motility and secretion and in mouse models mimicking symptoms of IBS-D. In vitro, the effects of methyl-orvinol on electrical field stimulated smooth muscle contractility and epithelial ion transport were characterized in the mouse colon. In vivo, the following tests were used to determine methyl-orvinol effect on mouse GI motility: colonic bead expulsion, whole GI transit and fecal pellet output. An antinociceptive action of methyl-orvinol was assessed in the mouse model of visceral pain induced by mustard oil. Methyl-orvinol (10 -10 to 10 -6 M) inhibited colonic smooth muscle contractions in a concentration-dependent manner. This effect was reversed by naloxone (non-selective opioid antagonist) and β-funaltrexamine (selective MOP antagonist). Experiments with a selective KOP receptor agonist, U50488 revealed that methyl-orvinol is a KOP receptor antagonist in the GI tract. Methyl-orvinol enhanced epithelial ion transport. In vivo, methyl-orvinol inhibited colonic bead expulsion and prolonged GI transit. Methyl-orvinol improved hypermotility and reduced abdominal pain in the mouse models mimicking IBS-D symptoms. Methyl-orvinol could become a promising drug candidate in chronic therapy of functional GI diseases such as IBS-D. Copyright © 2016 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  18. Computer Modeling of Human Delta Opioid Receptor

    Tatyana Dzimbova

    2013-04-01

    Full Text Available The development of selective agonists of δ-opioid receptor as well as the model of interaction of ligands with this receptor is the subjects of increased interest. In the absence of crystal structures of opioid receptors, 3D homology models with different templates have been reported in the literature. The problem is that these models are not available for widespread use. The aims of our study are: (1 to choose within recently published crystallographic structures templates for homology modeling of the human δ-opioid receptor (DOR; (2 to evaluate the models with different computational tools; and (3 to precise the most reliable model basing on correlation between docking data and in vitro bioassay results. The enkephalin analogues, as ligands used in this study, were previously synthesized by our group and their biological activity was evaluated. Several models of DOR were generated using different templates. All these models were evaluated by PROCHECK and MolProbity and relationship between docking data and in vitro results was determined. The best correlations received for the tested models of DOR were found between efficacy (erel of the compounds, calculated from in vitro experiments and Fitness scoring function from docking studies. New model of DOR was generated and evaluated by different approaches. This model has good GA341 value (0.99 from MODELLER, good values from PROCHECK (92.6% of most favored regions and MolProbity (99.5% of favored regions. Scoring function correlates (Pearson r = -0.7368, p-value = 0.0097 with erel of a series of enkephalin analogues, calculated from in vitro experiments. So, this investigation allows suggesting a reliable model of DOR. Newly generated model of DOR receptor could be used further for in silico experiments and it will give possibility for faster and more correct design of selective and effective ligands for δ-opioid receptor.

  19. Chimeric opioid peptides: tools for identifying opioid receptor types.

    Xie, G X; Miyajima, A; Yokota, T; Arai, K; Goldstein, A

    1990-01-01

    We synthesized several chimeric peptides in which the N-terminal nine residues of dynorphin-32, a peptide selective for the kappa opioid receptor, were replaced by opioid peptides selective for other opioid receptor types. Each chimeric peptide retained the high affinity and type selectivity characteristic of its N-terminal sequence. The common C-terminal two-thirds of the chimeric peptides served as an epitope recognized by the same monoclonal antibody. When bound to receptors on a cell surf...

  20. Structure of the [delta]-opioid receptor bound to naltrindole

    Granier, Sébastien; Manglik, Aashish; Kruse, Andrew C.; Kobilka, Tong Sun; Thian, Foon Sun; Weis, William I.; Kobilka, Brian K. (Stanford-MED)

    2012-07-11

    The opioid receptor family comprises three members, the {mu}-, {delta}- and {kappa}-opioid receptors, which respond to classical opioid alkaloids such as morphine and heroin as well as to endogenous peptide ligands like endorphins. They belong to the G-protein-coupled receptor (GPCR) superfamily, and are excellent therapeutic targets for pain control. The {delta}-opioid receptor ({delta}-OR) has a role in analgesia, as well as in other neurological functions that remain poorly understood. The structures of the {mu}-OR and {kappa}-OR have recently been solved. Here we report the crystal structure of the mouse {delta}-OR, bound to the subtype-selective antagonist naltrindole. Together with the structures of the {mu}-OR and {kappa}-OR, the {delta}-OR structure provides insights into conserved elements of opioid ligand recognition while also revealing structural features associated with ligand-subtype selectivity. The binding pocket of opioid receptors can be divided into two distinct regions. Whereas the lower part of this pocket is highly conserved among opioid receptors, the upper part contains divergent residues that confer subtype selectivity. This provides a structural explanation and validation for the 'message-address' model of opioid receptor pharmacology, in which distinct 'message' (efficacy) and 'address' (selectivity) determinants are contained within a single ligand. Comparison of the address region of the {delta}-OR with other GPCRs reveals that this structural organization may be a more general phenomenon, extending to other GPCR families as well.

  1. Chimeric opioid peptides: Tools for identifying opioid receptor types

    Xie, G.; Miyajima, A.; Yokota, T.; Arai, K.; Goldstein, A.

    1990-01-01

    The authors synthesized several chimeric [125J-labelled] peptides in which the N-terminal nine residues of dynorphin-32, a peptide selective for the κ opioid receptor, were replaced by opioid peptides selective for other opioid receptor types. Each chimeric peptide retained the high affinity and type selectivity characteristic of its N-terminal sequence. The common C-terminal two-thirds of the chimeric peptides served as an epitope recognized by the same monoclonal antibody. When bound to receptors on a cell surface or membrane preparation, these peptides could still bind specifically to the monoclonal antibody. These chimeric peptides should be useful for isolating μ, δ, and κ opioid receptors and for identifying opioid receptors on transfected cells in expression cloning procedures. The general approach using chimeric peptides should be applicable to other peptide receptors

  2. AMPA receptor ligands

    Strømgaard, Kristian; Mellor, Ian

    2004-01-01

    Alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors (AMPAR), subtype of the ionotropic glutamate receptors (IGRs), mediate fast synaptic transmission in the central nervous system (CNS), and are involved in many neurological disorders, as well as being a key player in the f......Alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors (AMPAR), subtype of the ionotropic glutamate receptors (IGRs), mediate fast synaptic transmission in the central nervous system (CNS), and are involved in many neurological disorders, as well as being a key player...... in the formation of memory. Hence, ligands affecting AMPARs are highly important for the study of the structure and function of this receptor, and in this regard polyamine-based ligands, particularly polyamine toxins, are unique as they selectively block Ca2+ -permeable AMPARs. Indeed, endogenous intracellular...

  3. Probes for narcotic receptor mediated phenomena 22. Pt.1: Synthesis and characterization of optically pure [{sup 3}H](+)-4-[({alpha}R)-{alpha}-((2S,5R)-4-propyl-2,5-dimethyl-1-pi perazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide, [{sup 3}H]SNC 121, a novel high affinity and selective ligand for delta opioid receptors

    Calderon, S.N.; Bertha, C.M.; Rice, K.C. [National Inst. of Diabetes and Digestive and Kidney Diseases, Medicinal Chemistry Lab., Bethesda, MD (United States); Gutkind, J.S. [National Inst. of Dental Research, Bethesda, MD (United States); Heng Xu; Partilla, J.S.; Rothman, R.B. [National Inst. on Drug Abuse, Clinical Psychopharmacology Section, Baltimore, MD (United States)

    1996-09-01

    The synthesis of unlabelled and labelled SNC 121, a selective nonpeptide ligand for the delta opioid receptor is reported. [{sup 3}H]SNC 121 of specific activity of 26.8 Ci/mmol, was synthesized by catalytic tritiation of the optically pure precursor SNC 80. (author).

  4. Probes for narcotic receptor mediated phenomena 22. Pt.1: Synthesis and characterization of optically pure [3H](+)-4-[(αR)-α-((2S,5R)-4-propyl-2,5-dimethyl-1-pi perazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide, [3H]SNC 121, a novel high affinity and selective ligand for delta opioid receptors

    Calderon, S.N.; Bertha, C.M.; Rice, K.C.; Gutkind, J.S.; Heng Xu; Partilla, J.S.; Rothman, R.B.

    1996-01-01

    The synthesis of unlabelled and labelled SNC 121, a selective nonpeptide ligand for the delta opioid receptor is reported. [ 3 H]SNC 121 of specific activity of 26.8 Ci/mmol, was synthesized by catalytic tritiation of the optically pure precursor SNC 80. (author)

  5. Binding-site analysis of opioid receptors using monoclonal anti-idiotypic antibodies

    Conroy, W.G.

    1988-01-01

    Structural relatedness between the variable region of anti-ligand antibodies and opioid binding sites allowed the generation of anti-idiotypic antibodies which recognized opioid receptors. The IgG 3 k antibodies which bound to opioid receptors were obtained when an anti-morphine antiserum was the idiotype. Both antibodies bound to opioid receptors, but only one of these blocked the binding of [ 3 H]naloxone. The antibody which did not inhibit the binding of [ 3 H]naloxone was itself displaced from the receptor by opioid ligands. The unique binding properties displayed by this antibody indicated that anti-idiotypic antibodies are not always a perfect image of the original ligand, and therefore may be more useful than typical ligands as probes for the receptor. An auto-anti-idiotypic technique was successfully used to obtain anti-opioid receptor antibodies. Another IgG 3 k antibody that blocked the binding of [ 3 H]naloxone to rat brain opioid receptors was obtained when a mouse was immunized with naloxone conjugated to bovine serum albumin. These data confirmed that an idiotype-anti-idiotype network which can generate an anti-receptor antibody normally functions when an opioid ligand is introduced into an animal in an immunogenic form

  6. The opioid receptors of the rat periaqueductal gray

    Fedynyshyn, J.P.

    1989-01-01

    The opioid binding characteristics of the rat (PAG) and the signal transduction mechanisms of the opioid receptors were examined with in vitro radioligand binding, GTPase, adenylyl cyclase, and inositol phosphate assays. The nonselective ligand {sup 3}H-ethylketocyclazocine (EKC), the {mu} and {delta} selective ligand {sup 3}H-(D-Ala{sup 2}, D-Leu{sup 5}) enkephalin (DADLE), the {mu} selective ligand {sup 3}H-(D-Ala{sup 2}, N-methyl Phe{sup 4}, Glyol{sup 5}) enkephalin (DAGO), and the {delta} selective ligand {sup 3}H-(D-Pen{sup 2}, D-Pen{sup 5}) enkephalin (DPDPE) were separately used as tracer ligands to label opioid binding sites in rat PAG enriched P{sub 2} membrane in competition with unlabeled DADLE, DAGO, DPDPE, or the {kappa} selective ligand trans-3,4-dichloro-N-(2-(1-pyrrolidinyl)cyclohexyl)benzeneacetamide, methane sulfonate, hydrate (U50, 488H). Only {mu} selective high affinity opioid binding was observed. No high affinity {delta} or {kappa} selective binding was detected. {sup 3}H-DAGO was used as a tracer ligand to label {mu} selective high affinity opioid binding sites in PAG enriched P{sub 2} membrane in competition with unlabeled {beta}-endorphin, dynorphin A (1-17), BAM-18, methionine enkephalin, dynorphin A (1-8), and leucine enkephalin. Of these endogenous opioid peptides only those with previously reported high affinity {mu} type opioid binding activity competed with {sup 3}H-DAGO for binding sites in rat PAG enriched P{sub 2} membrane with affinities similar to that of unlabeled DAGO.

  7. Glutamate receptor ligands

    Guldbrandt, Mette; Johansen, Tommy N; Frydenvang, Karla Andrea

    2002-01-01

    Homologation and substitution on the carbon backbone of (S)-glutamic acid [(S)-Glu, 1], as well as absolute stereochemistry, are structural parameters of key importance for the pharmacological profile of (S)-Glu receptor ligands. We describe a series of methyl-substituted 2-aminoadipic acid (AA...

  8. Synthesis of high specific activity tritium labelled 1S,2S-(-)-trans-2-isothiocyanato-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)benzene acetamide, a specific irreversible ligand for kappa opioid receptors

    Costa, B.R. de; Thurkauf, A.; Rothman, R.R. (National Inst. of Mental Health, Bethesda, MD (USA)); Jacobson, A.E.; Rice, K.C. (National Inst. of Digestive Diabetes, and Kidney Diseases, Bethesda, MD (USA))

    1990-11-01

    Optically pure tritium labeled 1S,2S-(-)-trans-2-isothiocyanato-N-methyl-N-(2-(1-pyrrolidinyl)cyclohexyl )benzeneacetamide, an affinity ligand specific for the kappa opioid receptor was synthesized from optically pure 1S,2S-(-)-trans-2-amino-N-methyl-N-(2-(1-pyrrolidinyl)cyclohexyl)benzeneacetamide via the sequence of dibromination (57%) followed by catalytic tritiation of the dibromide. The resulting tritium labelled aniline (14% yield, specific activity 31.2 Ci/mmol) was transformed to the title compound in 13.3% yield and 99+% radiochemical purity by treatment with thiophosgene. (author).

  9. Mu Opioid Receptor Gene: New Point Mutations in Opioid Addicts

    Amin Dinarvand

    2014-02-01

    Full Text Available Introduction: Association between single-nucleotide polymorphisms (SNPs in mu opioid receptor gene and drug addiction has been shown in various studies. Here, we have evaluated the existence of polymorphisms in exon 3 of this gene in Iranian population and investigated the possible association between these mutations and opioid addiction.  Methods: 79 opioid-dependent subjects (55 males, 24 females and 134 non-addict or control individuals (74 males, 60 females participated in the study. Genomic DNA was extracted from volunteers’ peripheral blood and exon 3 of the mu opioid receptor gene was amplified by polymerase chain reaction (PCR whose products were then sequenced.  Results: Three different heterozygote polymorphisms were observed in 3 male individuals: 759T>C and 877G>A mutations were found in 2 control volunteers and 1043G>C substitution was observed in an opioid-addicted subject. Association between genotype and opioid addiction for each mutation was not statistically significant.  Discussion: It seems that the sample size used in our study is not enough to confirm or reject any association between 759T>C, 877G>A and 1043G>C substitutions in exon 3 of the mu opioid receptor gene and opioid addiction susceptibility in Iranian population.

  10. Biased Agonism of Endogenous Opioid Peptides at the μ-Opioid Receptor.

    Thompson, Georgina L; Lane, J Robert; Coudrat, Thomas; Sexton, Patrick M; Christopoulos, Arthur; Canals, Meritxell

    2015-08-01

    Biased agonism is having a major impact on modern drug discovery, and describes the ability of distinct G protein-coupled receptor (GPCR) ligands to activate different cell signaling pathways, and to result in different physiologic outcomes. To date, most studies of biased agonism have focused on synthetic molecules targeting various GPCRs; however, many of these receptors have multiple endogenous ligands, suggesting that "natural" bias may be an unappreciated feature of these GPCRs. The μ-opioid receptor (MOP) is activated by numerous endogenous opioid peptides, remains an attractive therapeutic target for the treatment of pain, and exhibits biased agonism in response to synthetic opiates. The aim of this study was to rigorously assess the potential for biased agonism in the actions of endogenous opioids at the MOP in a common cellular background, and compare these to the effects of the agonist d-Ala2-N-MePhe4-Gly-ol enkephalin (DAMGO). We investigated activation of G proteins, inhibition of cAMP production, extracellular signal-regulated kinase 1 and 2 phosphorylation, β-arrestin 1/2 recruitment, and MOP trafficking, and applied a novel analytical method to quantify biased agonism. Although many endogenous opioids displayed signaling profiles similar to that of DAMGO, α-neoendorphin, Met-enkephalin-Arg-Phe, and the putatively endogenous peptide endomorphin-1 displayed particularly distinct bias profiles. These may represent examples of natural bias if it can be shown that they have different signaling properties and physiologic effects in vivo compared with other endogenous opioids. Understanding how endogenous opioids control physiologic processes through biased agonism can reveal vital information required to enable the design of biased opioids with improved pharmacological profiles and treat diseases involving dysfunction of the endogenous opioid system. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

  11. Mu opioid receptor binding sites in human brain

    Pilapil, C.; Welner, S.; Magnan, J.; Zamir, N.; Quirion, R.

    1986-01-01

    Our experiments focused on the examination of the distribution of mu opioid receptor binding sites in normal human brain using the highly selective ligand [ 3 H]DAGO, in both membrane binding assay and in vitro receptor autoradiography. Mu opioid binding sites are very discretely distributed in human brain with high densities of sites found in the posterior amygdala, caudate, putamen, hypothalamus and certain cortical areas. Moreover the autoradiographic distribution of [ 3 H]DAGO binding sites clearly reveals the discrete lamination (layers I and III-IV) of mu sites in cortical areas

  12. Development of concepts on the interaction of drugs with opioid receptors

    Kuzmina, N E; Kuzmin, V S

    2011-02-28

    The development of concepts on the molecular mechanisms of the action of medicinal drugs on the opioid receptors is briefly surveyed. The modern point of view on the mechanism of activation of opioid receptors is given based on the data from chimeric and site-directed mutagenesis of the cloned opioid receptors and the computer-aided simulations of the reception zone and ligand-receptor complexes. Three-dimensional models of the opioid pharmacophore derived by both conventional methods and a comparative analysis of molecular fields are described in detail.

  13. Combined autoradiographic-immunocytochemical analysis of opioid receptors and opioid peptide neuronal systems in brain

    Lewis, M.E.; Khachaturian, H.; Watson, S.J.

    1985-01-01

    Using adjacent section autoradiography-immunocytochemistry, the distribution of (TH)naloxone binding sites was studied in relation to neuronal systems containing (Leu)enkephalin, dynorphin A, or beta-endorphin immunoreactivity in rat brain. Brain sections from formaldehyde-perfused rats show robust specific binding of (TH)naloxone, the pharmacological (mu-like) properties of which appear unaltered. In contrast, specific binding of the delta ligand (TH)D-Ala2,D-Leu5-enkephalin was virtually totally eliminated as a result of formaldehyde perfusion. Using adjacent section analysis, the authors have noted associations between (TH)naloxone binding sites and one, two, or all three opioid systems in different brain regions; however, in some areas, no apparent relationship could be observed. Within regions, the relationship was complex. The complexity of the association between (TH)naloxone binding sites and the multiple opioid systems, and previous reports of co-localization of mu and kappa receptors in rat brain, are inconsistent with a simple-one-to-one relationship between a given opioid precursor and opioid receptor subtype. Instead, since differential processing of the three precursors gives rise to peptides of varying receptor subtype potencies and selectivities, the multiple peptide-receptor relationships may point to a key role of post-translational processing in determining the physiological consequences of opioid neurotransmission.

  14. Sex differences in opioid analgesia and addiction: interactions among opioid receptors and estrogen receptors

    2013-01-01

    Opioids are widely used as the pain reliever and also notorious for being addictive drugs. Sex differences in the opioid analgesia and addiction have been reported and investigated in human subjects and animal models. Yet, the molecular mechanism underlying the differences between males and females is still unclear. Here, we reviewed the literature describing the sex differences in analgesic responses and addiction liabilities to clinically relevant opioids. The reported interactions among opioids, estrogens, opioid receptors, and estrogen receptors are also evaluated. We postulate that the sex differences partly originated from the crosstalk among the estrogen and opioid receptors when stimulated by the exogenous opioids, possibly through common secondary messengers and the downstream gene transcriptional regulators. PMID:24010861

  15. Quantitative immunolocalization of {mu} opioid receptors: regulation by naltrexone

    Evans, C.J.; Lam, H.; To, T.; Anton, B. [Department of Psychiatry and Biobehavioral Sciences, Neuropsychiatric Institute, University of California, Los Angeles, CA (United States); Unterwald, E.M. [Department of Psychiatry, New York University Medical Center, New York, NY (United States)

    1998-04-24

    observable change in the total number of receptors. Quantitative receptor immunodetection together with ligand autoradiography provides a new approach for investigating the regulation of {mu} opioid receptors on tissue sections. (Copyright (c) 1998 Elsevier Science B.V., Amsterdam. All rights reserved.)

  16. Quantitative immunolocalization of μ opioid receptors: regulation by naltrexone

    Evans, C.J.; Lam, H.; To, T.; Anton, B.; Unterwald, E.M.

    1998-01-01

    of receptors. Quantitative receptor immunodetection together with ligand autoradiography provides a new approach for investigating the regulation of μ opioid receptors on tissue sections. (Copyright (c) 1998 Elsevier Science B.V., Amsterdam. All rights reserved.)

  17. Novel multiple opioid ligands based on 4-aminobenzazepinone (Aba), azepinoindole (Aia) and tetrahydroisoquinoline (Tic) scaffolds

    Ballet, Steven; Marczak, Ewa D.; Feytens, Debby; Salvadori, Severo; Sasaki, Yusuke; Abell, Andrew D.; Lazarus, Lawrence H.; Balboni, Gianfranco; Tourwé, Dirk

    2010-01-01

    The dimerization and trimerization of the Dmt-Tic, Dmt-Aia and Dmt-Aba pharmacophores provided multiple ligands which were evaluated in vitro for opioid receptor binding and functional activity. Whereas the Tic- and Aba multimers proved to be dual and balanced δ/μ antagonists, as determined by the functional [S35]GTPγS binding assay, the dimerization of potent Aia-based ‘parent’ ligands unexpectedly resulted in substantial less efficient receptor binding and non-active dimeric compounds. PMID:20137938

  18. The role of opioid antagonist efficacy and constitutive opioid receptor activity in the opioid withdrawal syndrome in mice

    Navani, Dipesh M.; Sirohi, Sunil; Madia, Priyanka A.; Yoburn, Byron C.

    2011-01-01

    On the basis of efficacy, opioid antagonists are classified as inverse opioid agonists (e.g. naltrexone) or neutral opioid antagonists (e.g. 6β-naltrexol). This study examined the interaction between naltrexone and 6β-naltrexol in the precipitated opioid withdrawal syndrome in morphine dependent mice. Furthermore, the possible contribution of constitutive opioid receptor activity to precipitated withdrawal was evaluated using increasing levels of morphine dependence. In the first experiment, ...

  19. Synthesis of opioid receptor imaging agent 7α-o-IA-DPN

    Wang Rongfu

    1997-01-01

    A new opioid receptor imaging agent is designed and synthesized. 7α-o-iodoallyl diprenorphine (7α-o-IA-DPN) was obtained in one step by radioiododestannylation, which involved in the selection of DPN as an opioid antagonist, the regioselective protection of the DPN phenol tertiary-OH using acetylation and the introduction of vinylstannane as prosthetic group into the tertiary alcohol group position in the 7α-side chain. The iodinated DPN derivation was possessed of high radiolabeled yield (>90%) with 80 TBq/mmol specific radioactivity and more than 95% radiochemical purity. In vitro opioid receptor binding analysis showed very high affinity (Ki = 0.4 nmol/L). This new radioiodinated opioid ligand is suitable for SPECT study of opioid receptor imaging

  20. Novel approaches for the treatment of psychostimulant and opioid abuse - focus on opioid receptor-based therapies.

    Bailey, Chris P; Husbands, Stephen M

    2014-11-01

    Psychostimulant and opioid addiction are poorly treated. The majority of abstinent users relapse back to drug-taking within a year of abstinence, making 'anti-relapse' therapies the focus of much current research. There are two fundamental challenges to developing novel treatments for drug addiction. First, there are three key stimuli that precipitate relapse back to drug-taking: stress, presentation of drug-conditioned cue, taking a small dose of drug. The most successful novel treatment would be effective against all three stimuli. Second, a large number of drug users are poly-drug users: taking more than one drug of abuse at a time. The ideal anti-addiction treatment would, therefore, be effective against all classes of drugs of abuse. In this review, the authors discuss the clinical need and animal models used to uncover potential novel treatments. There is a very broad range of potential treatment approaches and targets currently being examined as potential anti-relapse therapies. These broadly fit into two categories: 'memory-based' and 'receptor-based' and the authors discuss the key targets here within. Opioid receptors and ligands have been widely studied, and research into how different opioid subtypes affect behaviours related to addiction (reward, dysphoria, motivation) suggests that they are tractable targets as anti-relapse treatments. Regarding opioid ligands as novel 'anti-relapse' medication targets, research suggests that a 'non-selective' approach to targeting opioid receptors will be the most effective.

  1. Functional characteristics of the naked mole rat μ-opioid receptor.

    Melanie Busch-Dienstfertig

    Full Text Available While humans and most animals respond to µ-opioid receptor (MOR agonists with analgesia and decreased aggression, in the naked mole rat (NMR opioids induce hyperalgesia and severe aggression. Single nucleotide polymorphisms in the human mu-opioid receptor gene (OPRM1 can underlie altered behavioral responses to opioids. Therefore, we hypothesized that the primary structure of the NMR MOR may differ from other species. Sequencing of the NMR oprm1 revealed strong homology to other mammals, but exposed three unique amino acids that might affect receptor-ligand interactions. The NMR and rat oprm1 sequences were cloned into mammalian expression vectors and transfected into HEK293 cells. Radioligand binding and 3'-5'-cyclic adenosine monophosphate (cAMP enzyme immunoassays were used to compare opioid binding and opioid-mediated cAMP inhibition. At normalized opioid receptor protein levels we detected significantly lower [3H]DAMGO binding to NMR compared to rat MOR, but no significant difference in DAMGO-induced cAMP inhibition. Strong DAMGO-induced MOR internalization was detectable using radioligand binding and confocal imaging in HEK293 cells expressing rat or NMR receptor, while morphine showed weak or no effects. In summary, we found minor functional differences between rat and NMR MOR suggesting that other differences e.g. in anatomical distribution of MOR underlie the NMR's extreme reaction to opioids.

  2. Does the kappa opioid receptor system contribute to pain aversion?

    Catherine M Cahill

    2014-11-01

    Full Text Available The kappa opioid receptor (KOR and the endogenous peptide-ligand dynorphin have received significant attention due the involvement in mediating a variety of behavioral and neurophysiological responses, including opposing the rewarding properties of drugs of abuse including opioids. Accumulating evidence indicates this system is involved in regulating states of motivation and emotion. Acute activation of the KOR produces an increase in motivational behavior to escape a threat, however, KOR activation associated with chronic stress leads to the expression of symptoms indicative of mood disorders. It is well accepted that KOR can produce analgesia and is engaged in chronic pain states including neuropathic pain. Spinal studies have revealed KOR-induced analgesia in reversing pain hypersensitivities associated with peripheral nerve injury. While systemic administration of KOR agonists attenuates nociceptive sensory transmission, this effect appears to be a stress-induced effect as anxiolytic agents, including delta opioid receptor agonists, mitigate KOR agonist-induced analgesia. Additionally, while the role of KOR and dynorphin in driving the dysphoric and aversive components of stress and drug withdrawal has been well characterized, how this system mediates the negative emotional states associated with chronic pain is relatively unexplored. This review provides evidence that dynorphin and the KOR system contribute to the negative affective component of pain and that this receptor system likely contributes to the high comorbidity of mood disorders associated with chronic neuropathic pain.

  3. Sigma and opioid receptors in human brain tumors

    Thomas, G.E.; Szuecs, M.; Mamone, J.Y.; Bem, W.T.; Rush, M.D.; Johnson, F.E.; Coscia, C.J.

    1990-01-01

    Human brain tumors and nude mouse-borne human neuroblastomas and gliomas were analyzed for sigma and opioid receptor content. Sigma binding was assessed using [ 3 H] 1, 3-di-o-tolylguanidine (DTG), whereas opioid receptor subtypes were measured with tritiated forms of the following: μ, [D-ala 2 , mePhe 4 , gly-ol 5 ] enkephalin (DAMGE); κ, ethylketocyclazocine (EKC) or U69,593; δ, [D-pen 2 , D-pen 5 ] enkephalin (DPDPE) or [D-ala 2 , D-leu 5 ] enkephalin (DADLE) with μ suppressor present. Binding parameters were estimated by homologous displacement assays followed by analysis using the LIGAND program. Sigma binding was detected in 15 of 16 tumors examined with very high levels found in a brain metastasis from an adenocarcinoma of lung and a human neuroblastoma (SK-N-MC) passaged in nude mice. κ opioid receptor binding was detected in 4 of 4 glioblastoma multiforme specimens and 2 of 2 human astrocytoma cell lines tested but not in the other brain tumors analyzed

  4. Sigma and opioid receptors in human brain tumors

    Thomas, G.E.; Szuecs, M.; Mamone, J.Y.; Bem, W.T.; Rush, M.D.; Johnson, F.E.; Coscia, C.J. (St. Louis Univ. School of Medicine, MO (USA))

    1990-01-01

    Human brain tumors and nude mouse-borne human neuroblastomas and gliomas were analyzed for sigma and opioid receptor content. Sigma binding was assessed using ({sup 3}H) 1, 3-di-o-tolylguanidine (DTG), whereas opioid receptor subtypes were measured with tritiated forms of the following: {mu}, (D-ala{sup 2}, mePhe{sup 4}, gly-ol{sup 5}) enkephalin (DAMGE); {kappa}, ethylketocyclazocine (EKC) or U69,593; {delta}, (D-pen{sup 2}, D-pen{sup 5}) enkephalin (DPDPE) or (D-ala{sup 2}, D-leu{sup 5}) enkephalin (DADLE) with {mu} suppressor present. Binding parameters were estimated by homologous displacement assays followed by analysis using the LIGAND program. Sigma binding was detected in 15 of 16 tumors examined with very high levels found in a brain metastasis from an adenocarcinoma of lung and a human neuroblastoma (SK-N-MC) passaged in nude mice. {kappa} opioid receptor binding was detected in 4 of 4 glioblastoma multiforme specimens and 2 of 2 human astrocytoma cell lines tested but not in the other brain tumors analyzed.

  5. Radioiodinated ligands for dopamine receptors

    Kung, H.F.

    1994-01-01

    The dopamine receptor system is important for normal brain function; it is also the apparent action site for various neuroleptic drugs for the treatment of schizophrenia and other metal disorders. In the past few years radioiodinated ligands for single photon emission tomography (SPECT) have been successfully developed and tested in humans: [ 123 I]TISCH for D1 dopamine receptors; [ 123 I]IBZM, epidepride, IBF and FIDA2, four iodobenzamide derivatives, for D2/D3 dopamine receptors. In addition, [ 123 I]β-CIT (RTI-55) and IPT, cocaine derivatives, for the dopamine reuptake site are potentially useful for diagnosis of loss of dopamine neurons. The first iodinated ligand, (R)trans-7-OH-PIPAT, for D3 dopamine receptors, was synthesized and characterized with cloned cell lines (Spodoptera frugiperda, Sf9) expressing the D2 and D3 dopamine receptors and with rat basal forebrain membrane preparations. Most of the known iodobenzamides displayed similar potency in binding to both D2 and D3 dopamine receptors expressed in the cell lines. Initial studies appear to suggest that by fine tuning the structures it may be possible to develop agents specific for D2 and D3 dopamine receptors. It is important to investigate D2/D3 selectivity for this series of potent ligands

  6. Receptor binding properties and antinociceptive effects of chimeric peptides consisting of a micro-opioid receptor agonist and an ORL1 receptor antagonist.

    Kawano, Susumu; Ito, Risa; Nishiyama, Miharu; Kubo, Mai; Matsushima, Tomoko; Minamisawa, Motoko; Ambo, Akihiro; Sasaki, Yusuke

    2007-07-01

    Receptor binding properties and antinociceptive activities of chimeric peptides linked by spacers were investigated. The peptides consisted of the micro-opioid receptor ligand dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH(2)) or its analog YRFB (Tyr-D-Arg-Phe-betaAla-NH(2)) linked to the ORL1 receptor ligand Ac-Arg-Tyr-Tyr-Arg-Ile-Lys-NH(2) (Ac-RYYRIK-NH(2)). All chimeric peptides were found to possess high receptor binding affinities for both micro-opioid and ORL1 receptors in mouse brain membranes although their binding affinities for both receptors in spinal membranes were significantly lower. Among them, chimeric peptide 2, which consists of dermorphin and Ac-RYYRIK-NH(2) connected by a long spacer, had the highest binding affinity towards both receptors. In the tail-flick test following intrathecal (i.t.) administration to mice, all chimeric peptides showed potent and dose-dependent antinociceptive activities with an ED(50) of 1.34-4.51 (pmol/mouse), nearly comparable to dermorphin alone (ED(50); 1.08 pmol/mouse). In contrast to their micro-opioid receptor binding profiles, intracerebroventricular (i.c.v.) administration of the chimeric peptides resulted in much less potent antinociceptive activity (ED(50) 5.55-100peptides, and the regulation of mu-opioid receptor-mediated antinociception in brain. The present chimeric peptides may be useful as pharmacological tools for studies on micro-opioid receptor/ORL1 receptor heterodimers.

  7. PET imaging of human cardiac opioid receptors

    Villemagne, Patricia S.R.; Dannals, Robert F. [Department of Radiology, The Johns Hopkins University School of Medicine, 605 N Caroline St., Baltimore, Maryland (United States); Department of Environmental Health Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland (United States); Ravert, Hayden T. [Department of Radiology, The Johns Hopkins University School of Medicine, 605 N Caroline St., Baltimore, Maryland (United States); Frost, James J. [Department of Radiology, The Johns Hopkins University School of Medicine, 605 N Caroline St., Baltimore, Maryland (United States); Department of Environmental Health Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland (United States); Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, Maryland (United States)

    2002-10-01

    The presence of opioid peptides and receptors and their role in the regulation of cardiovascular function has been previously demonstrated in the mammalian heart. The aim of this study was to image {mu} and {delta} opioid receptors in the human heart using positron emission tomography (PET). Five subjects (three females, two males, 65{+-}8 years old) underwent PET scanning of the chest with [{sup 11}C]carfentanil ([{sup 11}C]CFN) and [{sup 11}C]-N-methyl-naltrindole ([{sup 11}C]MeNTI) and the images were analyzed for evidence of opioid receptor binding in the heart. Either [{sup 11}C]CFN or [{sup 11}C]MeNTI (20 mCi) was injected i.v. with subsequent dynamic acquisitions over 90 min. For the blocking studies, either 0.2 mg/kg or 1 mg/kg of naloxone was injected i.v. 5 min prior to the injection of [{sup 11}C]CFN and [{sup 11}C]MeNTI, respectively. Regions of interest were placed over the left ventricle, left ventricular chamber, lung and skeletal muscle. Graphical analysis demonstrated average baseline myocardial binding potentials (BP) of 4.37{+-}0.91 with [{sup 11}C]CFN and 3.86{+-}0.60 with [{sup 11}C]MeNTI. Administration of 0.2 mg/kg naloxone prior to [{sup 11}C]CFN produced a 25% reduction in BP in one subject in comparison with baseline values, and a 19% decrease in myocardial distribution volume (DV). Administration of 1 mg/kg of naloxone before [{sup 11}C]MeNTI in another subject produced a 14% decrease in BP and a 21% decrease in the myocardial DV. These results demonstrate the ability to image these receptors in vivo by PET. PET imaging of cardiac opioid receptors may help to better understand their role in cardiovascular pathophysiology and the effect of abuse of opioids and drugs on heart function. (orig.)

  8. Opioid and nicotine receptors affect growth regulation of human lung cancer cell lines

    Maneckjee, R.; Minna, J.D.

    1990-01-01

    Using specific radioactively-labeled ligands, the authors find that lung cancer cell lines of diverse histologic types express multiple, high-affinity membrane receptors for μ, δ, and κ opioid agonists and for nicotine and α-bungarotoxin. These receptors are biologically active because cAMP levels decreased in lung cancer cells after opioid and nicotine application. Nicotine at concentrations found in the blood of smokers had no effect on in vitro lung cancer cell growth, whereas μ, δ, and κ opioid agonists at low concentrations inhibited lung cancer growth in vitro. They also found that lung cancer cells expressed various combinations of immunoreactive opioid peptides (β-endorphin, enkephalin, or dynorphin), suggesting the participation of opioids in a negative autocrine loop or tumor-suppressing system. Due to the almost universal exposure of patients with lung cancer to nicotine, they tested whether nicotine affected the response of lung cancer cell growth to opioids and found that nicotine at concentrations of 100-200 nM partially or totally reversed opioid-induced growth inhibition in 9/14 lung cancer cell lines. These in vitro results for lung cancer cells suggest that opioids could function as part of a tumor suppressor system and that nicotine can function to circumvent this system in the pathogenesis of lung cancer

  9. Opioid and nicotine receptors affect growth regulation of human lung cancer cell lines

    Maneckjee, R.; Minna, J.D. (National Cancer Institute-Navy Medical Oncology Branch, Bethesda, MD (USA) Uniformed Services Univ. of the Health Sciences, Bethesda, MD (USA))

    1990-05-01

    Using specific radioactively-labeled ligands, the authors find that lung cancer cell lines of diverse histologic types express multiple, high-affinity membrane receptors for {mu}, {delta}, and {kappa} opioid agonists and for nicotine and {alpha}-bungarotoxin. These receptors are biologically active because cAMP levels decreased in lung cancer cells after opioid and nicotine application. Nicotine at concentrations found in the blood of smokers had no effect on in vitro lung cancer cell growth, whereas {mu}, {delta}, and {kappa} opioid agonists at low concentrations inhibited lung cancer growth in vitro. They also found that lung cancer cells expressed various combinations of immunoreactive opioid peptides ({beta}-endorphin, enkephalin, or dynorphin), suggesting the participation of opioids in a negative autocrine loop or tumor-suppressing system. Due to the almost universal exposure of patients with lung cancer to nicotine, they tested whether nicotine affected the response of lung cancer cell growth to opioids and found that nicotine at concentrations of 100-200 nM partially or totally reversed opioid-induced growth inhibition in 9/14 lung cancer cell lines. These in vitro results for lung cancer cells suggest that opioids could function as part of a tumor suppressor system and that nicotine can function to circumvent this system in the pathogenesis of lung cancer.

  10. Labeled receptor ligands for spect

    Kung, H.F.

    1989-01-01

    Receptor specific imaging agents for single photon emission computed tomography (SPECT) can potentially be useful in the understanding of basic biochemistry and pharmacology of receptors. SPECT images may also provide tools for evaluation of density and binding kinetics of a specific receptor, information important for diagnosis and patient management. Basic requirements for receptor imaging agents are: (a) they are labeled with short-lived isotopes, (b) they show high selectivity and specific uptake, (c) they exhibit high target/background ratio, and (d) they can be modeled to obtain quantitative information. Several good examples of CNS receptor specific ligands labeled with I-123 have been developed, including iodoQNB, iodoestrogen iodobenzadiazepine, iodobenazepine, iodobenzamides for muscarinic, estrogen benzadiazepine, D-1 and D-2 dopamine receptors. With the advent of newer and faster SPECT imaging devices, it may be feasible to quantitate the receptor density by in vivo imaging techniques. These new brain imaging agents can provide unique diagnostic information, which may not be available through other imaging modalities, such as CT and MRI

  11. Opioid receptor desensitization: mechanisms and its link to tolerance

    Stéphane eAllouche

    2014-12-01

    Full Text Available Opioid receptors are part of the class A of G-protein coupled receptors and the target of the opiates, the most powerful analgesic molecules used in clinic. During a protracted use, a tolerance to analgesic effect develops resulting in a reduction of the effectiveness. So understanding mechanisms of tolerance is a great challenge and may help to find new strategies to tackle this side effect. This review will summarize receptor-related mechanisms that could underlie tolerance especially receptor desensitization. We will focus on the latest data obtained on molecular mechanisms involved in opioid receptor desensitization: phosphorylation, receptor uncoupling, internalization and post-endocytic fate of the receptor.

  12. Sigma opioid receptor: characterization and co-identity with the phencyclidine receptor

    Mendelsohn, L.G.; Kalra, V.; Johnson, B.G.; Kerchner, G.A.

    1985-01-01

    The properties of the sigma opioid receptor of rat brain cortex have been characterized using the prototypic ligand (+)-[ 3 H] SKF 10,047. Binding to this receptor was rapid, and equilibrium was obtained within 30 min at 37 degrees C. Specific binding was linear with protein concentration up to 500 micrograms/2 ml and was dependent upon protein integrity. Denaturation by boiling destroyed over 95% of the specific binding. A high-affinity binding site with a KD of 150 +/- 40 nM and a maximum binding of 2.91 +/- 0.84 pmol/mg of protein was determined from a Scatchard plot of the binding data. The addition of salt, either NaCl or CaCl 2 , to the buffers markedly decreased binding, with CaCl 2 being more potent than NaCl. A broad pH optimum for specific binding was observed; maximum binding was at pH 9.0. The affinity of a number of ligands for the sigma site and the phencyclidine receptor were compared. The binding (IC50) of 13 ligands to the sigma site showed a correlation of 0.86 (P less than .01) with binding to the phencyclidine site. The data demonstrate that the biochemical properties of the sigma and phencyclidine receptors are similar and support the view that these receptors are one and the same site

  13. Fluorescent ligands for studying neuropeptide receptors by confocal microscopy

    A. Beaudet

    1998-11-01

    Full Text Available This paper reviews the use of confocal microscopy as it pertains to the identification of G-protein coupled receptors and the study of their dynamic properties in cell cultures and in mammalian brain following their tagging with specific fluorescent ligands. Principles that should guide the choice of suitable ligands and fluorophores are discussed. Examples are provided from the work carried out in the authors' laboratory using custom synthetized fluoresceinylated or BODIPY-tagged bioactive peptides. The results show that confocal microscopic detection of specifically bound fluorescent ligands permits high resolution appraisal of neuropeptide receptor distribution both in cell culture and in brain sections. Within the framework of time course experiments, it also allows for a dynamic assessment of the internalization and subsequent intracellular trafficking of bound fluorescent molecules. Thus, it was found that neurotensin, somatostatin and mu- and delta-selective opioid peptides are internalized in a receptor-dependent fashion and according to receptor-specific patterns into their target cells. In the case of neurotensin, this internalization process was found to be clathrin-mediated, to proceed through classical endosomal pathways and, in neurons, to result in a mobilization of newly formed endosomes from neural processes to nerve cell bodies and from the periphery of cell bodies towards the perinuclear zone. These mechanisms are likely to play an important role for ligand inactivation, receptor regulation and perhaps also transmembrane signaling.

  14. Chronic ethanol consumption in rats produces opioid antinociceptive tolerance through inhibition of mu opioid receptor endocytosis.

    Li He

    Full Text Available It is well known that the mu-opioid receptor (MOR plays an important role in the rewarding properties of ethanol. However, it is less clear how chronic ethanol consumption affects MOR signaling. Here, we demonstrate that rats with prolonged voluntary ethanol consumption develop antinociceptive tolerance to opioids. Signaling through the MOR is controlled at many levels, including via the process of endocytosis. Importantly, agonists at the MOR that promote receptor endocytosis, such as the endogenous peptides enkephalin and β-endorphin, show a reduced propensity to promote antinociceptive tolerance than do agonists, like morphine, which do not promote receptor endocytosis. These observations led us to examine whether chronic ethanol consumption produced opioid tolerance by interfering with MOR endocytosis. Indeed, here we show that chronic ethanol consumption inhibits the endocytosis of MOR in response to opioid peptide. This loss of endocytosis was accompanied by a dramatic decrease in G protein coupled receptor kinase 2 (GRK2 protein levels after chronic drinking, suggesting that loss of this component of the trafficking machinery could be a mechanism by which endocytosis is lost. We also found that MOR coupling to G-protein was decreased in ethanol-drinking rats, providing a functional explanation for loss of opioid antinociception. Together, these results suggest that chronic ethanol drinking alters the ability of MOR to endocytose in response to opioid peptides, and consequently, promotes tolerance to the effects of opioids.

  15. An investigation into the receptor-regulating effects of the acute administration of opioid agonists and an antagonist on beta adrenergic receptors in the rat cerebral cortex

    Roper, I.

    1987-01-01

    Past and current research indicated that biochemical deviations which might be involved in the etiology and pathophysiology of depression, included abnormalities or imbalances in the noradrenergic, serotonergic, hormonal and possibly in the endogenous opioid, dopaminergic, histaminergic, cholinergic and trace amine systems. In order to investigate a possible link between the noradrenergic system and opioids, it was decided to test the acute effects of opioid administration on cortical beta adrenoceptor numbers and affinity. As these receptors have been most consistently downregulated by antidepressant treatment, they may be involved in the mechanism of antidepressant action of these agents. It was decided to investigate beta adrenoceptor-regulatory effects of opioid treatment. Naloxone was tested alone, with a view to suppressing any possible endogenous opioid influences upon beta receptor status and revealing an effect which would possibly be the opposite of that brought about by the administration of opioid agonists. Naloxone was administered together with morphine to demonstrate that any beta receptor up- or downregulation which might be measured, had indeed been opioid-receptor mediated. It was found that the acute administration of four different mu opioid agonists, naloxone and naloxone plus morphine, did not cause any statistically significant alterations in cortical beta adrenergic receptor numbers or affinity in the rat. A radioactive ligand, the beta adrenoceptor-labelling compound referred to as DHA (L-dihydroalprenolol HCI) was used in this study

  16. Interaction of trimebutine and Jo-1196 (fedotozine) with opioid receptors in the canine ileum

    Allescher, H.D.; Ahmad, S.; Classen, M.; Daniel, E.E. (Technical Univ., Munich, (West Germany))

    1991-05-01

    Receptor binding of the opioid receptor antagonist, ({sup 3}H)diprenorphine, which has a similar affinity to the various opioid receptor subtypes, was characterized in subcellular fractions derived from either longitudinal or circular smooth muscle of the canine small intestine with their plexuses (myenteric plexus and deep muscular plexus, respectively) attached. The distribution of opioid binding activity showed a good correlation in the different fractions with the binding of the neuronal marker ({sup 3}H)saxitoxin but no correlation to the smooth muscle plasma membrane marker 5'-nucleotidase. The saturation data (Kd = 0.12 +/- 0.04 nM and maximum binding = 400 +/- 20 fmol/mg) and the data from kinetic experiments (Kd = 0.08 nmol) in the myenteric plexus were in good agreement with results obtained previously from the circular muscle/deep muscular plexus preparation. Competition experiments using selective drugs for mu (morphiceptin-analog (N-MePhe3-D-Pro4)-morphiceptin), delta (D-Pen2,5-enkephalin) and kappa (dynorphin 1-13, U50488-H) ligands showed the existence of all three receptor subtypes. The existence of kappa receptors was confirmed in saturation experiments using ({sup 3}H) ethylketocycloazocine as labeled ligand. Two putative opioid agonists, with effects on gastrointestinal motility, trimebutine and JO-1196 (fedotozin), were also examined. Trimebutine (Ki = 0.18 microM), Des-Met-trimebutine (Ki = 0.72 microM) and Jo-1196 (Ki = 0.19 microM) displaced specific opiate binding. The relative affinity for the opioid receptor subtypes was mu = 0.44, delta = 0.30 and kappa = 0.26 for trimebutine and mu = 0.25, delta = 0.22 and kappa = 0.52 for Jo-1196.

  17. Opioid regulation of mu receptor internalisation: relevance to the development of tolerance and dependence.

    Lopez-Gimenez, Juan F; Milligan, Graeme

    2010-11-01

    Internalisation of the mu opioid receptor from the surface of cells is generally achieved by receptor occupancy with agonist ligands of high efficacy. However, in many situations the potent analgesic morphine fails to promote internalisation effectively and whether there is a direct link between this and the propensity for the sustained use of morphine to result in both tolerance and dependence has been studied intensely. Although frequently described as a partial agonist, this characteristic appears insufficient to explain the poor capacity of morphine to promote internalisation of the mu opioid receptor. Experiments performed using both transfected cell systems and ex vivo/in vivo models have provided evidence that when morphine can promote internalisation of the mu receptor there is a decrease in the development of tolerance and dependence. Although aspects of this model are controversial, such observations suggest a number of approaches to further enhance the use of morphine as an analgesic.

  18. Delta-opioid receptor analgesia is independent of microglial activation in a rat model of neuropathic pain.

    Joanna Mika

    Full Text Available The analgesic effect of delta-opioid receptor (DOR ligands in neuropathic pain is not diminished in contrast to other opioid receptor ligands, which lose their effectiveness as analgesics. In this study, we examine whether this effect is related to nerve injury-induced microglial activation. We therefore investigated the influence of minocycline-induced inhibition of microglial activation on the analgesic effects of opioid receptor agonists: morphine, DAMGO, U50,488H, DPDPE, Deltorphin II and SNC80 after chronic constriction injury (CCI to the sciatic nerve in rats. Pre-emptive and repeated administration of minocycline (30 mg/kg, i.p. over 7 days significantly reduced allodynia and hyperalgesia as measured on day 7 after CCI. The antiallodynic and antihyperalgesic effects of intrathecally (i.t. administered morphine (10-20 µg, DAMGO (1-2 µg and U50,488H (25-50 µg were significantly potentiated in rats after minocycline, but no such changes were observed after DPDPE (10-20 µg, deltorphin II (1.5-15 µg and SNC80 (10-20 µg administration. Additionally, nerve injury-induced down-regulation of all types of opioid receptors in the spinal cord and dorsal root ganglia was not influenced by minocycline, which indicates that the effects of opioid ligands are dependent on other changes, presumably neuroimmune interactions. Our study of rat primary microglial cell culture using qRT-PCR, Western blotting and immunocytochemistry confirmed the presence of mu-opioid receptors (MOR and kappa-opioid receptors (KOR, further we provide the first evidence for the lack of DOR on microglial cells. In summary, DOR analgesia is different from analgesia induced by MOR and KOR receptors because it does not dependent on injury-induced microglial activation. DOR agonists appear to be the best candidates for new drugs to treat neuropathic pain.

  19. Sex Differences in Kappa Opioid Receptor Function and Their Potential Impact on Addiction

    Chartoff, Elena H.; Mavrikaki, Maria

    2015-01-01

    Behavioral, biological, and social sequelae that lead to drug addiction differ between men and women. Our efforts to understand addiction on a mechanistic level must include studies in both males and females. Stress, anxiety, and depression are tightly linked to addiction, and whether they precede or result from compulsive drug use depends on many factors, including biological sex. The neuropeptide dynorphin (DYN), an endogenous ligand at kappa opioid receptors (KORs), is necessary for stress...

  20. [11C]-MeJDTic: a novel radioligand for κ-opioid receptor positron emission tomography imaging

    Poisnel, Geraldine; Oueslati, Farhana; Dhilly, Martine; Delamare, Jerome; Perrio, Cecile; Debruyne, Daniele; Barre, Louisa

    2008-01-01

    Introduction: Radiopharmaceuticals that can bind selectively the κ-opioid receptor may present opportunities for staging clinical brain disorders and evaluating the efficiency of new therapies related to stroke, neurodegenerative diseases or opiate addiction. The N-methylated derivative of JDTic (named MeJDTic), which has been recently described as a potent and selective antagonist of κ-opioid receptor in vitro, was labeled with carbon-11 and evaluated for in vivo imaging the κ-opioid receptor in mice. Methods: [ 11 C]-MeJDTic was prepared by methylation of JDTic with [ 11 C]-methyl triflate. The binding of [ 11 C]-MeJDTic to κ-opioid receptor was investigated ex vivo by biodistribution and competition studies using nonfasted male CD1 mice. Results: [ 11 C]-MeJDTic exhibited a high and rapid distribution in peripheral organs. The uptake was maximal in lung where the κ receptor is largely expressed. [ 11 C]-MeJDTic rapidly crossed the blood-brain barrier and accumulated in the brain regions of interest (hypothalamus). The parent ligand remained the major radioactive compound in brain during the experiment. Chase studies with U50,488 (a κ referring agonist), morphine (a μ agonist) and naltrindole (a δ antagonist) demonstrated that this uptake was the result of specific binding to the κ-opioid receptor. Conclusion: These findings suggested that [ 11 C]-MeJDTic appeared to be a promising selective 'lead' radioligand for κ-opioid receptor PET imaging

  1. Characteristic molecular vibrations of adenosine receptor ligands.

    Chee, Hyun Keun; Yang, Jin-San; Joung, Je-Gun; Zhang, Byoung-Tak; Oh, S June

    2015-02-13

    Although the regulation of membrane receptor activation is known to be crucial for molecular signal transduction, the molecular mechanism underlying receptor activation is not fully elucidated. Here we study the physicochemical nature of membrane receptor behavior by investigating the characteristic molecular vibrations of receptor ligands using computational chemistry and informatics methods. By using information gain, t-tests, and support vector machines, we have identified highly informative features of adenosine receptor (AdoR) ligand and corresponding functional amino acid residues such as Asn (6.55) of AdoR that has informative significance and is indispensable for ligand recognition of AdoRs. These findings may provide new perspectives and insights into the fundamental mechanism of class A G protein-coupled receptor activation. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  2. Internalisation of the mu-opioid receptor by endomorphin-1 and leu-enkephalin is dependant on aromatic amino acid residues.

    Del Borgo, Mark P; Blanchfield, Joanne T; Toth, Istvan

    2008-04-15

    The opioid receptor system in the central nervous system controls a number of physiological processes, most notably pain. However, most opioids currently available have a variety of side-effects as well as exhibiting tolerance. Tolerance is most likely to be a complex phenomenon, however, the role of receptor internalisation is thought to play a crucial role. In this study, we examined the role of aromaticity in ligand-mediated receptor internalisation of the mu-opioid receptor (MOPR). These studies show that the amount of receptor internalisation may be dependant on the amphiphilicity of the ligand. Specifically, deletion of the C-terminus aromatic residues of endomorphin 1, particularly tryptophan reduces receptor-mediated internalisation whilst the addition of tryptophan within the enkephalin sequence increases receptor internalisation and decreases tolerance.

  3. Functional μ-Opioid-Galanin Receptor Heteromers in the Ventral Tegmental Area.

    Moreno, Estefanía; Quiroz, César; Rea, William; Cai, Ning-Sheng; Mallol, Josefa; Cortés, Antoni; Lluís, Carme; Canela, Enric I; Casadó, Vicent; Ferré, Sergi

    2017-02-01

    The neuropeptide galanin has been shown to interact with the opioid system. More specifically, galanin counteracts the behavioral effects of the systemic administration of μ-opioid receptor (MOR) agonists. Yet the mechanism responsible for this galanin-opioid interaction has remained elusive. Using biophysical techniques in mammalian transfected cells, we found evidence for selective heteromerization of MOR and the galanin receptor subtype Gal1 (Gal1R). Also in transfected cells, a synthetic peptide selectively disrupted MOR-Gal1R heteromerization as well as specific interactions between MOR and Gal1R ligands: a negative cross talk, by which galanin counteracted MAPK activation induced by the endogenous MOR agonist endomorphin-1, and a cross-antagonism, by which a MOR antagonist counteracted MAPK activation induced by galanin. These specific interactions, which represented biochemical properties of the MOR-Gal1R heteromer, could then be identified in situ in slices of rat ventral tegmental area (VTA) with MAPK activation and two additional cell signaling pathways, AKT and CREB phosphorylation. Furthermore, in vivo microdialysis experiments showed that the disruptive peptide selectively counteracted the ability of galanin to block the dendritic dopamine release in the rat VTA induced by local infusion of endomorphin-1, demonstrating a key role of MOR-Gal1R heteromers localized in the VTA in the direct control of dopamine cell function and their ability to mediate antagonistic interactions between MOR and Gal1R ligands. The results also indicate that MOR-Gal1R heteromers should be viewed as targets for the treatment of opioid use disorders. The μ-opioid receptor (MOR) localized in the ventral tegmental area (VTA) plays a key role in the reinforcing and addictive properties of opioids. With parallel in vitro experiments in mammalian transfected cells and in situ and in vivo experiments in rat VTA, we demonstrate that a significant population of these MORs form

  4. Ligand-receptor Interactions by NMR Spectroscopy

    Novak. P.

    2008-04-01

    Full Text Available Today NMR spectroscopy is a method of choice for elucidation of interactions between biomolecules and the potential ligands. Knowledge on these interactions is an essential prerequisite for the rational drug design. The most important contribution of NMR to drug design a few years ago was the 3D structure determination of proteins. Besides delivering the 3D structures of the free proteins as a raw material for the modeling studies on ligand binding, NMR can directly yield valuable experimental data on the biologically important protein-ligand complexes. In addition to X-ray diffraction, NMR spectroscopy can provide information on the internal protein dynamics ordynamics of intermolecular interactions. Changes in NMR parameters allow us to detect ("SAR by NMR" and quantitatively determine binding affinities (titration, diffusion NMR experiments, etc. of potential ligands. Also, it is possible to determine the binding site and conformations of ligands, receptors and receptor-ligand complexes with the help of NMR methods such as tr-NOESY. Epitopes or functional groups responsible for binding of ligands to the receptor can be identified by employing STD or WaterLOGSY experiments. In this review are described some of the most frequent NMR methods for the characterization of the interactions between biomolecules and ligands, together with their advantages and disadvantages.

  5. Site-directed alkylation of multiple opioid receptors. I. Binding selectivity

    James, I.F.; Goldstein, A.

    1984-01-01

    A method for measuring and expressing the binding selectivity of ligands for mu, delta, and kappa opioid binding sites is reported. Radioligands are used that are partially selective for these sites in combination with membrane preparations enriched in each site. Enrichment was obtained by treatment of membranes with the alkylating agent beta-chlornaltrexamine in the presence of appropriate protecting ligands. After enrichment for mu receptors, [ 3 H] dihydromorphine bound to a single type of site as judged by the slope of competition binding curves. After enrichment for delta or kappa receptors, binding sites for [ 3 H] [D-Ala2, D-Leu5]enkephalin and [3H]ethylketocyclazocine, respectively, were still not homogeneous. There were residual mu sites in delta-enriched membranes but no evidence for residual mu or delta sites in kappa-enriched membranes were found. This method was used to identify ligands that are highly selective for each of the three types of sites

  6. GRK2 Constitutively Governs Peripheral Delta Opioid Receptor Activity

    Allison Doyle Brackley

    2016-09-01

    Full Text Available Opioids remain the standard for analgesic care; however, adverse effects of systemic treatments contraindicate long-term administration. While most clinical opioids target mu opioid receptors (MOR, those that target the delta class (DOR also demonstrate analgesic efficacy. Furthermore, peripherally restrictive opioids represent an attractive direction for analgesia. However, opioid receptors including DOR are analgesically incompetent in the absence of inflammation. Here, we report that G protein-coupled receptor kinase 2 (GRK2 naively associates with plasma membrane DOR in peripheral sensory neurons to inhibit analgesic agonist efficacy. This interaction prevents optimal Gβ subunit association with the receptor, thereby reducing DOR activity. Importantly, bradykinin stimulates GRK2 movement away from DOR and onto Raf kinase inhibitory protein (RKIP. protein kinase C (PKC-dependent RKIP phosphorylation induces GRK2 sequestration, restoring DOR functionality in sensory neurons. Together, these results expand the known function of GRK2, identifying a non-internalizing role to maintain peripheral DOR in an analgesically incompetent state.

  7. Methamphetamine-induced changes in the striatal dopamine pathway in μ-opioid receptor knockout mice

    Park Sang Won

    2011-11-01

    Full Text Available Abstract Background Repeated exposure to methamphetamine (METH can cause not only neurotoxicity but also addiction. Behavioral sensitization is widely used as an animal model for the study of drug addiction. We previously reported that the μ-opioid receptor knockout mice were resistant to METH-induced behavioral sensitization but the mechanism is unknown. Methods The present study determined whether resistance of the μ-opioid receptor (μ-OR knockout mice to behavioral sensitization is due to differential expression of the stimulatory G protein α subunit (Gαs or regulators of G-protein signaling (RGS coupled to the dopamine D1 receptor. Mice received daily intraperitoneal injections of saline or METH (10 mg/kg for 7 consecutive days to induce sensitization. On day 11(following 4 abstinent days, mice were either given a test dose of METH (10 mg/kg for behavioral testing or sacrificed for neurochemical assays without additional METH treatment. Results METH challenge-induced stereotyped behaviors were significantly reduced in the μ-opioid receptor knockout mice when compared with those in wild-type mice. Neurochemical assays indicated that there is a decrease in dopamine D1 receptor ligand binding and an increase in the expression of RGS4 mRNA in the striatum of METH-treated μ-opioid receptor knockout mice but not of METH-treated wild-type mice. METH treatment had no effect on the expression of Gαs and RGS2 mRNA in the striatum of either strain of mice. Conclusions These results indicate that down-regulation of the expression of the dopamine D1 receptor and up-regulation of RGS4 mRNA expression in the striatum may contribute to the reduced response to METH-induced stereotypy behavior in μ-opioid receptor knockout mice. Our results highlight the interactions of the μ-opioid receptor system to METH-induced behavioral responses by influencing the expression of RGS of dopamine D1 receptors.

  8. Non-analgesic effects of opioids: management of opioid-induced constipation by peripheral opioid receptor antagonists: prevention or withdrawal?

    Holzer, Peter

    2012-01-01

    The therapeutic action of opioid analgesics is compromised by peripheral adverse effects among which opioid-induced constipation (OIC) is the most disabling, with a prevalence reported to vary between 15 and 90 %. Although OIC is usually treated with laxatives, there is insufficient clinical evidence that laxatives are efficacious in this indication. In contrast, there is ample evidence from double- blind, randomized and placebo-controlled trials that peripheral opioid receptor antagonists (PORAs) counteract OIC. This specific treatment modality is currently based on subcutaneous methylnaltrexone for the interruption of OIC in patients with advanced illness, and a fixed combination of oral prolonged-release naloxone with prolonged-release oxycodone for the prevention of OIC in the treatment of non-cancer and cancer pain. Both drugs counteract OIC while the analgesic effect of opioids remains unabated. The clinical studies show that more than 50 % of the patients with constipation under opioid therapy may benefit from the use of PORAs, while PORA-resistant patients are likely to suffer from non-opioid-induced constipation, the prevalence of which increases with age. While the addition of naloxone to oxycodone seems to act by preventing OIC, the intermittent dosing of methylnaltrexone every other day seems to stimulate defaecation by provoking an intestinal withdrawal response. The availability of PORAs provides a novel opportunity to specifically control OIC and other peripheral adverse effects of opioid analgesics (e.g., urinary retention and pruritus). The continuous dosing of a PORA has the advantage of few adverse effects, while intermittent dosing of a PORA can be associated with abdominal cramp-like pain.

  9. Distribution of kappa opioid receptors in the brain of young and old male rats

    Maggi, R.; Limonta, P.; Dondi, D.; Martini, L.; Piva, F.

    1989-01-01

    The experiments to be described have been designed in order to: (a) provide new information on the concentrations of opioid kappa receptors in different regions of the brain of the male rats; and (b) to analyze whether the density of brain kappa receptors might be modified by the process of aging. The concentration of kappa receptors was investigated in the hypothalamus, amygdala, mesencephalon, corpus striatum, hippocampus, thalamus, frontal poles, anterior and posterior cortex collected from male rats of 2 and 19 months of age. 3 H-bremazocine (BRZ) was used as the ligand of kappa receptors, after protection of mu and delta receptors respectively with dihydromorphine and d-ala-d-leu-enkephalin. The results obtained show that: (1) in young male rats, the number of kappa opioid receptors is different in the various brain areas examined. (2) Aging exerts little influence on the number of kappa receptors in the majority of the brain structures considered. However in the amygdala and in the thalamus the number of kappa receptors was increased in old animals

  10. Sigma-2 receptor ligands QSAR model dataset

    Antonio Rescifina

    2017-08-01

    Full Text Available The data have been obtained from the Sigma-2 Receptor Selective Ligands Database (S2RSLDB and refined according to the QSAR requirements. These data provide information about a set of 548 Sigma-2 (σ2 receptor ligands selective over Sigma-1 (σ1 receptor. The development of the QSAR model has been undertaken with the use of CORAL software using SMILES, molecular graphs and hybrid descriptors (SMILES and graph together. Data here reported include the regression for σ2 receptor pKi QSAR models. The QSAR model was also employed to predict the σ2 receptor pKi values of the FDA approved drugs that are herewith included.

  11. Opioid receptor mediated anticonvulsant effect of pentazocine.

    Khanna, N; Khosla, R; Kohli, J

    1998-01-01

    Intraperitoneal (i.p.) administration of (+/-) pentazocine (10, 30 & 50 mg/kg), a Sigma opioid agonist, resulted in a dose dependent anticonvulsant action against maximal electroshock seizures in mice. This anticonvulsant effect of pentazocine was not antagonized by both the doses of naloxone (1 and 10 mg/kg) suggesting thereby that its anticonvulsant action is probably mediated by Sigma opiate binding sites. Its anticonvulsant effect was potentiated by both the anticonvulsant drugs viz. diazepam and diphenylhydantoin. Morphine, mu opioid agonist, on the other hand, failed to protect the animals against maximal electroshock seizures when it was given in doses of 10-40 mg/kg body wt.

  12. Peripheral δ-opioid receptors attenuate the exercise pressor reflex.

    Leal, Anna K; Yamauchi, Katsuya; Kim, Joyce; Ruiz-Velasco, Victor; Kaufman, Marc P

    2013-10-15

    In rats with ligated femoral arteries, the exercise pressor reflex is exaggerated, an effect that is attenuated by stimulation of peripheral μ-opioid receptors on group IV metabosensitive afferents. In contrast, δ-opioid receptors are expressed mostly on group III mechanosensitive afferents, a finding that prompted us to determine whether stimulation of these opioid receptors could also attenuate the exaggerated exercise pressor reflex in "ligated" rats. We found femoral arterial injection of [D-Pen2,D-Pen5]enkephalin (DPDPE; 1.0 μg), a δ-opioid agonist, significantly attenuated the pressor and cardioaccelerator components of the exercise pressor reflex evoked by hindlimb muscle contraction in both rats with ligated and patent femoral arteries. DPDPE significantly decreased the pressor responses to muscle mechanoreflex activation, evoked by tendon stretch, in ligated rats only. DPDPE (1.0 μg) had no effect in either group on the pressor and cardioaccelerator responses to capsaicin (0.2 μg), which primarily stimulates group IV afferents. DPDPE (1.0 μg) had no effect on the pressor and cardioaccelerator responses to lactic acid (24 mM), which stimulates group III and IV afferents, in rats with patent femoral arteries but significantly decreased the pressor response in ligated rats. Western blots revealed the amount of protein comprising the δ-opioid receptor was greater in dorsal root ganglia innervating hindlimbs with ligated femoral arteries than in dorsal root ganglia innervating hindlimbs with patent femoral arteries. Our findings support the hypothesis that stimulation of δ-opioid receptors on group III afferents attenuated the exercise pressor reflex.

  13. Opioid receptors in midbrain dopaminergic regions of the rat. 1. Mu receptor autoradiography

    German, D.C.; Speciale, S.G.; Manaye, K.F.; Sadeq, M.

    1993-01-01

    Several lines of evidence indicate that an interaction exists between opioid peptides and midbrain dopaminergic neurons. The purpose of this study was to map and quantify the density of the mu opioid receptor subtype relative to the location of the dopaminergic (DA) neurons in the retrorubral field (nucleus A8), substantia nigra (nucleus A9), and ventral tegmental area and related nuclei (nucleus A10) in the rat. Sections through the rostral-caudal extent of the midbrain were stained with an antibody against tyrosine hydroxylase, as a DA cell marker, and comparable sections were processed for in vitro receptor autoradiography using the mu-selective ligand, 3 H-Tyr-D-Ala-N-MePhe-Gyl-ol enkephalin. In the nucleus A8 region, there were low levels of mu binding. In the rostral portion of nucleus A9, there was prominent mu binding both in the ventral pars compacta, which contains numerous DA neurons, and in regions that correspond to the location of the DA dendrites which project ventrally into the underlying substantia nigra pars reticulata. In the caudal portion of nucleus A9, mu binding was greatest in the substantia nigra pars reticulata, but also in the same region that contains DA neurons. In nucleus A10, mu receptor densities differed depending upon the nucleus A10 subdivision, and the rostral-caudal position in the nucleus. Low receptor densities were observed in rostral portions of the ventral tegmental area and interfascicular nucleus, and there was negligible binding in the parabrachial pigmented nucleus and paranigral nucleus at the level of the interpeduncular nucleus; all regions where there are high densities of DA somata. Mu binding was relatively high in the central linear nucleus, and in the dorsal and medial divisions of the medial terminal nucleus of the accessory optic system, which has been shown to contain DA dendrites. These data indicate that mu opioid receptors are located in certain regions occupied by all three midbrain DA nuclei, but in a

  14. Effect of prenatal methadone and ethanol on opioid receptor development in rats

    Peters, M.A.; Braun, R.L. (Loma Linda Univ., CA (United States))

    1991-03-11

    The current literature shows that the offspring of female rats exposed to methadone or ethanol display similar neurochemical and neurobehavioral alterations, and suggests that these drugs may be operating through a common mechanism. If this hypothesis is true, their effect on the endogenous opioid systems should be qualitatively similar. In this study virgin females were treated with methadone or 10% ethanol oral solution starting prior to conception and continued throughout gestation. When the offspring had reached 15 or 30 days of age they were sacrificed, the brain was removed and prepared for opioid receptor binding studies. ({sup 3}H)DAGO and ({sup 3}H)DADLE were used as ligands for the mu and delta receptors, respectively. These studies show significant treatment-related differences in both the number of mu and delta binding sites as well as in apparent receptor affinity. Significant sex- and age-related differences between treatments were also observed. These data show that methadone and ethanol, while manifesting some similar neurochemical and behavioral effects, have unique effects on opioid receptor binding, suggesting that they may be acting by different mechanisms.

  15. TRV0109101, a G Protein-Biased Agonist of the µ-Opioid Receptor, Does Not Promote Opioid-Induced Mechanical Allodynia following Chronic Administration.

    Koblish, Michael; Carr, Richard; Siuda, Edward R; Rominger, David H; Gowen-MacDonald, William; Cowan, Conrad L; Crombie, Aimee L; Violin, Jonathan D; Lark, Michael W

    2017-08-01

    Prescription opioids are a mainstay in the treatment of acute moderate to severe pain. However, chronic use leads to a host of adverse consequences including tolerance and opioid-induced hyperalgesia (OIH), leading to more complex treatment regimens and diminished patient compliance. Patients with OIH paradoxically experience exaggerated nociceptive responses instead of pain reduction after chronic opioid usage. The development of OIH and tolerance tend to occur simultaneously and, thus, present a challenge when studying the molecular mechanisms driving each phenomenon. We tested the hypothesis that a G protein-biased µ -opioid peptide receptor (MOPR) agonist would not induce symptoms of OIH, such as mechanical allodynia, following chronic administration. We observed that the development of opioid-induced mechanical allodynia (OIMA), a model of OIH, was absent in β -arrestin1 -/- and β -arrestin2 -/- mice in response to chronic administration of conventional opioids such as morphine, oxycodone and fentanyl, whereas tolerance developed independent of OIMA. In agreement with the β -arrestin knockout mouse studies, chronic administration of TRV0109101, a G protein-biased MOPR ligand and structural analog of oliceridine, did not promote the development of OIMA but did result in drug tolerance. Interestingly, following induction of OIMA by morphine or fentanyl, TRV0109101 was able to rapidly reverse allodynia. These observations establish a role for β -arrestins in the development of OIH, independent of tolerance, and suggest that the use of G protein-biased MOPR ligands, such as oliceridine and TRV0109101, may be an effective therapeutic avenue for managing chronic pain with reduced propensity for opioid-induced hyperalgesia. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

  16. MDAN-21: A Bivalent Opioid Ligand Containing mu-Agonist and Delta-Antagonist Pharmacophores and Its Effects in Rhesus Monkeys

    Mario D. Aceto

    2012-01-01

    Full Text Available MDAN-21, 7′-{2-[(7-{2-[({(5α,6α-4,5-Epoxy-3,14-dihydroxy-17-methylmorphin-6-yl}-aminocarbonylmetoxy]-acetylamino}-heptylaminocarbonyl-methoxy]-acetylamino}-naltrindole, a bivalent opioid ligand containing a mu-opioid receptor agonist (derived from oxymorphone linked to the delta-opioid receptor antagonist (related to naltrindole by a spacer of 21 atoms, was reported to have potent analgesic properties in mice. Tolerance, physical dependence, and conditioned place preference were not evident in that species. The finding that bivalent ligands in this series, with spacers 19 atoms or greater, were devoid of tolerance and dependence led to the proposal that MDAN-21 targets heteromeric mu-delta-opioid receptors. The present study focused on its effects in nonhuman primates (Macaca mulatta, a species with a physiology and behavioral repertoire not unlike humans. With regard to opioids, this species usually better predicts clinical outcomes. MDAN-21 substituted for morphine in morphine-dependent monkeys in the remarkably low dose range 0.006–0.032 mg/kg, subcutaneously. Although MDAN-21 failed to produce reliable thermal analgesia in the dose range 0.0032–0.032 mg/kg, intramuscularly, it was active in the same dose range and by the same route of administration, in the capsaicin-induced thermal allodynia assay. The results suggest that MDAN-21 may be useful in the treatment of opioid dependence and allodynia. The data provide additional evidence that opioid withdrawal is associated with sensitized pain.

  17. 3D-QSAR comparative molecular field analysis on opioid receptor antagonists: pooling data from different studies.

    Peng, Youyi; Keenan, Susan M; Zhang, Qiang; Kholodovych, Vladyslav; Welsh, William J

    2005-03-10

    Three-dimensional quantitative structure-activity relationship (3D-QSAR) models were constructed using comparative molecular field analysis (CoMFA) on a series of opioid receptor antagonists. To obtain statistically significant and robust CoMFA models, a sizable data set of naltrindole and naltrexone analogues was assembled by pooling biological and structural data from independent studies. A process of "leave one data set out", similar to the traditional "leave one out" cross-validation procedure employed in partial least squares (PLS) analysis, was utilized to study the feasibility of pooling data in the present case. These studies indicate that our approach yields statistically significant and highly predictive CoMFA models from the pooled data set of delta, mu, and kappa opioid receptor antagonists. All models showed excellent internal predictability and self-consistency: q(2) = 0.69/r(2) = 0.91 (delta), q(2) = 0.67/r(2) = 0.92 (mu), and q(2) = 0.60/r(2) = 0.96 (kappa). The CoMFA models were further validated using two separate test sets: one test set was selected randomly from the pooled data set, while the other test set was retrieved from other published sources. The overall excellent agreement between CoMFA-predicted and experimental binding affinities for a structurally diverse array of ligands across all three opioid receptor subtypes gives testimony to the superb predictive power of these models. CoMFA field analysis demonstrated that the variations in binding affinity of opioid antagonists are dominated by steric rather than electrostatic interactions with the three opioid receptor binding sites. The CoMFA steric-electrostatic contour maps corresponding to the delta, mu, and kappa opioid receptor subtypes reflected the characteristic similarities and differences in the familiar "message-address" concept of opioid receptor ligands. Structural modifications to increase selectivity for the delta over mu and kappa opioid receptors have been predicted on the

  18. Striatal μ-opioid receptor availability predicts cold pressor pain threshold in healthy human subjects

    Hagelberg, Nora; Aalto, Sargo; Tuominen, Lauri

    2012-01-01

    the potential associations between μ-opioid receptor BP(ND) and psychophysical measures. The results show that striatal μ-opioid receptor BP(ND) predicts cold pressor pain threshold, but not cold pressor pain tolerance or tactile sensitivity. This finding suggests that striatal μ-opioid receptor density......Previous PET studies in healthy humans have shown that brain μ-opioid receptor activation during experimental pain is associated with reductions in the sensory and affective ratings of the individual pain experience. The aim of this study was to find out whether brain μ-opioid receptor binding...... at the resting state, in absence of painful stimulation, can be a long-term predictor of experimental pain sensitivity. We measured μ-opioid receptor binding potential (BP(ND)) with μ-opioid receptor selective radiotracer [(11)C]carfentanil and positron emission tomography (PET) in 12 healthy male subjects...

  19. Cholinergic, opioid and glycine receptor binding sites localized in human spinal cord by in vitro autoradiography

    Gillberg, P.-G.; Aquilonius, S.-M.

    1985-01-01

    Binding sites for the receptor ligands 3 H-quinuclidinylbenzilate, 3 H-alpha-bungarotoxin ( 3 H-alpha-Btx), 3 H-etorphine and 3 H-strychnine were localized autoradiographically at cervical, thoracic and lumbar levels of spinal cords from post-mortem human control subjects and subjects with amyotrophic lateral sclerosis (ALS). The highest densities of muscarinic binding sites were found in the motor neuron areas and in the substantia gelatinosa, while the grey matter binding was very low within Clarke's column. Both 3 H-alpha-Btx and opioid receptor binding sites were numerous within the substantia gelatinosa, while glycine receptor binding sites were more uniformly distribute within the spinal grey matter. In ALS cases, muscarinic receptor binding sites were markedly reduced in motor neuron areas and slightly reduced in the dorsal horn, while the other binding sites studied were relatively unchanged. (author)

  20. Crystal structure of the[mu]-opioid receptor bound to a morphinan antagonist

    Manglik, Aashish; Kruse, Andrew C.; Kobilka, Tong Sun; Thian, Foon Sun; Mathiesen, Jesper M.; Sunahara, Roger K.; Pardo, Leonardo; Weis, William I.; Kobilka, Brian K.; Granier, Sébastien (Michigan-Med); (Stanford-MED); (UAB, Spain)

    2012-06-27

    Opium is one of the world's oldest drugs, and its derivatives morphine and codeine are among the most used clinical drugs to relieve severe pain. These prototypical opioids produce analgesia as well as many undesirable side effects (sedation, apnoea and dependence) by binding to and activating the G-protein-coupled {mu}-opioid receptor ({mu}-OR) in the central nervous system. Here we describe the 2.8 {angstrom} crystal structure of the mouse {mu}-OR in complex with an irreversible morphinan antagonist. Compared to the buried binding pocket observed in most G-protein-coupled receptors published so far, the morphinan ligand binds deeply within a large solvent-exposed pocket. Of particular interest, the {mu}-OR crystallizes as a two-fold symmetrical dimer through a four-helix bundle motif formed by transmembrane segments 5 and 6. These high-resolution insights into opioid receptor structure will enable the application of structure-based approaches to develop better drugs for the management of pain and addiction.

  1. [3H]naloxone as an opioid receptor label: Analysis of binding site heterogeneity and use for determination of opioid affinities of casomorphin analogues

    Schnittler, M.; Repke, H.; Liebmann, C.; Schrader, U.; Schulze, H.P.; Neubert, K.

    1990-01-01

    The nonselective antagonist [ 3 H]naloxone was used to identify opioid receptors in rat brain membranes. The multiple naloxone binding sites were related to different opioid receptors by means of selective opiod ligands as well as various β-casomorphin analogues. Analysis of binding site heterogeneity was performed using several computer curve fitting methods. The results indicate that structurally modified casomorphin peptides are able to discriminate between μ 1 and μ 2 binding sites. The affinities to the μ sites obtained with [ 3 H]naloxone as label are in a good agreement with those from experiments with the μ selective radioligand [ 3 H]DAGO. The μ 1 site affinities of these casomorphin derivatives are well correlated with their antinociceptive potencies. This finding suggests the mediation of the analgesic activity via the high-affinity μ 1 subtype. (author)

  2. Dark chocolate receptors: epicatechin-induced cardiac protection is dependent on delta-opioid receptor stimulation.

    Panneerselvam, Mathivadhani; Tsutsumi, Yasuo M; Bonds, Jacqueline A; Horikawa, Yousuke T; Saldana, Michelle; Dalton, Nancy D; Head, Brian P; Patel, Piyush M; Roth, David M; Patel, Hemal H

    2010-11-01

    Epicatechin, a flavonoid, is a well-known antioxidant linked to a variety of protective effects in both humans and animals. In particular, its role in protection against cardiovascular disease has been demonstrated by epidemiologic studies. Low-dose epicatechin, which does not have significant antioxidant activity, is also protective; however, the mechanism by which low-dose epicatechin induces this effect is unknown. Our laboratory tested the hypothesis that low-dose epicatechin mediates cardiac protection via opioid receptor activation. C57BL/6 mice were randomly assigned to 1 of 10 groups: control, epicatechin, naloxone (nonselective opioid receptor antagonist), epicatechin + naloxone, naltrindole (δ-specific opioid receptor antagonist), epicatechin + naltrindole, norbinaltorphimine (nor-BNI, κ-specific opioid receptor antagonist), epicatechin + nor-BNI, 5-hydroxydecanoic acid [5-HD, ATP-sensitive potassium channel antagonist], and epicatechin + 5-HD. Epicatechin (1 mg/kg) or other inhibitors (5 mg/kg) were administered by oral gavage or intraperitoneal injection, respectively, daily for 10 days. Mice were subjected to 30 min coronary artery occlusion followed by 2 h of reperfusion, and infarct size was determined via planimetry. Whole heart homogenates were assayed for downstream opioid receptor signaling targets. Infarct size was significantly reduced in epicatechin- and epicatechin + nor-BNI-treated mice compared with control mice. This protection was blocked by naloxone, naltrindole, and 5-HD. Epicatechin and epicatechin + nor-BNI increased the phosphorylation of Src, Akt, and IκBα, while simultaneously decreasing the expression of c-Jun NH(2)-terminal kinase and caspase-activated DNase. All signaling effects are consistent with opioid receptor stimulation and subsequent cardiac protection. Naloxone, naltrindole, and 5-HD attenuated these effects. In conclusion, epicatechin acts via opioid receptors and more specifically through the δ-opioid receptor to

  3. Gold Nanorods Targeted to Delta Opioid Receptor: Plasmon-Resonant Contrast and Photothermal Agents

    Kvar C. Black

    2008-01-01

    Full Text Available Molecularly targeted gold nanorods were investigated for applications in both diagnostic imaging and disease treatment with cellular resolution. The nanorods were tested in two genetically engineered cell lines derived from the human colon carcinoma HCT-116, a model for studying ligand-receptor interactions. One of these lines was modified to express delta opioid receptor (δOR and green fluorescent protein, whereas the other was receptor free and expressed a red fluorescent protein, to serve as the control. Deltorphin, a high-affinity ligand for δOR, was stably attached to the gold nanorods through a thiol-terminated linker. In a mixed population of cells, we demonstrated selective imaging and destruction of receptor-expressing cells while sparing those cells that did not express the receptor. The molecularly targeted nanorods can be used as an in vitro ligand-binding and cytotoxic treatment assay platform and could potentially be applied in vivo for diagnostic and therapeutic purposes with endoscopic technology.

  4. Biotinylated human. beta. -endorphins as probes for the opioid receptor

    Hochhaus, G.; Gibson, B.W.; Sadee, W.

    1988-01-05

    The reaction of human ..beta..-endorphin and biotinyl N-hydroxysuccinimide with or without spacer arm, afforded a series of products that were separated by high performance liquid chromatography (HPLC). Liquid secondary ion mass spectrometry of the biotinylated products and their tryptic digests produced abundant protonated molecular ions (MH/sup +/), which specified the number and location of biotinylation. Between 1 and 4 biotinyl residues were incorporated per human ..beta..-endorphin molecule, at Lys-9, -19, -24, -28, and -29, but not at the amino-terminal Try-1. Three HPLC fractions were isolated for receptor binding studies monobiotinylation of Lys-9, Lys-19, and a mixture of Lys-24, Lys-28, and Lys-29 derivatives. IC/sub 50/ values for binding to ..mu.. and delta opioid receptor sites were 3-8 times higher for monobiotinylated derivatives than for the parent human ..beta..-endorphin. Association with avidin decreased opioid receptor affinities for the C/sub 6/ spacer derivative biotinylated at position Lys-9, which is close to the (1-5) enkephalin receptor region. In contrast, avidin did not affect or even increased apparent affinities to ..mu.. and delta sites for derivatives biotinylated at the ..cap alpha..-helical part of the molecule (Lys-19, -24, -28, and -29). Biotinylated human ..beta..-endorphins also bound to low affinity nonopioid binding sites on NG-108-15 cells; however, affinities to these sites were considerably reduced when derivatives were bound to avidin. The ability of biotinylated human ..beta..-endorphin to cross-link the ..mu.. and delta opioid receptors to avidin allows application of the biotin-avidin system as a molecular probe of the opioid receptor.

  5. [{sup 11}C]-MeJDTic: a novel radioligand for {kappa}-opioid receptor positron emission tomography imaging

    Poisnel, Geraldine; Oueslati, Farhana; Dhilly, Martine; Delamare, Jerome [Groupe de Developpements Methodologiques en Tomographie par Emission de Positons, DSV/DRM UMR CEA 2E, Universite de Caen-Basse Normandie, Centre Cyceron, 14074 Caen Cedex (France); Perrio, Cecile [Groupe de Developpements Methodologiques en Tomographie par Emission de Positons, DSV/DRM UMR CEA 2E, Universite de Caen-Basse Normandie, Centre Cyceron, 14074 Caen Cedex (France)], E-mail: perrio@cyceron.fr; Debruyne, Daniele [Groupe de Developpements Methodologiques en Tomographie par Emission de Positons, DSV/DRM UMR CEA 2E, Universite de Caen-Basse Normandie, Centre Cyceron, 14074 Caen Cedex (France)], E-mail: debruyne@cyceron.fr; Barre, Louisa [Groupe de Developpements Methodologiques en Tomographie par Emission de Positons, DSV/DRM UMR CEA 2E, Universite de Caen-Basse Normandie, Centre Cyceron, 14074 Caen Cedex (France)

    2008-07-15

    Introduction: Radiopharmaceuticals that can bind selectively the {kappa}-opioid receptor may present opportunities for staging clinical brain disorders and evaluating the efficiency of new therapies related to stroke, neurodegenerative diseases or opiate addiction. The N-methylated derivative of JDTic (named MeJDTic), which has been recently described as a potent and selective antagonist of {kappa}-opioid receptor in vitro, was labeled with carbon-11 and evaluated for in vivo imaging the {kappa}-opioid receptor in mice. Methods: [{sup 11}C]-MeJDTic was prepared by methylation of JDTic with [{sup 11}C]-methyl triflate. The binding of [{sup 11}C]-MeJDTic to {kappa}-opioid receptor was investigated ex vivo by biodistribution and competition studies using nonfasted male CD1 mice. Results: [{sup 11}C]-MeJDTic exhibited a high and rapid distribution in peripheral organs. The uptake was maximal in lung where the {kappa} receptor is largely expressed. [{sup 11}C]-MeJDTic rapidly crossed the blood-brain barrier and accumulated in the brain regions of interest (hypothalamus). The parent ligand remained the major radioactive compound in brain during the experiment. Chase studies with U50,488 (a {kappa} referring agonist), morphine (a {mu} agonist) and naltrindole (a {delta} antagonist) demonstrated that this uptake was the result of specific binding to the {kappa}-opioid receptor. Conclusion: These findings suggested that [{sup 11}C]-MeJDTic appeared to be a promising selective 'lead' radioligand for {kappa}-opioid receptor PET imaging.

  6. Opioid adjuvant strategy: improving opioid effectiveness.

    Bihel, Frédéric

    2016-01-01

    Opioid analgesics continue to be the mainstay of pharmacologic treatment of moderate to severe pain. Many patients, particularly those suffering from chronic pain, require chronic high-dose analgesic therapy. Achieving clinical efficacy and tolerability of such treatment regimens is hampered by the appearance of opioid-induced side effects such as tolerance, hyperalgesia and withdrawal syndrome. Among the therapeutic options to improve the opioid effectiveness, this current review focuses on strategies combining opioids to other drugs that can modulate opioid-mediated effects. We will discuss about experimental evidences reported for several potential opioid adjuvants, including N-methyl-D-aspartate receptor antagonists, 5-HT7 agonists, sigma-1 antagonists, I2-R ligands, cholecystokinin antagonists, neuropeptide FF-R antagonists and toll-like receptor 4 antagonists.

  7. Atypical Opioid Mechanisms of Control of Injury-Induced Cutaneous Pain by Delta Receptors

    2016-07-01

    treat, and current opioids (i.e. mu opioid receptor agonists such as morphine) cause unacceptable side effects including addiction . Injuries suffered...treat, and current opioids that act on mu opioid receptors such as morphine generate significant side effects including addiction . War-related...al., J Neurosci Methods, 1994), starting with 0.1 g and ending with 2.0 g filament as cutoff value. As shown in Figure 2, our preliminary experiments

  8. Enhanced efficacy (intrinsic activity) of cyclic opioid peptide analogs at the μ-receptor

    Schiller, P.W.; Lemieux, C.; Nguyen, T.M.D.; Maziak, L.A.

    1986-01-01

    Side-chain to end group cyclized enkephalin analogs (e.g. H-Tyr-cyclo[-D-Lys-Gly-Phe-Leu-] and cyclic opioid peptide analogs obtained through covalent linkage of two side-chains (e.g. H-Tyr-D-Cys-Gly-Phe-Cys-NH 2 or H-Tyr-D-Lys-Gly-Phe-Glu-NH 3 ) were tested in the μ-receptor-representative guinea pig ileum (GPI) bioassay and in a binding assay based on displacement of the μ-ligand [ 3 H]DAGO from rat brain membranes. The cyclic analogs were 5 to 70 times more potent in the GPI assay than in the binding assay, whereas linear analogs showed equal potency in the two assays. These results suggest that the efficacy (intrinsic activity) of cyclic opioid peptide analogs at the μ-receptor is increased as a consequence of the conformation constraint imposed through ring closure. This effect was most pronounced in analogs containing a long hydrophobic sidechain as part of the ring structure in the 2-position of the peptide sequence. Further experimental evidence ruled out the possibilities that these potency discrepancies may be due to differences in enzymatic degradation, dissimilar exposure of the receptors in their lipid environment or interaction with different receptor types in the two assay systems. It can be hypothesized that the semi-rigid cyclic analogs may induce a more productive conformational change in the receptor protein than the linear peptides

  9. Identification of rat brain opioid (enkephalin) receptor by photoaffinity labeling

    Yeung, C.W.

    1986-01-01

    A photoreactive, radioactive enkephalin derivative was prepared and purified by high performance liquid chromatography. Rat brain and spinal cord plasma membranes were incubated with this radioiodinated photoprobe and were subsequently photolysed. Autoradiography of the sodium dodecyl sulfate gel electrophoresis of the solubilized and reduced membranes showed that a protein having an apparent molecular weight of 46,000 daltons was specifically labeled, suggesting that this protein may be the opioid (enkephalin) receptor

  10. Identification of endogenous opioid receptor components in rat brain using a monoclonal antibody

    Bero, L.A.; Roy, S.; Lee, N.M.

    1988-11-01

    A monoclonal antibody generated against the tertiary structure of a partially purified opioid binding protein was used to probe the structure of the dynorphin and beta-endorphin receptors. The Fab fragment 3B4F11 inhibited completely the binding of 125I-beta-endorphin and (3H)dynorphin to rat brain P2 membranes with IC50 values of 26 ng/ml and 40 ng/ml, respectively. To explore further the interaction of 3B4F11 with the beta-endorphin receptor, the effect of the Fab fragment on 125I-beta-endorphin cross-linking to rat brain membranes was examined. 125I-beta-endorphin was covalently bound to three major species of approximate molecular weights 108,000, 73,000, and 49,000. The delta-selective ligand D-Pen2, D-pen5enkephalin was least effective at inhibiting the cross-linking of beta-endorphin, whereas the micro-selective ligand Tyr-D-Ala-Gly-NMe-Phe-Gly-ol and kappa-selective ligand U50488 inhibited beta-endorphin cross-linking to the 108,000 and 73,000 Da species. Both 3B4F11 and beta-endorphin prevented the covalent binding of 125I-beta-endorphin to all three labeled species. These findings suggest that micro and kappa receptor types might have some structural similarities, whereas the delta receptor type might differ in molecular size. In addition, the micro, kappa, and delta ligands might have different primary sequences, whereas their tertiary structures might share regions of molecular homology with all three receptor constituents labeled by 125I-beta-endorphin. 3B4F11 will be a valuable tool for the purification and isolation of the several components of the beta-endorphin receptor complex.

  11. Opioid modulation of immunocompetence: Receptor characterization and second messenger involvement

    Hemmick, L.M.

    1989-01-01

    The purpose of this thesis was to examine the effects of opioids on several indices of immunocompetence, determined the receptor specificity of these effects, and ascertain whether the actions of opioids on lymphocytes could be correlated with activation of second messenger systems. By measuring 45 Ca 2+ uptake into lymphocytes, it was demonstrated that β-endorphin 1-31 (β-END 1-31) enhanced rat thymocyte Ca 2+ uptake in response to concanavalin A (Con A) but not phytohemagglutinin (PHA). Related opioid peptides and alkaloids were unable to mimic the effect, and naloxone did not block it, suggesting that β-END 1-31 acted by binding to specific, non-opioid receptors on the thymocytes. Rat splenocyte Con A-stimulated Ca 2+ uptake was not affected by β-END 1-31. β-END 1-31 did not affect basal Ca 2+ uptake by either cell type. Using [ 3 H]thymidine uptake as an index of lymphocyte proliferation, β-END 1-31 and several related opioid peptides reversed prostaglandin E 1 (PGE 1 ) suppression of rat lymph node cell Con A- and PHA-stimulated proliferation. Naloxone did not block the reversal. β-END 1-31 was unable to reverse forskolin and cholera toxin suppression of proliferation, indicating that the lowering of cyclic AMP levels was not the mechanism involved. Verapamil inhibition of proliferation was also not reversed by β-END 1-31, suggesting that promotion of Ca 2+ influx was not a major mechanism involved

  12. Opioid modulation of immunocompetence: Receptor characterization and second messenger involvement

    Hemmick, L.M.

    1989-01-01

    The purpose of this thesis was to examine the effects of opioids on several indices of immunocompetence, determined the receptor specificity of these effects, and ascertain whether the actions of opioids on lymphocytes could be correlated with activation of second messenger systems. By measuring {sup 45}Ca{sup 2+} uptake into lymphocytes, it was demonstrated that {beta}-endorphin 1-31 ({beta}-END 1-31) enhanced rat thymocyte Ca{sup 2+} uptake in response to concanavalin A (Con A) but not phytohemagglutinin (PHA). Related opioid peptides and alkaloids were unable to mimic the effect, and naloxone did not block it, suggesting that {beta}-END 1-31 acted by binding to specific, non-opioid receptors on the thymocytes. Rat splenocyte Con A-stimulated Ca{sup 2+} uptake was not affected by {beta}-END 1-31. {beta}-END 1-31 did not affect basal Ca{sup 2+} uptake by either cell type. Using ({sup 3}H)thymidine uptake as an index of lymphocyte proliferation, {beta}-END 1-31 and several related opioid peptides reversed prostaglandin E{sub 1} (PGE{sub 1}) suppression of rat lymph node cell Con A- and PHA-stimulated proliferation. Naloxone did not block the reversal. {beta}-END 1-31 was unable to reverse forskolin and cholera toxin suppression of proliferation, indicating that the lowering of cyclic AMP levels was not the mechanism involved. Verapamil inhibition of proliferation was also not reversed by {beta}-END 1-31, suggesting that promotion of Ca{sup 2+} influx was not a major mechanism involved.

  13. Opioid withdrawal increases transient receptor potential vanilloid 1 activity in a protein kinase A-dependent manner.

    Spahn, Viola; Fischer, Oliver; Endres-Becker, Jeannette; Schäfer, Michael; Stein, Christoph; Zöllner, Christian

    2013-04-01

    Hyperalgesia is a cardinal symptom of opioid withdrawal. The transient receptor potential vanilloid 1 (TRPV1) is a ligand-gated ion channel expressed on sensory neurons responding to noxious heat, protons, and chemical stimuli such as capsaicin. TRPV1 can be inhibited via μ-opioid receptor (MOR)-mediated reduced activity of adenylyl cyclases (ACs) and decreased cyclic adenosine monophosphate (cAMP) levels. In contrast, opioid withdrawal following chronic activation of MOR uncovers AC superactivation and subsequent increases in cAMP and protein kinase A (PKA) activity. Here we investigated (1) whether an increase in cAMP during opioid withdrawal increases the activity of TRPV1 and (2) how opioid withdrawal modulates capsaicin-induced nocifensive behavior in rats. We applied whole-cell patch clamp, microfluorimetry, cAMP assays, radioligand binding, site-directed mutagenesis, and behavioral experiments. Opioid withdrawal significantly increased cAMP levels and capsaicin-induced TRPV1 activity in both transfected human embryonic kidney 293 cells and dissociated dorsal root ganglion (DRG) neurons. Inhibition of AC and PKA, as well as mutations of the PKA phosphorylation sites threonine 144 and serine 774, prevented the enhanced TRPV1 activity. Finally, capsaicin-induced nocifensive behavior was increased during opioid withdrawal in vivo. In summary, our results demonstrate an increased activity of TRPV1 in DRG neurons as a new mechanism contributing to opioid withdrawal-induced hyperalgesia. Copyright © 2013 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.

  14. Affinity of the enantiomers of. alpha. - and. beta. -cyclazocine for binding to the phencyclidine and. mu. opioid receptors

    Todd, S.L.; Balster, R.L.; Martin, B.R. (Virginia Commonwealth Univ., Richmond (USA))

    1990-01-01

    The enantiomers in the {alpha} and {beta} series of cyclazocine were evaluated for their ability to bind to phencyclidine (PCP) and {mu}-opioid receptors in order to determine their receptor selectivity. The affinity of (-)-{beta}-cyclazocine for the PCP receptor was 1.5 greater than PCP itself. In contrast, (-)-{alpha}-cyclazocine, (+)-{alpha}-cyclazocine, and (+)-{beta}-cyclazocine were 3-, 5- and 138-fold less potent than PCP, respectively. Scatchard analysis of saturable binding of ({sup 3}H)Tyr-D-Ala-Gly-N-MePhe-Gly-ol (DAMGO) also exhibited a homogeneous population of binding sites with an apparent K{sub D} of 1.9 nM and an estimated Bmax of 117 pM. (3H)Tyr-D-Ala-Gly-N-MePhe-Gly-ol (DAMGO) binding studies revealed that (-)-{alpha}-cyclazocine (K{sub D} = 0.48 nM) was 31-, 1020- and 12,600-fold more potent than (-)-{beta}-cyclazocine, (+)-{alpha}-cyclazocine and (+)-{beta}-cyclazocine, respectively, for binding to the {mu}-opioid receptor. These data show that, although (-)-{beta}-cyclazocine is a potent PCP receptor ligand consistent with its potent PCP-like discriminative stimulus effects, it shows little selectivity for PCP receptor since it also potently displaces {mu}-opioid binding. However, these cyclazocine isomers, due to their extraordinary degree of stereoselectivity, may be useful in characterizing the structural requirements for benzomorphans having activity at the PCP receptor.

  15. Binge eating disorder and morbid obesity are associated with lowered mu-opioid receptor availability in the brain.

    Joutsa, Juho; Karlsson, Henry K; Majuri, Joonas; Nuutila, Pirjo; Helin, Semi; Kaasinen, Valtteri; Nummenmaa, Lauri

    2018-03-09

    Both morbid obesity and binge eating disorder (BED) have previously been linked with aberrant brain opioid function. Behaviorally these two conditions are however different suggesting also differences in neurotransmitter function. Here we directly compared mu-opioid receptor (MOR) availability between morbidly obese and BED subjects. Seven BED and nineteen morbidly obese (non-BED) patients, and thirty matched control subjects underwent positron emission tomography (PET) with MOR-specific ligand [ 11 C]carfentanil. Both subjects with morbid obesity and BED had widespread reduction in [ 11 C]carfentanil binding compared to control subjects. However, there was no significant difference in brain MOR binding between subjects with morbid obesity and BED. Thus, our results indicate that there is common brain opioid abnormality in behaviorally different eating disorders involving obesity. Copyright © 2018 Elsevier B.V. All rights reserved.

  16. Opioid mediated activity and expression of mu and delta opioid receptors in isolated human term non-labouring myometrium.

    Fanning, Rebecca A

    2013-01-05

    The existence of opioid receptors in mammalian myometrial tissue is now widely accepted. Previously enkephalin degrading enzymes have been shown to be elevated in pregnant rat uterus and a met-enkephalin analogue has been shown to alter spontaneous contractility of rat myometrium. Here we have undertaken studies to determine the effects of met-enkephalin on in vitro human myometrial contractility and investigate the expression of opioid receptors in pregnant myometrium. Myometrial biopsies were taken from women undergoing elective caesarean delivery at term. Organ bath experiments were used to investigate the effect of the met-enkephalin analogue [d-Ala 2, d-met 5] enkephalin (DAMEA) on spontaneous contractility. A confocal immunofluorescent technique and real time PCR were used to determine the expression of protein and mRNA, respectively for two opioid receptor subtypes, mu and delta. DAMEA had a concentration dependent inhibitory effect on contractile activity (1 × 10(-7)M-1 × 10(-4)M; 54% reduction in contractile activity, P<0.001 at 1 × 10(-4)M concentration). Mu and delta opioid receptor protein sub-types and their respective mRNA were identified in all tissues sampled. This is the first report of opioid receptor expression and of an opioid mediated uterorelaxant action in term human non-labouring myometrium in vitro.

  17. Adrenergic Agonists Bind to Adrenergic-Receptor-Like Regions of the Mu Opioid Receptor, Enhancing Morphine and Methionine-Enkephalin Binding: A New Approach to "Biased Opioids"?

    Root-Bernstein, Robert; Turke, Miah; Subhramanyam, Udaya K Tiruttani; Churchill, Beth; Labahn, Joerg

    2018-01-17

    Extensive evidence demonstrates functional interactions between the adrenergic and opioid systems in a diversity of tissues and organs. While some effects are due to receptor and second messenger cross-talk, recent research has revealed an extracellular, allosteric opioid binding site on adrenergic receptors that enhances adrenergic activity and its duration. The present research addresses whether opioid receptors may have an equivalent extracellular, allosteric adrenergic binding site that has similar enhancing effects on opioid binding. Comparison of adrenergic and opioid receptor sequences revealed that these receptors share very significant regions of similarity, particularly in some of the extracellular and transmembrane regions associated with adrenergic binding in the adrenergic receptors. Five of these shared regions from the mu opioid receptor (muOPR) were synthesized as peptides and tested for binding to adrenergic, opioid and control compounds using ultraviolet spectroscopy. Adrenergic compounds bound to several of these muOPR peptides with low micromolar affinity while acetylcholine, histamine and various adrenergic antagonists did not. Similar studies were then conducted with purified, intact muOPR with similar results. Combinations of epinephrine with methionine enkephalin or morphine increased the binding of both by about half a log unit. These results suggest that muOPR may be allosterically enhanced by adrenergic agonists.

  18. Analgesic tone conferred by constitutively active mu opioid receptors in mice lacking β-arrestin 2

    Hales Tim G

    2011-04-01

    Full Text Available Abstract Hedonic reward, dependence and addiction are unwanted effects of opioid analgesics, linked to the phasic cycle of μ opioid receptor activation, tolerance and withdrawal. In vitro studies of recombinant G protein coupled receptors (GPCRs over expressed in cell lines reveal an alternative tonic signaling mechanism that is independent of agonist. Such studies demonstrate that constitutive GPCR signaling can be inhibited by inverse agonists but not by neutral antagonists. However, ligand-independent activity has been difficult to examine in vivo, at the systems level, due to relatively low levels of constitutive activity of most GPCRs including μ receptors, often necessitating mutagenesis or pharmacological manipulation to enhance basal signaling. We previously demonstrated that the absence of β-arrestin 2 (β-arr2 augments the constitutive coupling of μ receptors to voltage-activated Ca2+ channels in primary afferent dorsal root ganglion neurons from β-arr2-/- mice. We used this in vitro approach to characterize neutral competitive antagonists and inverse agonists of the constitutively active wild type μ receptors in neurons. We administered these agents to β-arr2-/- mice to explore the role of constitutive μ receptor activity in nociception and hedonic tone. This study demonstrates that the induction of constitutive μ receptor activity in vivo in β-arr2-/- mice prolongs tail withdrawal from noxious heat, a phenomenon that was reversed by inverse agonists, but not by antagonists that lack negative efficacy. By contrast, the aversive effects of inverse agonists were similar in β-arr2-/- and β-arr2+/+ mice, suggesting that hedonic tone was unaffected.

  19. Dermorphin-related peptides from the skin of Phyllomedusa bicolor and their amidated analogs activate two mu opioid receptor subtypes that modulate antinociception and catalepsy in the rat.

    Negri, L; Erspamer, G F; Severini, C; Potenza, R L; Melchiorri, P; Erspamer, V

    1992-08-01

    Three naturally occurring dermorphin-like peptides from the skin of the frog Phyllomedusa bicolor, the related carboxyl-terminal amides, and some substituted analogs were synthesized, their binding profiles to opioid receptors were determined, and their biological activities were studied in isolated organ preparations and intact animals. The opioid binding profile revealed a very high selectivity of these peptides for mu sites and suggested the existence of two receptor subtypes, of high and low affinity. The peptides tested acted as potent mu opioid agonists on isolated organ preparations. They were several times more active in inhibiting electrically evoked contractions in guinea pig ileum than in mouse vas deferens. When injected into the lateral brain ventricle or peritoneum of rats, the high-affinity-site-preferring ligand, [Lys7-NH2]dermorphin, behaved as a potent analgesic agent. By contrast, the low-affinity-site-preferring ligand, [Trp4,Asn7-NH2]dermorphin, produced a weak antinociception but an intense catalepsy.

  20. Novel Somatostatin Receptor Ligands Therapies for Acromegaly

    Rosa Maria Paragliola

    2018-03-01

    Full Text Available Surgery is considered the treatment of choice in acromegaly, but patients with persistent disease after surgery or in whom surgery cannot be considered require medical therapy. Somatostatin receptor ligands (SRLs octreotide (OCT, lanreotide, and the more recently approved pasireotide, characterized by a broader receptor ligand binding profile, are considered the mainstay in the medical management of acromegaly. However, in the attempt to offer a more efficacious and better tolerated medical approach, recent research has been aimed to override some limitations related to the use of currently approved drugs and novel SRLs therapies, with potential attractive features, have been proposed. These include both new formulation of older molecules and new molecules. Novel OCT formulations are aimed in particular to improve patients’ compliance and to reduce injection discomfort. They include an investigational ready-to-use subcutaneous depot OCT formulation (CAM2029, delivered via prefilled syringes and oral OCT that uses a “transient permeability enhancer” technology, which allows for OCT oral absorption. Another new delivery system is a long-lasting OCT implant (VP-003, which provide stable doses of OCT throughout a period of several months. Finally, a new SRL DG3173 (somatoprim seems to be more selective for GH secretion, suggesting possible advantages in the presence of hyperglycemia or diabetes. How much these innovations will actually be beneficial to acromegaly patients in real clinical practice remains to be seen.

  1. Substrate coated with receptor and labelled ligand for assays

    1980-01-01

    Improvements in the procedures for assaying ligands are described. The assay consists of a polystyrene tube on which receptors are present for both the ligand to be assayed and a radioactively labelled form of the ligand. The receptors on the bottom portion of the tube are also coated with labelled ligands, thus eliminating the necessity for separate addition of the labelled ligand and sample during an assay. Examples of ligands to which this method is applicable include polypeptides, nucleotides, nucleosides and proteins. Specific examples are given in which the ligand to be assayed is digoxin, the labelled form of the ligand is 3-0-succinyl digoxyigenin tyrosine ( 125 I) and the receptor is digoxin antibody. (U.K.)

  2. Potent μ-Opioid Receptor Agonists from Cyclic Peptides Tyr-c[D-Lys-Xxx-Tyr-Gly]: Synthesis, Biological, and Structural Evaluation.

    Li, Yangmei; Cazares, Margret; Wu, Jinhua; Houghten, Richard A; Toll, Laurence; Dooley, Colette

    2016-02-11

    To optimize the structure of a μ-opioid receptor ligand, analogs H-Tyr-c[D-Lys-Xxx-Tyr-Gly] were synthesized and their biological activity was tested. The analog containing a Phe(3) was identified as not only exhibiting binding affinity 14-fold higher than the original hit but also producing agonist activity 3-fold more potent than morphine. NMR study suggested that a trans conformation at D-Lys(2)-Xxx(3) is crucial for these cyclic peptides to maintain high affinity, selectivity, and functional activity toward the μ-opioid receptor.

  3. Mu-opioid receptor knockout mice show diminished food-anticipatory activity

    Kas, Martien J H; van den Bos, Ruud; Baars, Annemarie M; Lubbers, Marianne; Lesscher, Heidi M B; Hillebrand, Jacquelien J G; Schuller, Alwin G; Pintar, John E; Spruijt, Berry M

    We have previously suggested that during or prior to activation of anticipatory behaviour to a coming reward, mu-opioid receptors are activated. To test this hypothesis schedule induced food-anticipatory activity in mu-opioid receptor knockout mice was measured using running wheels. We hypothesized

  4. Orexin Receptor Multimerization versus Functional Interactions: Neuropharmacological Implications for Opioid and Cannabinoid Signalling and Pharmacogenetics

    Miles D. Thompson

    2017-10-01

    Full Text Available Orexins/hypocretins are neuropeptides formed by proteolytic cleavage of a precursor peptide, which are produced by neurons found in the lateral hypothalamus. The G protein-coupled receptors (GPCRs for these ligands, the OX1 and OX2 orexin receptors, are more widely expressed throughout the central nervous system. The orexin/hypocretin system has been implicated in many pathways, and its dysregulation is under investigation in a number of diseases. Disorders in which orexinergic mechanisms are being investigated include narcolepsy, idiopathic sleep disorders, cluster headache and migraine. Human narcolepsy has been associated with orexin deficiency; however, it has only rarely been attributed to mutations in the gene encoding the precursor peptide. While gene variations within the canine OX2 gene hcrtr2 have been directly linked with narcolepsy, the majority of human orexin receptor variants are weakly associated with diseases (the idiopathic sleep disorders, cluster headache and polydipsia-hyponatremia in schizophrenia or are of potential pharmacogenetic significance. Evidence for functional and/or heterodimerization between wild-type variant orexin receptors and opioid and cannabinoid receptors is discussed in the context of its relevance to depression and epilepsy.

  5. Morphine-induced internalization of the L83I mutant of the rat μ-opioid receptor.

    Cooke, A E; Oldfield, S; Krasel, C; Mundell, S J; Henderson, G; Kelly, E

    2015-01-01

    Naturally occurring single-nucleotide polymorphisms (SNPs) within GPCRs can result in alterations in various pharmacological parameters. Understanding the regulation and function of endocytic trafficking of the μ-opioid receptor (MOP receptor) is of great importance given its implication in the development of opioid tolerance. This study has compared the agonist-dependent trafficking and signalling of L83I, the rat orthologue of a naturally occurring variant of the MOP receptor. Cell surface elisa, confocal microscopy and immunoprecipitation assays were used to characterize the trafficking properties of the MOP-L83I variant in comparison with the wild-type receptor in HEK 293 cells. Functional assays were used to compare the ability of the L83I variant to signal to several downstream pathways. Morphine-induced internalization of the L83I MOP receptor was markedly increased in comparison with the wild-type receptor. The altered trafficking of this variant was found to be specific to morphine and was both G-protein receptor kinase- and dynamin-dependent. The enhanced internalization of L83I variant in response to morphine was not due to increased phosphorylation of serine 375, arrestin association or an increased ability to signal. These results suggest that morphine promotes a specific conformation of the L83I variant that makes it more liable to internalize in response to morphine, unlike the wild-type receptor that undergoes significantly less morphine-stimulated internalization, providing an example of a ligand-selective biased receptor. The presence of this SNP within an individual may consequently affect the development of tolerance and analgesic responses. This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2. © 2014 The British Pharmacological Society.

  6. Using [11C]diprenorphine to image opioid receptor occupancy by methadone in opioid addiction: clinical and preclinical studies.

    Melichar, Jan K; Hume, Susan P; Williams, Tim M; Daglish, Mark R C; Taylor, Lindsay G; Ahmad, Rabia; Malizia, Andrea L; Brooks, David J; Myles, Judith S; Lingford-Hughes, Anne; Nutt, David J

    2005-01-01

    Substitute methadone prescribing is one of the main modes of treatment for opioid dependence with established evidence for improved health and social outcomes. However, the pharmacology underpinning the effects of methadone is little studied despite controversies about dosing in relation to outcome. We therefore examined the relationship between methadone dose and occupation of opioid receptors in brain using the positron emission tomography (PET) radioligand [(11)C]diprenorphine in humans and rats. Eight opioid-dependent subjects stable on their substitute methadone (18-90 mg daily) had an [(11)C]diprenorphine PET scan at predicted peak plasma levels of methadone. These were compared with eight healthy controls. No difference in [(11)C]diprenorphine binding was found between the groups, with no relationship between methadone dose and occupancy. Adult male Sprague-Dawley rats that had been given an acute i.v. injection of methadone hydrochloride (0.35, 0.5, 0.7, or 1.0 mg kg(-1)) before [(11)C]diprenorphine showed a dose-dependent increase in biodistribution but no reduction in [(11)C]diprenorphine binding. We suggest that the lack of a dose-dependent relationship between methadone dose, either given chronically in human or acutely in rat, and occupancy of opioid receptor measured with [(11)C]diprenorphine PET is related to efficacy of this opioid agonist at very low levels of opioid receptor occupancy. This has implications for understanding the actions of methadone in comparison with other opioid drugs such as partial agonists and antagonists.

  7. Autoradiographic localization of mu and delta opioid receptors in the mesocorticolimbic dopamine system

    Dilts, R.P. Jr.

    1989-01-01

    In vitro autoradiographic techniques were coupled with selective chemical lesions of the A10 dopamine cells and intrinsic perikarya of the region to delineate the anatomical localization of mu and delta opioid receptors, as well as, neurotensin receptors. Mu opioid receptors were labeled with {sup 125}I-DAGO. Delta receptors were labeled with {sup 125}I-DPDPE. Neurotensin receptors were labeled with {sup 125}I-NT3. Unilateral lesions of the dopamine perikarya were produced by injections of 6-OHDA administered in the ventral mesencephalon. Unilateral lesions of intrinsic perikarya were induced by injections of quinolinic acid in to the A10 dopamine cell region. Unilateral lesions produced with 6-OHDA resulted in the loss of neurotensin receptors in the A10 region and within the terminal fields. Mu opioid receptors were unaffected by this treatment, but delta opioid receptors increased in the contralateral striatum and nucleus accumbens following 6-OHDA administration. Quinolinic acid produced a reduction of mu opioid receptors within the A10 region with a concomitant reduction in neurotensin receptors in both the cell body region and terminal fields. These results are consistent with a variety of biochemical and behavioral data which suggest the indirect modulation of dopamine transmission by the opioids. In contrast these results strongly indicate a direct modulation of the mesolimbic dopamine system by neurotensin.

  8. [Effect of opioid receptors on acute stress-induced changes in recognition memory].

    Liu, Ying; Wu, Yu-Wei; Qian, Zhao-Qiang; Yan, Cai-Fang; Fan, Ka-Min; Xu, Jin-Hui; Li, Xiao; Liu, Zhi-Qiang

    2016-12-25

    Although ample evidence has shown that acute stress impairs memory, the influences of acute stress on different phases of memory, such as acquisition, consolidation and retrieval, are different. Experimental data from both human and animals support that endogenous opioid system plays a role in stress, as endogenous opioid release is increased and opioid receptors are activated during stress experience. On the other hand, endogenous opioid system mediates learning and memory. The aim of the present study was to investigate the effect of acute forced swimming stress on recognition memory of C57 mice and the role of opioid receptors in this process by using a three-day pattern of new object recognition task. The results showed that 15-min acute forced swimming damaged the retrieval of recognition memory, but had no effect on acquisition and consolidation of recognition memory. No significant change of object recognition memory was found in mice that were given naloxone, an opioid receptor antagonist, by intraperitoneal injection. But intraperitoneal injection of naloxone before forced swimming stress could inhibit the impairment of recognition memory retrieval caused by forced swimming stress. The results of real-time PCR showed that acute forced swimming decreased the μ opioid receptor mRNA levels in whole brain and hippocampus, while the injection of naloxone before stress could reverse this change. These results suggest that acute stress may impair recognition memory retrieval via opioid receptors.

  9. Dopamine and μ-opioid receptor dysregulation in the brains of binge-eating female rats - possible relevance in the psychopathology and treatment of binge-eating disorder.

    Heal, David J; Hallam, Michelle; Prow, Michael; Gosden, Jane; Cheetham, Sharon; Choi, Yong K; Tarazi, Frank; Hutson, Peter

    2017-06-01

    Adult, female rats given irregular, limited access to chocolate develop binge-eating behaviour with normal bodyweight and compulsive/perseverative and impulsive behaviours similar to those in binge-eating disorder. We investigated whether (a) dysregulated central nervous system dopaminergic and opioidergic systems are part of the psychopathology of binge-eating and (b) these neurotransmitter systems may mediate the actions of drugs ameliorating binge-eating disorder psychopathology. Binge-eating produced a 39% reduction of striatal D 1 receptors with 22% and 23% reductions in medial and lateral caudate putamen and a 22% increase of striatal μ-opioid receptors. There was no change in D 1 receptor density in nucleus accumbens, medial prefrontal cortex or dorsolateral frontal cortex, striatal D 2 receptors and dopamine reuptake transporter sites, or μ-opioid receptors in frontal cortex. There were no changes in ligand affinities. The concentrations of monoamines, metabolites and estimates of dopamine (dopamine/dihydroxyphenylacetic acid ratio) and serotonin/5-hydroxyindolacetic acid ratio turnover rates were unchanged in striatum and frontal cortex. However, turnover of dopamine and serotonin in the hypothalamus was increased ~20% and ~15%, respectively. Striatal transmission via D 1 receptors is decreased in binge-eating rats while μ-opioid receptor signalling may be increased. These changes are consistent with the attenuation of binge-eating by lisdexamfetamine, which increases catecholaminergic neurotransmission, and nalmefene, a μ-opioid antagonist.

  10. Structure of the Nanobody-Stabilized Active State of the Kappa Opioid Receptor.

    Che, Tao; Majumdar, Susruta; Zaidi, Saheem A; Ondachi, Pauline; McCorvy, John D; Wang, Sheng; Mosier, Philip D; Uprety, Rajendra; Vardy, Eyal; Krumm, Brian E; Han, Gye Won; Lee, Ming-Yue; Pardon, Els; Steyaert, Jan; Huang, Xi-Ping; Strachan, Ryan T; Tribo, Alexandra R; Pasternak, Gavril W; Carroll, F Ivy; Stevens, Raymond C; Cherezov, Vadim; Katritch, Vsevolod; Wacker, Daniel; Roth, Bryan L

    2018-01-11

    The κ-opioid receptor (KOP) mediates the actions of opioids with hallucinogenic, dysphoric, and analgesic activities. The design of KOP analgesics devoid of hallucinatory and dysphoric effects has been hindered by an incomplete structural and mechanistic understanding of KOP agonist actions. Here, we provide a crystal structure of human KOP in complex with the potent epoxymorphinan opioid agonist MP1104 and an active-state-stabilizing nanobody. Comparisons between inactive- and active-state opioid receptor structures reveal substantial conformational changes in the binding pocket and intracellular and extracellular regions. Extensive structural analysis and experimental validation illuminate key residues that propagate larger-scale structural rearrangements and transducer binding that, collectively, elucidate the structural determinants of KOP pharmacology, function, and biased signaling. These molecular insights promise to accelerate the structure-guided design of safer and more effective κ-opioid receptor therapeutics. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Acute stimulation of brain mu opioid receptors inhibits glucose-stimulated insulin secretion via sympathetic innervation.

    Tudurí, Eva; Beiroa, Daniel; Stegbauer, Johannes; Fernø, Johan; López, Miguel; Diéguez, Carlos; Nogueiras, Rubén

    2016-11-01

    Pancreatic insulin-secreting β-cells express opioid receptors, whose activation by opioid peptides modulates hormone secretion. Opioid receptors are also expressed in multiple brain regions including the hypothalamus, where they play a role in feeding behavior and energy homeostasis, but their potential role in central regulation of glucose metabolism is unknown. Here, we investigate whether central opioid receptors participate in the regulation of insulin secretion and glucose homeostasis in vivo. C57BL/6J mice were acutely treated by intracerebroventricular (i.c.v.) injection with specific agonists for the three main opioid receptors, kappa (KOR), delta (DOR) and mu (MOR) opioid receptors: activation of KOR and DOR did not alter glucose tolerance, whereas activation of brain MOR with the specific agonist DAMGO blunted glucose-stimulated insulin secretion (GSIS), reduced insulin sensitivity, increased the expression of gluconeogenic genes in the liver and, consequently, impaired glucose tolerance. Pharmacological blockade of α2A-adrenergic receptors prevented DAMGO-induced glucose intolerance and gluconeogenesis. Accordingly, DAMGO failed to inhibit GSIS and to impair glucose tolerance in α2A-adrenoceptor knockout mice, indicating that the effects of central MOR activation on β-cells are mediated via sympathetic innervation. Our results show for the first time a new role of the central opioid system, specifically the MOR, in the regulation of insulin secretion and glucose metabolism. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Polymorphisms of the Kappa Opioid Receptor and Prodynorphin Genes: HIV risk and HIV Natural History

    Proudnikov, Dmitri; Randesi, Matthew; Levran, Orna; Yuferov, Vadim; Crystal, Howard; Ho, Ann; Ott, Jurg; Kreek, Mary Jeanne

    2013-01-01

    Objective Studies indicate cross-desensitization between opioid receptors (e.g., kappa opioid receptor, OPRK1), and chemokine receptors (e.g., CXCR4) involved in HIV infection. We tested whether gene variants of OPRK1 and its ligand, prodynorphin (PDYN), influence the outcome of HIV therapy. Methods Three study points, admission to the Women’s Interagency HIV Study (WIHS), initiation of highly active antiretroviral therapy (HAART) and the most recent visit were chosen for analysis as crucial events in the clinical history of the HIV patients. Regression analyses of 17 variants of OPRK1, and 11 variants of PDYN with change of viral load (VL) and CD4 count between admission and initiation of HAART, and initiation of HAART to the most recent visit to WIHS were performed in 598 HIV+ subjects including African Americans, Hispanics and Caucasians. Association with HIV status was done in 1009 subjects. Results Before HAART, greater VL decline (improvement) in carriers of PDYN IVS3+189C>T, and greater increase of CD4 count (improvement) in carriers of OPRK1 −72C>T, were found in African Americans. Also, greater increase of CD4 count in carriers of OPRK1 IVS2+7886A>G, and greater decline of CD4 count (deterioration) in carriers of OPRK1 −1205G>A, were found in Caucasians. After HAART, greater decline of VL in carriers of OPRK1 IVS2+2225G>A, and greater increase of VL in carriers of OPRK1 IVS2+10658G>T and IVS2+10963A>G, were found in Caucasians. Also, a lesser increase of CD4 count was found in Hispanic carriers of OPRK1 IVS2+2225G>A. Conclusion OPRK1 and PDYN polymorphisms may alter severity of HIV infection and response to treatment. PMID:23392455

  13. Effects of combining opioids and clinically available NMDA receptor antagonists in the treatment of pain.

    Snijdelaar, D.G.

    2005-01-01

    This thesis concerns the effects of combining opioids with clinically available NMDA receptor antagonists in the treatment of acute and chronic pain. There are a number of problems with the use of opioids, such as, the development of tolerance/hyperalgesia, the reduced effectiveness in (central)

  14. Morphine withdrawal enhances constitutive μ-opioid receptor activity in the ventral tegmental area

    Meye, F.J.; van Zessen, R.; Smidt, M.P.; Adan, R.A.H.; Ramakers, G.M.J.

    2012-01-01

    μ-opioid receptors (MORs) in the ventral tegmental area (VTA) are pivotally involved in addictive behavior. While MORs are typically activated by opioids, they can also become constitutively active in the absence of any agonist. In the current study, we present evidence that MOR constitutive

  15. Mycobacteria attenuate nociceptive responses by formyl peptide receptor triggered opioid peptide release from neutrophils.

    Heike L Rittner

    2009-04-01

    Full Text Available In inflammation, pain is regulated by a balance of pro- and analgesic mediators. Analgesic mediators include opioid peptides which are secreted by neutrophils at the site of inflammation, leading to activation of opioid receptors on peripheral sensory neurons. In humans, local opioids and opioid peptides significantly downregulate postoperative as well as arthritic pain. In rats, inflammatory pain is induced by intraplantar injection of heat inactivated Mycobacterium butyricum, a component of complete Freund's adjuvant. We hypothesized that mycobacterially derived formyl peptide receptor (FPR and/or toll like receptor (TLR agonists could activate neutrophils, leading to opioid peptide release and inhibition of inflammatory pain. In complete Freund's adjuvant-induced inflammation, thermal and mechanical nociceptive thresholds of the paw were quantified (Hargreaves and Randall-Selitto methods, respectively. Withdrawal time to heat was decreased following systemic neutrophil depletion as well as local injection of opioid receptor antagonists or anti-opioid peptide (i.e. Met-enkephalin, beta-endorphin antibodies indicating an increase in pain. In vitro, opioid peptide release from human and rat neutrophils was measured by radioimmunoassay. Met-enkephalin release was triggered by Mycobacterium butyricum and formyl peptides but not by TLR-2 or TLR-4 agonists. Mycobacterium butyricum induced a rise in intracellular calcium as determined by FURA loading and calcium imaging. Opioid peptide release was blocked by intracellular calcium chelation as well as phosphoinositol-3-kinase inhibition. The FPR antagonists Boc-FLFLF and cyclosporine H reduced opioid peptide release in vitro and increased inflammatory pain in vivo while TLR 2/4 did not appear to be involved. In summary, mycobacteria activate FPR on neutrophils, resulting in tonic secretion of opioid peptides from neutrophils and in a decrease in inflammatory pain. Future therapeutic strategies may aim

  16. Ligand recognition by RAR and RXR receptors: binding and selectivity.

    Sussman, Fredy; de Lera, Angel R

    2005-10-06

    Fundamental biological functions, most notably embriogenesis, cell growth, cell differentiation, and cell apoptosis, are in part regulated by a complex genomic network that starts with the binding (and activation) of retinoids to their cognate receptors, members of the superfamily of nuclear receptors. We have studied ligand recognition of retinoic receptors (RXRalpha and RARgamma) using a molecular-mechanics-based docking method. The protocol used in this work is able to rank the affinity of pairs of ligands for a single retinoid receptor, the highest values corresponding to those that adapt better to the shape of the binding site and generate the optimal set of electrostatic and apolar interactions with the receptor. Moreover, our studies shed light onto some of the energetic contributions to retinoid receptor ligand selectivity. In this regard we show that there is a difference in polarity between the binding site regions that anchor the carboxylate in RAR and RXR, which translates itself into large differences in the energy of interaction of both receptors with the same ligand. We observe that the latter energy change is canceled off by the solvation energy penalty upon binding. This energy compensation is borne out as well by experiments that address the effect of site-directed mutagenesis on ligand binding to RARgamma. The hypothesis that the difference in binding site polarity might be exploited to build RXR-selective ligands is tested with some compounds having a thiazolidinedione anchoring group.

  17. INTERACTION BETWEEN DELTA OPIOID RECEPTORS AND BENZODIAZEPINES IN CO2- INDUCED RESPIRATORY RESPONSES IN MICE

    Borkowski, Anne H.; Barnes, Dylan C.; Blanchette, Derek R.; Castellanos, F. Xavier; Klein, Donald F.; Wilson, Donald A.

    2011-01-01

    The false-suffocation hypothesis of panic disorder (Klein, 1993) suggested δ-opioid receptors as a possible source of the respiratory dysfunction manifested in panic attacks occurring in panic disorder (Preter and Klein, 2008). This study sought to determine if a lack of δ-opioid receptors in a mouse model affects respiratory response to elevated CO2, and whether the response is modulated by benzodiazepines, which are widely used to treat panic disorder. In a whole-body plethysmograph, respiratory responses to 5% CO2 were compared between δ-opioid receptor knockout mice and wild-type mice after saline, diazepam (1 mg/kg), and alprazolam (0.3 mg/kg) injection. The results show that lack of δ-opioid receptors does not affect normal response to elevated CO2, but does prevent benzodiazepines from modulating that response. Thus, in the presence of benzodiazepine agonists, respiratory responses to elevated CO2 were enhanced in δ-opioid receptor knockout mice compared to wild-type mice. This suggests an interplay between benzodiazepine receptors and δ-opioid receptors in regulating the respiratory effects of elevated CO2, which might be related to CO2 induced panic. PMID:21561601

  18. Epibatidine-derivatives: ligands for the neuronal nicotinic acetylcholine receptor

    Westera, G.; Patt, J.T.; Jankowski, K.; Bertrand, D.; Spang, J.; Schubiger, P.A.

    1997-01-01

    Epibatidine, isolated from the Ecuadorian frog Epipedobates tricolar, has been synthesized. 11 C-N-methyl derivate is investigated as useful nicotinergic receptor ligand by electrophysiological methods and in vivo mice experiments. (author) 2 figs., 7 refs

  19. Optimisation of in silico derived 2-aminobenzimidazole hits as unprecedented selective kappa opioid receptor agonists

    Sasmal, Pradip K; Krishna, C Vamsee; Sudheerkumar Adabala, S

    2015-01-01

    Kappa opioid receptor (KOR) is an important mediator of pain signaling and it is targeted for the treatment of various pains. Pharmacophore based mining of databases led to the identification of 2-aminobenzimidazole derivative as KOR agonists with selectivity over the other opioid receptors DOR a...... of novel benzimidazole derivatives as KOR agonists are described. The in vivo proof of principle for anti-nociceptive effect with a lead compound from this series is exemplified....

  20. Biotechnological Fluorescent Ligands of the Bradykinin B1 Receptor: Protein Ligands for a Peptide Receptor.

    Xavier Charest-Morin

    Full Text Available The bradykinin (BK B1 receptor (B1R is a peculiar G protein coupled receptor that is strongly regulated to the point of being inducible in immunopathology. Limited clinical evidence suggests that its expression in peripheral blood mononuclear cells is a biomarker of active inflammatory states. In an effort to develop a novel imaging/diagnostic tool, we report the rational design and testing of a fusion protein that is a ligand of the human B1R but not likely to label peptidases. This ligand is composed of a fluorescent protein (FP (enhanced green FP [EGFP] or mCherry prolonged at its N-terminus by a spacer peptide and a classical peptide agonist or antagonist (des-Arg9-BK, [Leu8]des-Arg9-BK, respectively. The design of the spacer-ligand joint peptide was validated by a competition assay for [3H]Lys-des-Arg9-BK binding to the human B1R applied to 4 synthetic peptides of 18 or 19 residues. The labeling of B1R-expressing cells with EGFP or mCherry fused with 7 of such peptides was performed in parallel (microscopy. Both assays indicated that the best design was FP-(Asn-Glyn-Lys-des-Arg9-BK; n = 15 was superior to n = 5, suggesting benefits from minimizing steric hindrance between the FP and the receptor. Cell labeling concerned mostly plasma membranes and was inhibited by a B1R antagonist. EGFP-(Asn-Gly15-Lys-des-Arg9-BK competed for the binding of [3H]Lys-des-Arg9-BK to human recombinant B1R, being only 10-fold less potent than the unlabeled form of Lys-des-Arg9-BK to do so. The fusion protein did not label HEK 293a cells expressing recombinant human BK B2 receptors or angiotensin converting enzyme. This study identifies a modular C-terminal sequence that can be adapted to protein cargoes, conferring high affinity for the BK B1R, with possible applications in diagnostic cytofluorometry, histology and drug delivery (e.g., in oncology.

  1. Tissue distribution of the death ligand TRAIL and its receptors

    Spierings, DC; de Vries, EG; Vellenga, E; van den Heuvel, FA; Koornstra, JJ; Wesseling, J; Hollema, H; de Jong, S

    Recombinant human (rh) TNF-related apoptosis-inducing ligand (TRAIL) harbors potential as an anticancer agent. RhTRAIL induces apoptosis via the TRAIL receptors TRAIL-R1 and TRAIL-R2 in tumors and is non-toxic to nonhuman primates. Because limited data are available about TRAIL receptor

  2. Kappa opioid receptors stimulate phosphoinositide turnover in rat brain

    Periyasamy, S.; Hoss, W. (Univ. of Toledo, OH (USA))

    1990-01-01

    The effects of various subtype-selective opioid agonists and antagonists on the phosphoinositide (PI) turnover response were investigated in the rat brain. The {kappa}-agonists U-50,488H and ketocyclazocine produced a concentration-dependent increase in the accumulation of IP's in hippocampal slices. The other {kappa}-agonists Dynorphin-A (1-13) amide, and its protected analog D(Ala){sup 2}-dynorphin-A (1-13) amide also produced a significant increase in the formation of ({sup 3}H)-IP's, whereas the {mu}-selective agonists (D-Ala{sup 2}-N-Me-Phe{sup 4}-Gly{sup 5}-ol)-enkephalin and morphine and the {delta}-selective agonist (D-Pen{sup 2,5})-enkephalin were ineffective. The increase in IP's formation elicited by U-50,488H was partially antagonized by naloxone and more completely antagonized by the {kappa}-selective antagonists nor-binaltorphimine and MR 2266. The formation of IP's induced by U-50,488H varies with the regions of the brain used, being highest in hippocampus and amygdala, and lowest in striatum and pons-medullar. The results indicate that brain {kappa}- but neither {mu}- nor {delta}- receptors are coupled to the PI turnover response.

  3. Stimulation of accumbal GABAA receptors inhibits delta2-, but not delta1-, opioid receptor-mediated dopamine efflux in the nucleus accumbens of freely moving rats.

    Aono, Yuri; Kiguchi, Yuri; Watanabe, Yuriko; Waddington, John L; Saigusa, Tadashi

    2017-11-15

    The nucleus accumbens contains delta-opioid receptors that may reduce inhibitory neurotransmission. Reduction in GABA A receptor-mediated inhibition of accumbal dopamine release due to delta-opioid receptor activation should be suppressed by stimulating accumbal GABA A receptors. As delta-opioid receptors are divided into delta2- and delta1-opioid receptors, we analysed the effects of the GABA A receptor agonist muscimol on delta2- and delta1-opioid receptor-mediated accumbal dopamine efflux in freely moving rats using in vivo microdialysis. Drugs were administered intracerebrally through the dialysis probe. Doses of compounds indicate total amount administered (mol) during 25-50min infusions. The delta2-opioid receptor agonist deltorphin II (25.0nmol)- and delta1-opioid receptor agonist DPDPE (5.0nmol)-induced increases in dopamine efflux were inhibited by the delta2-opioid receptor antagonist naltriben (1.5nmol) and the delta1-opioid receptor antagonist BNTX (150.0pmol), respectively. Muscimol (250.0pmol) inhibited deltorphin II (25.0nmol)-induced dopamine efflux. The GABA A receptor antagonist bicuculline (50.0pmol), which failed to affect deltorphin II (25.0nmol)-induced dopamine efflux, counteracted the inhibitory effect of muscimol on deltorphin II-induced dopamine efflux. Neither muscimol (250.0pmol) nor bicuculline (50.0 and 500.0pmol) altered DPDPE (5.0nmol)-induced dopamine efflux. The present results show that reduction in accumbal GABA A receptor-mediated inhibition of dopaminergic activity is necessary to produce delta2-opioid receptor-induced increase in accumbal dopamine efflux. This study indicates that activation of delta2- but not delta1-opioid receptors on the cell bodies and/or terminals of accumbal GABAergic interneurons inhibits GABA release and, accordingly, decreases GABA A receptor-mediated inhibition of dopaminergic terminals, resulting in enhanced accumbal dopamine efflux. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Inhibition of GABAergic Neurotransmission by HIV-1 Tat and Opioid Treatment in the Striatum Involves μ-opioid Receptors

    Changqing Xu

    2016-11-01

    Full Text Available Due to combined antiretroviral therapy (cART, human immunodeficiency virus type 1 (HIV-1 is considered a chronic disease with high prevalence of mild forms of neurocognitive impairments, also referred to as HIV-associated neurocognitive disorders (HAND. Although opiate drug use can exacerbate HIV-1 Tat-induced neuronal damage, it remains unknown how and to what extent opioids interact with Tat on the GABAergic system. We conducted whole-cell recordings in mouse striatal slices and examined the effects of HIV-1 Tat in the presence and absence of morphine (1 μM and damgo (1 μM on GABAergic neurotransmission. Results indicated a decrease in the frequency and amplitude of spontaneous inhibitory postsynaptic currents (sIPSCs and miniature IPSCs (mIPSCs by Tat (5 – 50 nM in a concentration-dependent manner. The significant Tat-induced decrease in IPSCs was abolished when removing extracellular and/or intracellular calcium. Treatment with morphine or damgo alone significantly decreased the frequency, but not amplitude of IPSCs. Interestingly, morphine but not damgo indicated an additional downregulation of the mean frequency of mIPSCs in combination with Tat. Pretreatment with naloxone (1 μM and CTAP (1 μM prevented the Tat-induced decrease in sIPSCs frequency but only naloxone prevented the combined Tat and morphine effect on mIPSCs frequency. Results indicate a Tat- or opioid-induced decrease in GABAergic neurotransmission via µ-opioid receptors with combined Tat and morphine effects involving additional opioid receptor-related mechanisms. Exploring the interactions between Tat and opioids on the GABAergic system may help to guide future research on HAND in the context of opiate drug use.

  5. Hypothalamic kappa opioid receptor mediates both diet- and MCH-induced liver damage through inflammation and ER stress

    Imbernon, Monica; Sanchez-Rebordelo, Estrella; Romero-Picó, Amparo; Kalló, Imre; Chee, Melissa J; Porteiro, Begoña; Al-Massadi, Omar; Contreras, Cristina; Fernø, Johan; Senra, Ana; Gallego, Rosalia; Folgueira, Cintia; Seoane, Luisa M; van Gestel, Margriet; Adan, Roger A; Liposits, Zsolt; Dieguez, Carlos; Lopez, Miguel; Nogueiras, Ruben

    2016-01-01

    The opioid system is widely known to modulate the brain reward system and thus affect human and animal behaviour, including feeding. We hypothesized that the hypothalamic opioid system might also control energy metabolism in peripheral tissues. Mice lacking the kappa opioid receptor (κOR) and

  6. Purification and characterization of mu-specific opioid receptor from rat brain

    Hasegawa, J.; Cho, T.M.; Ge, B.L.; Loh, H.H.

    1986-03-05

    A mu-specific opioid receptor was purified to apparent homogeneity from rat brain membranes by 6-succinylmorphine affinity chromatography, Ultrogel filtration, wheat germ agglutinin affinity chromatography, and isoelectric focusing. The purified receptor had a molecular weight of 58,000 as determined by polyacrylamide gel electrophoresis, and was judged to be homogeneous by the following criteria: (1) a single band on the SDS gel; and (2) a specific opioid binding activity of 17,720 pmole/mg protein, close to the theoretical value. In addition, the 58,000 molecular weight value agrees closely with that determined by covalently labelling purified receptor with bromoacetyl-/sup 3/H-dihydromorphine or with /sup 125/I-beta-endorphin and dimethyl suberimidate. To their knowledge, this is the first complete purification of an opioid receptor that retains its ability to bind opiates.

  7. Antinociceptive Action of Isolated Mitragynine from Mitragyna Speciosa through Activation of Opioid Receptor System

    Mohamad Aris Mohd Moklas

    2012-09-01

    Full Text Available Cannabinoids and opioids systems share numerous pharmacological properties and antinociception is one of them. Previous findings have shown that mitragynine (MG, a major indole alkaloid found in Mitragyna speciosa (MS can exert its antinociceptive effects through the opioids system. In the present study, the action of MG was investigated as the antinociceptive agent acting on Cannabinoid receptor type 1 (CB1 and effects on the opioids receptor. The latency time was recorded until the mice showed pain responses such as shaking, licking or jumping and the duration of latency was measured for 2 h at every 15 min interval by hot plate analysis. To investigate the beneficial effects of MG as antinociceptive agent, it was administered intraperitoneally 15 min prior to pain induction with a single dosage (3, 10, 15, 30, and 35 mg/kg b.wt. In this investigation, 35 mg/kg of MG showed significant increase in the latency time and this dosage was used in the antagonist receptor study. The treated groups were administered with AM251 (cannabinoid receptor-1 antagonist, naloxone (non-selective opioid antagonist, naltrindole (δ-opioid antagonist naloxonazine (µ1-receptor antagonist and norbinaltorpimine (κ-opioid antagonist respectively, prior to administration of MG (35 mg/kg. The results showed that the antinociceptive effect of MG was not antagonized by AM251; naloxone and naltrindole were effectively blocked; and norbinaltorpimine partially blocked the antinociceptive effect of MG. Naloxonazine did inhibit the effect of MG, but it was not statistically significant. These results demonstrate that CB1 does not directly have a role in the antinociceptive action of MG where the effect was observed with the activation of opioid receptor.

  8. Dehydroepiandrosterone: an ancestral ligand of neurotrophin receptors.

    Pediaditakis, Iosif; Iliopoulos, Ioannis; Theologidis, Ioannis; Delivanoglou, Nickoleta; Margioris, Andrew N; Charalampopoulos, Ioannis; Gravanis, Achille

    2015-01-01

    Dehydroepiandosterone (DHEA), the most abundant steroid in humans, affects multiple cellular functions of the endocrine, immune, and nervous systems. However, up to quite recently, no receptor has been described specifically for it, whereas most of its physiological actions have been attributed to its conversion to either androgens or estrogens. DHEA interacts and modulate a variety of membrane and intracellular neurotransmitter and steroid receptors. We have recently reported that DHEA protects neuronal cells against apoptosis, interacting with TrkA, the high-affinity prosurvival receptor of the neurotrophin, nerve growth factor. Intrigued by its pleiotropic effects in the nervous system of a variety of species, we have investigated the ability of DHEA to interact with the other two mammalian neurotrophin receptors, ie, the TrkB and TrkC, as well as their invertebrate counterparts (orthologs) in mollusks Lymnaea and Aplysia and in cephalochordate fish Amphioxus. Amazingly, DHEA binds to all Trk receptors, although with lower affinity by 2 orders of magnitude compared with that of the polypeptidic neurotrophins. DHEA effectively induced the first step of the TrkA and TrkC receptors activation (phosphorylation at tyrosine residues), including the vertebrate neurotrophin nonresponding invertebrate Lymnaea and Aplysia receptors. Based on our data, we hypothesize that early in evolution, DHEA may have acted as a nonspecific neurotrophic factor promoting neuronal survival. The interaction of DHEA with all types of neurotrophin receptors offers new insights into the largely unidentified mechanisms of its actions on multiple tissues and organs known to express neurotrophin receptors.

  9. Amygdala mu-opioid receptors mediate the motivating influence of cue-triggered reward expectations.

    Lichtenberg, Nina T; Wassum, Kate M

    2017-02-01

    Environmental reward-predictive stimuli can retrieve from memory a specific reward expectation that allows them to motivate action and guide choice. This process requires the basolateral amygdala (BLA), but little is known about the signaling systems necessary within this structure. Here we examined the role of the neuromodulatory opioid receptor system in the BLA in such cue-directed action using the outcome-specific Pavlovian-to-instrumental transfer (PIT) test in rats. Inactivation of BLA mu-, but not delta-opioid receptors was found to dose-dependently attenuate the ability of a reward-predictive cue to selectively invigorate the performance of actions directed at the same unique predicted reward (i.e. to express outcome-specific PIT). BLA mu-opioid receptor inactivation did not affect the ability of a reward itself to similarly motivate action (outcome-specific reinstatement), suggesting a more selective role for the BLA mu-opioid receptor in the motivating influence of currently unobservable rewarding events. These data reveal a new role for BLA mu-opioid receptor activation in the cued recall of precise reward memories and the use of this information to motivate specific action plans. © 2016 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

  10. Evaluation of Analgesic Activity of Papaver libanoticum Extract in Mice: Involvement of Opioids Receptors

    Mohamad Ali Hijazi

    2017-01-01

    Full Text Available Papaver libanoticum is an endemic plant to Lebanese region (family Papaveraceae that has not been investigated before. The present study aimed to explore the analgesic activity of dried ethanolic extract of Papaver libanoticum (PLE using tail flick, hot plate, and acetic acid induced writhing models in mice. The involvement of opioid receptors in the analgesic mechanism was investigated using naloxone antagonism. Results demonstrated that PLE exhibited a potent dose dependent analgesic activity in all tested models for analgesia. The analgesic effect involved activation of opioid receptors in the central nervous system, where both spinal and supraspinal components might be involved. The time course for analgesia revealed maximum activity after three hours in both tail flick and hot plate methods, which was prolonged to 24 hours. Metabolites of PLE could be responsible for activation of opioid receptors. The EC50 of PLE was 79 and 50 mg/kg in tail flick and hot plate tests, respectively. The total coverage of analgesia by PLE was double that of morphine in both tests. In conclusion, PLE proved to have opioid agonistic activity with a novel feature of slow and prolonged effect. The present study could add a potential tool in the armaments of opioid drugs as a natural potent analgesic and for treatment of opioid withdrawal syndrome.

  11. Sex Differences in Kappa Opioid Receptor Function and Their Potential Impact on Addiction

    Chartoff, Elena H.; Mavrikaki, Maria

    2015-01-01

    Behavioral, biological, and social sequelae that lead to drug addiction differ between men and women. Our efforts to understand addiction on a mechanistic level must include studies in both males and females. Stress, anxiety, and depression are tightly linked to addiction, and whether they precede or result from compulsive drug use depends on many factors, including biological sex. The neuropeptide dynorphin (DYN), an endogenous ligand at kappa opioid receptors (KORs), is necessary for stress-induced aversive states and is upregulated in the brain after chronic exposure to drugs of abuse. KOR agonists produce signs of anxiety, fear, and depression in laboratory animals and humans, findings that have led to the hypothesis that drug withdrawal-induced DYN release is instrumental in negative reinforcement processes that drive addiction. However, these studies were almost exclusively conducted in males. Only recently is evidence available that there are sex differences in the effects of KOR activation on affective state. This review focuses on sex differences in DYN and KOR systems and how these might contribute to sex differences in addictive behavior. Much of what is known about how biological sex influences KOR systems is from research on pain systems. The basic molecular and genetic mechanisms that have been discovered to underlie sex differences in KOR function in pain systems may apply to sex differences in KOR function in reward systems. Our goals are to discuss the current state of knowledge on how biological sex contributes to KOR function in the context of pain, mood, and addiction and to explore potential mechanisms for sex differences in KOR function. We will highlight evidence that the function of DYN-KOR systems is influenced in a sex-dependent manner by: polymorphisms in the prodynorphin (pDYN) gene, genetic linkage with the melanocortin-1 receptor (MC1R), heterodimerization of KORs and mu opioid receptors (MORs), and gonadal hormones. Finally, we

  12. Sex differences in kappa opioid receptor function and their potential impact on addiction

    Elena eChartoff

    2015-12-01

    Full Text Available Behavioral, biological and social sequelae that lead to drug addiction differ between men and women. Our efforts to understand addiction on a mechanistic level must include studies in both males and females. Stress, anxiety, and depression are tightly linked to addiction, and whether they precede or result from compulsive drug use depends on many factors, including biological sex. The neuropeptide dynorphin (DYN, an endogenous ligand at kappa opioid receptors (KORs, is necessary for stress-induced aversive states and is upregulated in the brain after chronic exposure to drugs of abuse. KOR agonists produce signs of anxiety, fear, and depression in laboratory animals and humans, findings that have led to the hypothesis that drug withdrawal-induced DYN release is instrumental in negative reinforcement processes that drive addiction. However, these studies were almost exclusively conducted in males. Only recently is evidence available that there are sex differences in the effects of KOR activation on affective state. This review focuses on sex differences in DYN and KOR systems and how these might contribute to sex differences in addictive behavior. Much of what is known about how biological sex influences KOR systems is from research on pain systems. The basic molecular and genetic mechanisms that have been discovered to underlie sex differences in KOR function in pain systems may apply to sex differences in KOR function in reward systems. Our goals are to discuss the current state of knowledge on how biological sex contributes to KOR function in the context of pain,mood and addiction and to explore potential mechanisms for sex differences in KOR function. We will highlight evidence that the function of DYN-KOR systems is influenced in a sex-dependent manner by: polymorphisms in the prodynorphin (pDYN gene, genetic linkage with the melanocortin-1 receptor (MC1R, heterodimerization of KORs and mu opioid receptors (MORs, and gonadal hormones

  13. Sex Differences in Kappa Opioid Receptor Function and Their Potential Impact on Addiction.

    Chartoff, Elena H; Mavrikaki, Maria

    2015-01-01

    Behavioral, biological, and social sequelae that lead to drug addiction differ between men and women. Our efforts to understand addiction on a mechanistic level must include studies in both males and females. Stress, anxiety, and depression are tightly linked to addiction, and whether they precede or result from compulsive drug use depends on many factors, including biological sex. The neuropeptide dynorphin (DYN), an endogenous ligand at kappa opioid receptors (KORs), is necessary for stress-induced aversive states and is upregulated in the brain after chronic exposure to drugs of abuse. KOR agonists produce signs of anxiety, fear, and depression in laboratory animals and humans, findings that have led to the hypothesis that drug withdrawal-induced DYN release is instrumental in negative reinforcement processes that drive addiction. However, these studies were almost exclusively conducted in males. Only recently is evidence available that there are sex differences in the effects of KOR activation on affective state. This review focuses on sex differences in DYN and KOR systems and how these might contribute to sex differences in addictive behavior. Much of what is known about how biological sex influences KOR systems is from research on pain systems. The basic molecular and genetic mechanisms that have been discovered to underlie sex differences in KOR function in pain systems may apply to sex differences in KOR function in reward systems. Our goals are to discuss the current state of knowledge on how biological sex contributes to KOR function in the context of pain, mood, and addiction and to explore potential mechanisms for sex differences in KOR function. We will highlight evidence that the function of DYN-KOR systems is influenced in a sex-dependent manner by: polymorphisms in the prodynorphin (pDYN) gene, genetic linkage with the melanocortin-1 receptor (MC1R), heterodimerization of KORs and mu opioid receptors (MORs), and gonadal hormones. Finally, we

  14. Distinct roles of exogenous opioid agonists and endogenous opioid peptides in the peripheral control of neuropathy-triggered heat pain.

    Labuz, Dominika; Celik, Melih Ö; Zimmer, Andreas; Machelska, Halina

    2016-09-08

    Neuropathic pain often results from peripheral nerve damage, which can involve immune response. Local leukocyte-derived opioid peptides or exogenous opioid agonists inhibit neuropathy-induced mechanical hypersensitivity in animal models. Since neuropathic pain can also be augmented by heat, in this study we investigated the role of opioids in the modulation of neuropathy-evoked heat hypersensitivity. We used a chronic constriction injury of the sciatic nerve in wild-type and opioid peptide-knockout mice, and tested opioid effects in heat and mechanical hypersensitivity using Hargreaves and von Frey tests, respectively. We found that although perineural exogenous opioid agonists, including peptidergic ligands, were effective, the endogenous opioid peptides β-endorphin, Met-enkephalin and dynorphin A did not alleviate heat hypersensitivity. Specifically, corticotropin-releasing factor, an agent triggering opioid peptide secretion from leukocytes, applied perineurally did not attenuate heat hypersensitivity in wild-type mice. Exogenous opioids, also shown to release opioid peptides via activation of leukocyte opioid receptors, were equally analgesic in wild-type and opioid peptide-knockout mice, indicating that endogenous opioids do not contribute to exogenous opioid analgesia in heat hypersensitivity. Furthermore, exogenously applied opioid peptides were ineffective as well. Conversely, opioid peptides relieved mechanical hypersensitivity. Thus, both opioid type and sensory modality may determine the outcome of neuropathic pain treatment.

  15. Endogenous opioid peptide-mediated neurotransmission in central and pericentral nuclei of the inferior colliculus recruits μ1-opioid receptor to modulate post-ictal antinociception.

    Felippotti, Tatiana Tocchini; de Freitas, Renato Leonardo; Coimbra, Norberto Cysne

    2012-02-01

    The aim of the present work was to investigate the involvement of the μ1-endogenous opioid peptide receptor-mediated system in post-ictal antinociception. Antinociceptive responses were determined by the tail-flick test after pre-treatment with the selective μ1-opioid receptor antagonist naloxonazine, peripherally or centrally administered at different doses. Peripheral subchronic (24 h) pre-treatment with naloxonazine antagonised the antinociception elicited by tonic-clonic seizures. Acute (10 min) pre-treatment, however, did not have the same effect. In addition, microinjections of naloxonazine into the central, dorsal cortical and external cortical nuclei of the inferior colliculus antagonised tonic-clonic seizure-induced antinociception. Neither acute (10-min) peripheral pre-treatment with naloxonazine nor subchronic intramesencephalic blockade of μ1-opioid receptors resulted in consistent statistically significant differences in the severity of tonic-clonic seizures shown by Racine's index (1972), although the intracollicular specific antagonism of μ1-opioid receptor decreased the duration of seizures. μ1-Opioid receptors and the inferior colliculus have been implicated in several endogenous opioid peptide-mediated responses such as antinociception and convulsion. The present findings suggest the involvement of μ1-opiate receptors of central and pericentral nuclei of the inferior colliculus in the modulation of tonic-clonic seizures and in the organisation of post-ictal antinociception. Copyright © 2011 Elsevier Ltd. All rights reserved.

  16. Opioid and GABAB receptors differentially couple to an adenylyl cyclase/protein kinase A downstream effector after chronic morphine treatment.

    Elena Elizabeth Bagley

    2014-06-01

    Full Text Available Opioids are intensely addictive, and cessation of their chronic use is associated with a highly aversive withdrawal syndrome. A cellular hallmark of withdrawal is an opioid sensitive protein kinase A-dependent increase in GABA transporter-1 (GAT-1 currents in periaqueductal gray (PAG neurons. Elevated GAT-1 activity directly increases GABAergic neuronal excitability and synaptic GABA release, which will enhance GABAergic inhibition of PAG output neurons. This reduced activity of PAG output neurons to several brain regions, including the hypothalamus and medulla, contributes to many of the PAG-mediated signs of opioid withdrawal. The GABAB receptor agonist baclofen reduces some of the PAG mediated signs of opioid withdrawal. Like the opioid receptors the GABAB receptor is a Gi/Go coupled G-protein coupled receptor. This suggests it could be modulating GAT-1 activity in PAG neurons through its inhibition of the adenylyl cyclase/protein kinase A pathway. Opioid modulation of the GAT-1 activity can be detected by changes in the reversal potential of opioid membrane currents. We found that when opioids are reducing the GAT-1 cation conductance and increasing the GIRK conductance the opioid agonist reversal potential is much more negative than Ek. Using this approach for GABAB receptors we show that the GABAB receptor agonist, baclofen, does not couple to inhibition of GAT-1 currents during opioid withdrawal. It is possible this differential signaling of the two Gi/Go coupled G-protein coupled receptors is due to the strong compartmentalization of the GABAB receptor that does not favor signaling to the adenylyl cyclase/protein kinase A/GAT-1 pathway. This highlights the importance of studying the effects of G-protein coupled receptors in native tissue with endogenous G-protein coupled receptors and the full complement of relevant proteins and signaling molecules. This study suggests that baclofen reduces opioid withdrawal symptoms through a non-GAT-1

  17. Plasticity of Signaling by Spinal Estrogen Receptor α, κ-Opioid Receptor, and Metabotropic Glutamate Receptors over the Rat Reproductive Cycle Regulates Spinal Endomorphin 2 Antinociception: Relevance of Endogenous-Biased Agonism.

    Liu, Nai-Jiang; Murugaiyan, Vijaya; Storman, Emiliya M; Schnell, Stephen A; Kumar, Arjun; Wessendorf, Martin W; Gintzler, Alan R

    2017-11-15

    We previously showed that intrathecal application of endomorphin 2 [EM2; the highly specific endogenous μ-opioid receptor (MOR) ligand] induces antinociception that varies with stage of the rat estrous cycle: minimal during diestrus and prominent during proestrus. Earlier studies, however, did not identify proestrus-activated signaling strategies that enable spinal EM2 antinociception. We now report that in female rats, increased spinal dynorphin release and κ-opioid receptor (KOR) signaling, as well as the emergence of glutamate-activated metabotropic glutamate receptor 1 (mGluR 1 ) signaling, are critical to the transition from an EM2 nonresponsive state (during diestrus) to an analgesically responsive state (during proestrus). Differential signaling by mGluR 1 , depending on its activation by membrane estrogen receptor α (mERα; during diestrus) versus glutamate (during proestrus), concomitant with the ebb and flow of spinal dynorphin/KOR signaling, functions as a switch, preventing or promoting, respectively, spinal EM2 antinociception. Importantly, EM2 and glutamate-containing varicosities appose spinal neurons that express MOR along with mGluRs and mERα, suggesting that signaling mechanisms regulating analgesic effectiveness of intrathecally applied EM2 also pertain to endogenous EM2. Regulation of spinal EM2 antinociception by both the nature of the endogenous mGluR 1 activator (i.e., endogenous biased agonism at mGluR 1 ) and changes in spinal dynorphin/KOR signaling represent a novel mechanism for modulating analgesic responsiveness to endogenous EM2 (and perhaps other opioids). This points the way for developing noncanonical pharmacological approaches to pain management by harnessing endogenous opioids for pain relief. SIGNIFICANCE STATEMENT The current prescription opioid abuse epidemic underscores the urgency to develop alternative pharmacotherapies for managing pain. We find that the magnitude of spinal endomorphin 2 (EM2) antinociception not only

  18. Neuroanatomical patterns of the mu, delta, and kappa opioid receptors of rat brain as determined by quantitative in vitro autoradiography

    Tempel, A.; Zukin, R.S.

    1987-01-01

    Highly specific radioligands and quantitative autoradiography reveal strikingly different neuroanatomical patterns for the mu, delta, and kappa opioid receptors of rat brain. The mu receptors are most densely localized in patches in the striatum, layers I and III of the cortex, the pyramidal cell layer of the hippocampal formation, specific nuclei of the thalamus, the pars reticulata of the substantia nigra, the interpeduncular nucleus, and the locus coeruleus. In contrast, delta receptors are highly confined, exhibiting selective localization in layers I, II, and VIa of the neocortex, a diffuse pattern in the striatum, and moderate concentration in the pars reticulata of the substantia nigra and in the interpeduncular nucleus. delta receptors are absent in most other brain structures. This distribution is unexpected in that the enkephalins, the putative endogenous ligands of the delta receptor, occur essentially throughout the brain. The kappa receptors of rat brain exhibit a third pattern distinct from that of the mu and delta receptors. kappa receptors occur at low density in patches in the striatum and at particularly high density in the nucleus accumbens, along the pyramidal and molecular layers of the hippocampus, in the granular cell layer of the dentate gyrus, specific midline nuclei of the thalamus, and hindbrain regions. kappa receptors appear to be uniformly distributed across regions in the neocortex with the exception of layer III, which revealed only trace levels of binding. An important conclusion of the present study is that delta receptors occur at high density only in the forebrain and in two midbrain structures, whereas mu and kappa receptors exhibit discrete patterns in most major brain regions

  19. New features of the delta opioid receptor: conformational properties of deltorphin I analogues.

    Balboni, G; Marastoni, M; Picone, D; Salvadori, S; Tancredi, T; Temussi, P A; Tomatis, R

    1990-06-15

    Deltorphin I is an opioid peptide of sequence H-Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2, recently isolated from the skin of Phyllomedusa bicolor. Its enormous selectivity towards the delta opioid receptor and the similarity of the conformation of the N-terminal part of the sequence with that of dermorphin (H-Tyr-D-Ala-he-Gly-Tyr-Pro-Ser-NH2), a mu selective peptide, prompted the synthesis, biological evaluation and comparative conformational study of four analogs. A 1H-NMR study showed that the conformational preferences of the N-terminal sequences of all peptides are similar. The different selectivities towards opioid receptors have been interpreted in terms of charge effects in the interaction with the membrane and at the receptor site and of hydrophobicity of the C-terminal part, when structured in a folded conformation.

  20. On the Modularity of the Intrinsic Flexibility of the µ Opioid Receptor: A Computational Study

    Fossépré, Mathieu; Leherte, Laurence; Laaksonen, Aatto; Vercauteren, Daniel P.

    2014-01-01

    The µ opioid receptor (µOR), the principal target to control pain, belongs to the G protein-coupled receptors (GPCRs) family, one of the most highlighted protein families due to their importance as therapeutic targets. The conformational flexibility of GPCRs is one of their essential characteristics as they take part in ligand recognition and subsequent activation or inactivation mechanisms. It is assessed that the intrinsic mechanical properties of the µOR, more specifically its particular flexibility behavior, would facilitate the accomplishment of specific biological functions, at least in their first steps, even in the absence of a ligand or any chemical species usually present in its biological environment. The study of the mechanical properties of the µOR would thus bring some indications regarding the highly efficient ability of the µOR to transduce cellular message. We therefore investigate the intrinsic flexibility of the µOR in its apo-form using all-atom Molecular Dynamics simulations at the sub-microsecond time scale. We particularly consider the µOR embedded in a simplified membrane model without specific ions, particular lipids, such as cholesterol moieties, or any other chemical species that could affect the flexibility of the µOR. Our analyses highlighted an important local effect due to the various bendability of the helices resulting in a diversity of shape and volume sizes adopted by the µOR binding site. Such property explains why the µOR can interact with ligands presenting highly diverse structural geometry. By investigating the topology of the µOR binding site, a conformational global effect is depicted: the correlation between the motional modes of the extra- and intracellular parts of µOR on one hand, along with a clear rigidity of the central µOR domain on the other hand. Our results show how the modularity of the µOR flexibility is related to its pre-ability to activate and to present a basal activity. PMID:25549261

  1. Regulation of extinction-related plasticity by opioid receptors in the ventrolateral periaqueductal gray matter

    Ryan Parsons

    2010-08-01

    Full Text Available Recent work has led to a better understanding of the neural mechanisms underlying the extinction of Pavlovian fear conditioning. Long-term synaptic changes in the medial prefrontal cortex (mPFC are critical for extinction learning, but very little is currently known about how the mPFC and other brain areas interact during extinction. The current study examined the effect of drugs that impair the extinction of fear conditioning on the activation of the extracellular-related kinase/mitogen-activated protein kinase (ERK/MAPK in brain regions that likely participate in the consolidation of extinction learning. Inhibitors of opioid and N-methyl-D-aspartic acid (NMDA receptors were applied to the ventrolateral periaqueductal gray matter (vlPAG and amygdala shortly before extinction training. Results from these experiments show that blocking opioid receptors in the vlPAG prevented the formation of extinction memory, whereas NMDA receptor blockade had no effect. Conversely, blocking NMDA receptors in the amygdala disrupted the formation of fear extinction memory, but opioid receptor blockade in the same brain area did not. Subsequent experiments tested the effect of these drug treatments on the activation of the ERK/MAPK signaling pathway in various brain regions following extinction training. Only opioid receptor blockade in the vlPAG disrupted ERK phosphorylation in the mPFC and amygdala. These data support the idea that opiodergic signaling derived from the vlPAG affects plasticity across the brain circuit responsible for the formation of extinction memory.

  2. Electroacupuncture-Induced Dynamic Processes of Gene Expression Levels of Endogenous Opioid Peptide Precursors and Opioid Receptors in the CNS of Goats

    Li-Li Cheng

    2013-01-01

    Full Text Available In order to investigate the dynamic processes of mRNA levels of proenkephalin, proopiomelanocortin, prodynorphin, and opioid receptors (δ-, μ-, and κ-receptor induced by electroacupuncture (EA in the central nerve system, goats were stimulated by EA of 60 Hz for 0.5 h at a set of Baihui, Santai, Ergen, and Sanyangluo points. The pain threshold was measured using the method of potassium iontophoresis. The mRNA levels of the three opioid peptide precursors and three opioid receptors were determined with quantitative real-time PCR and the levels of Met-enkephalin with SABC immunohistochemistry at 0.5 h before and at 0, 2, 4, 6, 8, 12, and 24 h after EA. The results showed that the pain threshold correlated (P<0.01 with Met-enkephalin immunoactivities in the measured nuclei and areas of goats. The analgesic aftereffect lasted for 12 h at least. The mRNA levels of the three opioid peptide precursors and three opioid receptors began to increase at 0 h, reached the peak during the time from 4 h to 6 h or at 12 h, and remained higher at 24 h after EA was discontinued. These results suggested that the initiation of gene expression of opioid peptides and the three receptors may be associated with EA-induced analgesic aftereffect.

  3. Kinetic analysis of transport and opioid receptor binding of ( sup 3 H)(-)-cyclofoxy in rat brain in vivo: Implications for human studies

    Sawada, Y.; Kawai, R.; McManaway, M.; Otsuki, H.; Rice, K.C.; Patlak, C.S.; Blasberg, R.G. (National Institutes of Health, Bethesda, MD (USA))

    1991-03-01

    (3H)Cyclofoxy (CF: 17-cyclopropylmethyl-3,14-dihydroxy-4,5-alpha-epoxy-6-beta-fluoromorp hinan) is an opioid antagonist with affinity to both mu and kappa subtypes that was synthesized for quantitative evaluation of opioid receptor binding in vivo. Two sets of experiments in rats were analyzed. The first involved determining the metabolite-corrected blood concentration and tissue distribution of CF in brain 1 to 60 min after i.v. bolus injection. The second involved measuring brain washout for 15 to 120 s following intracarotid artery injection of CF. A physiologically based model and a classical compartmental pharmacokinetic model were compared. The models included different assumptions for transport across the blood-brain barrier (BBB); estimates of nonspecific tissue binding and specific binding to a single opiate receptor site were found to be essentially the same with both models. The nonspecific binding equilibrium constant varied modestly in different brain structures (Keq = 3-9), whereas the binding potential (BP) varied over a much broader range (BP = 0.6-32). In vivo estimates of the opioid receptor dissociation constant were similar for different brain structures (KD = 2.1-5.2 nM), whereas the apparent receptor density (Bmax) varied between 1 (cerebellum) and 78 (thalamus) pmol/g of brain. The receptor dissociation rate constants in cerebrum (k4 = 0.08-0.16 min-1; koff = 0.16-0.23 min-1) and brain vascular permeability (PS = 1.3-3.4 ml/min/g) are sufficiently high to achieve equilibrium conditions within a reasonable period of time. Graphical analysis of the data is inappropriate due to the high tissue-loss rate constant for CF in brain. From these findings, CF should be a very useful opioid receptor ligand for the estimation of the receptor binding parameters in human subjects using (18F)CF and positron emission tomography.

  4. Kinetic analysis of transport and opioid receptor binding of [3H](-)-cyclofoxy in rat brain in vivo: Implications for human studies

    Sawada, Y.; Kawai, R.; McManaway, M.; Otsuki, H.; Rice, K.C.; Patlak, C.S.; Blasberg, R.G.

    1991-01-01

    [3H]Cyclofoxy (CF: 17-cyclopropylmethyl-3,14-dihydroxy-4,5-alpha-epoxy-6-beta-fluoromorp hinan) is an opioid antagonist with affinity to both mu and kappa subtypes that was synthesized for quantitative evaluation of opioid receptor binding in vivo. Two sets of experiments in rats were analyzed. The first involved determining the metabolite-corrected blood concentration and tissue distribution of CF in brain 1 to 60 min after i.v. bolus injection. The second involved measuring brain washout for 15 to 120 s following intracarotid artery injection of CF. A physiologically based model and a classical compartmental pharmacokinetic model were compared. The models included different assumptions for transport across the blood-brain barrier (BBB); estimates of nonspecific tissue binding and specific binding to a single opiate receptor site were found to be essentially the same with both models. The nonspecific binding equilibrium constant varied modestly in different brain structures (Keq = 3-9), whereas the binding potential (BP) varied over a much broader range (BP = 0.6-32). In vivo estimates of the opioid receptor dissociation constant were similar for different brain structures (KD = 2.1-5.2 nM), whereas the apparent receptor density (Bmax) varied between 1 (cerebellum) and 78 (thalamus) pmol/g of brain. The receptor dissociation rate constants in cerebrum (k4 = 0.08-0.16 min-1; koff = 0.16-0.23 min-1) and brain vascular permeability (PS = 1.3-3.4 ml/min/g) are sufficiently high to achieve equilibrium conditions within a reasonable period of time. Graphical analysis of the data is inappropriate due to the high tissue-loss rate constant for CF in brain. From these findings, CF should be a very useful opioid receptor ligand for the estimation of the receptor binding parameters in human subjects using [18F]CF and positron emission tomography

  5. Synthesis of [3]DIPPA: a potent irreversible antagonist selective for the κ opioid receptor

    Chang, Anchih; Portoghese, P.S.

    1995-01-01

    2-(3,4-Dichlorophenyl)-N-methyl-N-[(1S)-1-(3-isothiocyanatophe nyl)-2-(1-pyrrolidinyl)ethyl]acetamide (1,DIPPA) has been previously reported to be an opioid receptor affinity label that produces selective and long-lasting κ opioid receptor antagonism in mice. High specific activity [ 3 H]DIPPA (39.7 Ci/mmol) was prepared by bromination and catalytic tritiation of the amino precursor of DIPPA followed by conversion to the isothiocyanate with thiophosgene. (Author)

  6. Mu receptor binding of some commonly used opioids and their metabolites

    Chen, Zhaorong; Irvine, R.J.; Somogyi, A.A.; Bochner, F.

    1991-01-01

    The binding affinity to the μ receptor of some opioids chemically related to morphine and some of their metabolites was examined in rat brain homogenates with 3 H-DAMGO. The chemical group at position 6 of the molecule had little effect on binding. Decreasing the length of the alkyl group at position 3 decreased the K i values (morphine < codeine < ethylmorphine < pholcodine). Analgesics with high clinical potency containing a methoxyl group at position 3 had relatively weak receptor binding, while their O-demethylated metabolites had much stronger binding. Many opioids may exert their pharmacological actions predominantly through metabolites

  7. Mu receptor binding of some commonly used opioids and their metabolites

    Chen, Zhaorong; Irvine, R.J. (Univ. of Adelaide (Australia)); Somogyi, A.A.; Bochner, F. (Univ. of Adelaide (Australia) Royal Adelaide Hospital (Australia))

    1991-01-01

    The binding affinity to the {mu} receptor of some opioids chemically related to morphine and some of their metabolites was examined in rat brain homogenates with {sup 3}H-DAMGO. The chemical group at position 6 of the molecule had little effect on binding. Decreasing the length of the alkyl group at position 3 decreased the K{sub i} values (morphine < codeine < ethylmorphine < pholcodine). Analgesics with high clinical potency containing a methoxyl group at position 3 had relatively weak receptor binding, while their O-demethylated metabolites had much stronger binding. Many opioids may exert their pharmacological actions predominantly through metabolites.

  8. Mu opioid receptor availability in people with psychiatric disorders who died by suicide: a case control study

    Scarr Elizabeth

    2012-08-01

    Full Text Available Abstract Background Mu opioid receptors have previously been shown to be altered in people with affective disorders who died as a result of suicide. We wished to determine whether these changes were more widespread and independent of psychiatric diagnoses. Methods Mu receptor levels were determined using [3 H]DAMGO binding in BA24 from 51 control subjects; 38 people with schizophrenia (12 suicides; 20 people with major depressive disorder (15 suicides; 13 people with bipolar disorder (5 suicides and 9 people who had no history of psychiatric disorders but who died as a result of suicide. Mu receptor levels were further determined in BA9 and caudate-putamen from 38 people with schizophrenia and 20 control subjects using [3 H]DAMGO binding and, in all three regions, using Western blots. Data was analysed using one-way ANOVAs with Bonferroni’s Multiple Comparison Test or, where data either didn’t approximate to a binomial distribution or the sample size was too small to determine distribution, a Kruskal-Wallis test with Dunn’s Multiple Comparison Test. Results [3 H]DAMGO binding density was lower in people who had died as a result of suicide (p3 H]DAMGO binding densities, but not mu protein levels, were significantly decreased in BA9 from people with schizophrenia who died as a result of suicide (p Conclusions Overall these data suggest that mu opioid receptor availability is decreased in the brains of people with schizophrenia who died as a result of suicide, which would be consistent with increased levels of endogenous ligands occupying these receptors.

  9. Supersensitive Kappa Opioid Receptors Promotes Ethanol Withdrawal-Related Behaviors and Reduce Dopamine Signaling in the Nucleus Accumbens.

    Rose, Jamie H; Karkhanis, Anushree N; Chen, Rong; Gioia, Dominic; Lopez, Marcelo F; Becker, Howard C; McCool, Brian A; Jones, Sara R

    2016-05-01

    Chronic ethanol exposure reduces dopamine transmission in the nucleus accumbens, which may contribute to the negative affective symptoms associated with ethanol withdrawal. Kappa opioid receptors have been implicated in withdrawal-induced excessive drinking and anxiety-like behaviors and are known to inhibit dopamine release in the nucleus accumbens. The effects of chronic ethanol exposure on kappa opioid receptor-mediated changes in dopamine transmission at the level of the dopamine terminal and withdrawal-related behaviors were examined. Five weeks of chronic intermittent ethanol exposure in male C57BL/6 mice were used to examine the role of kappa opioid receptors in chronic ethanol-induced increases in ethanol intake and marble burying, a measure of anxiety/compulsive-like behavior. Drinking and marble burying were evaluated before and after chronic intermittent ethanol exposure, with and without kappa opioid receptor blockade by nor-binaltorphimine (10mg/kg i.p.). Functional alterations in kappa opioid receptors were assessed using fast scan cyclic voltammetry in brain slices containing the nucleus accumbens. Chronic intermittent ethanol-exposed mice showed increased ethanol drinking and marble burying compared with controls, which was attenuated with kappa opioid receptor blockade. Chronic intermittent ethanol-induced increases in behavior were replicated with kappa opioid receptor activation in naïve mice. Fast scan cyclic voltammetry revealed that chronic intermittent ethanol reduced accumbal dopamine release and increased uptake rates, promoting a hypodopaminergic state of this region. Kappa opioid receptor activation with U50,488H concentration-dependently decreased dopamine release in both groups; however, this effect was greater in chronic intermittent ethanol-treated mice, indicating kappa opioid receptor supersensitivity in this group. These data suggest that the chronic intermittent ethanol-induced increase in ethanol intake and anxiety

  10. Dopamine D1 receptor gene variation modulates opioid dependence risk by affecting transition to addiction.

    Feng Zhu

    Full Text Available Dopamine D1 receptor (DRD1 modulates opioid reinforcement, reward, and opioid-induced neuroadaptation. We propose that DRD1 polymorphism affects susceptibility to opioid dependence (OD, the efficiency of transition to OD, and opioid-induced pleasure response. We analyzed potential association between seven DRD1 polymorphisms with the following traits: duration of transition from the first use to dependence (DTFUD, subjective pleasure responses to opioid on first use and post-dependence use, and OD risk in 425 Chinese with OD and 514 healthy controls. DTFUD and level of pleasure responses were examined using a semi-structured interview. The DTFUD of opioid addicts ranged from 5 days to 11 years. Most addicts (64.0% reported non-comfortable response upon first opioid use, while after dependence, most addicts (53.0% felt strong opioid-induced pleasure. Survival analysis revealed a correlation of prolonged DTFUD with the minor allele-carrying genotypes of DRD1 rs4532 (hazard ratios (HR = 0.694; p = 0.001 and rs686 (HR = 0.681, p = 0.0003. Binary logistic regression indicated that rs10063995 GT genotype (vs. GG+TT, OR = 0.261 could predict decreased pleasure response to first-time use and the minor alleles of rs686 (OR = 0.535 and rs4532 (OR = 0.537 could predict decreased post-dependence pleasure. Moreover, rs686 minor allele was associated with a decreased risk for rapid transition from initial use to dependence (DTFUD≤30 days; OR = 0.603 or post-dependence euphoria (OR = 0.603 relative to major allele. In conclusion, DRD1 rs686 minor allele decreases the OD risk by prolonging the transition to dependence and attenuating opioid-induced pleasure in Chinese.

  11. Mu and delta opioid receptors oppositely regulate motor impulsivity in the signaled nose poke task.

    Mary C Olmstead

    Full Text Available Impulsivity is a primary feature of many psychiatric disorders, most notably attention deficit hyperactivity disorder and drug addiction. Impulsivity includes a number of processes such as the inability to delay gratification, the inability to withhold a motor response, or acting before all of the relevant information is available. These processes are mediated by neural systems that include dopamine, serotonin, norepinephrine, glutamate and cannabinoids. We examine, for the first time, the role of opioid systems in impulsivity by testing whether inactivation of the mu- (Oprm1 or delta- (Oprd1 opioid receptor gene alters motor impulsivity in mice. Wild-type and knockout mice were examined on either a pure C57BL6/J (BL6 or a hybrid 50% C57Bl/6J-50% 129Sv/pas (HYB background. Mice were trained to respond for sucrose in a signaled nose poke task that provides independent measures of associative learning (responses to the reward-paired cue and motor impulsivity (premature responses. Oprm1 knockout mice displayed a remarkable decrease in motor impulsivity. This was observed on the two genetic backgrounds and did not result from impaired associative learning, as responses to the cue signaling reward did not differ across genotypes. Furthermore, mutant mice were insensitive to the effects of ethanol, which increased disinhibition and decreased conditioned responding in wild-type mice. In sharp contrast, mice lacking the Oprd1 gene were more impulsive than controls. Again, mutant animals showed no deficit in associative learning. Ethanol completely disrupted performance in these animals. Together, our results suggest that mu-opioid receptors enhance, whereas delta-opioid receptors inhibit, motor impulsivity. This reveals an unanticipated contribution of endogenous opioid receptor activity to disinhibition. In a broader context, these data suggest that alterations in mu- or delta-opioid receptor function may contribute to impulse control disorders.

  12. Evolution of ligand specificity in vertebrate corticosteroid receptors

    Deitcher David L

    2011-01-01

    Full Text Available Abstract Background Corticosteroid receptors include mineralocorticoid (MR and glucocorticoid (GR receptors. Teleost fishes have a single MR and duplicate GRs that show variable sensitivities to mineralocorticoids and glucocorticoids. How these receptors compare functionally to tetrapod MR and GR, and the evolutionary significance of maintaining two GRs, remains unclear. Results We used up to seven steroids (including aldosterone, cortisol and 11-deoxycorticosterone [DOC] to compare the ligand specificity of the ligand binding domains of corticosteroid receptors between a mammal (Mus musculus and the midshipman fish (Porichthys notatus, a teleost model for steroid regulation of neural and behavioral plasticity. Variation in mineralocorticoid sensitivity was considered in a broader phylogenetic context by examining the aldosterone sensitivity of MR and GRs from the distantly related daffodil cichlid (Neolamprologus pulcher, another teleost model for neurobehavioral plasticity. Both teleost species had a single MR and duplicate GRs. All MRs were sensitive to DOC, consistent with the hypothesis that DOC was the initial ligand of the ancestral MR. Variation in GR steroid-specificity corresponds to nine identified amino acid residue substitutions rather than phylogenetic relationships based on receptor sequences. Conclusion The mineralocorticoid sensitivity of duplicate GRs in teleosts is highly labile in the context of their evolutionary phylogeny, a property that likely led to neo-functionalization and maintenance of two GRs.

  13. Receptor-ligand binding sites and virtual screening.

    Hattotuwagama, Channa K; Davies, Matthew N; Flower, Darren R

    2006-01-01

    Within the pharmaceutical industry, the ultimate source of continuing profitability is the unremitting process of drug discovery. To be profitable, drugs must be marketable: legally novel, safe and relatively free of side effects, efficacious, and ideally inexpensive to produce. While drug discovery was once typified by a haphazard and empirical process, it is now increasingly driven by both knowledge of the receptor-mediated basis of disease and how drug molecules interact with receptors and the wider physiome. Medicinal chemistry postulates that to understand a congeneric ligand series, or set thereof, is to understand the nature and requirements of a ligand binding site. Likewise, structural molecular biology posits that to understand a binding site is to understand the nature of ligands bound therein. Reality sits somewhere between these extremes, yet subsumes them both. Complementary to rules of ligand design, arising through decades of medicinal chemistry, structural biology and computational chemistry are able to elucidate the nature of binding site-ligand interactions, facilitating, at both pragmatic and conceptual levels, the drug discovery process.

  14. DMPD: Endogenous ligands of Toll-like receptors. [Dynamic Macrophage Pathway CSML Database

    Full Text Available 15178705 Endogenous ligands of Toll-like receptors. Tsan MF, Gao B. J Leukoc Biol. ...2004 Sep;76(3):514-9. Epub 2004 Jun 3. (.png) (.svg) (.html) (.csml) Show Endogenous ligands of Toll-like re...ceptors. PubmedID 15178705 Title Endogenous ligands of Toll-like receptors. Authors Tsan MF, Gao B. Publicat

  15. Panicolytic-like effect of tramadol is mediated by opioid receptors in the dorsal periaqueductal grey.

    Fiaes, Gislaine Cardoso de Souza; Roncon, Camila Marroni; Sestile, Caio Cesar; Maraschin, Jhonatan Christian; Souza, Rodolfo Luis Silva; Porcu, Mauro; Audi, Elisabeth Aparecida

    2017-05-30

    Tramadol is a synthetic opioid prescribed for the treatment of moderate to severe pain, acting as agonist of μ-opioid receptors and serotonin (5-HT) and noradrenaline (NE) reuptake inhibitor. This study evaluated the effects of tramadol in rats submitted to the elevated T-maze (ETM), an animal model that evaluates behavioural parameters such as anxiety and panic. Male Wistar rats were intraperitoneally (i.p.) treated acutely with tramadol (16 and 32mg/kg) and were submitted to the ETM. Tramadol (32mg/kg) promoted a panicolytic-like effect. Considering that dorsal periaqueductal grey (dPAG) is the main brain structure related to the pathophysiology of panic disorder (PD), this study also evaluated the participation of 5-HT and opioid receptors located in the dPAG in the panicolytic-like effect of tramadol. Seven days after stereotaxic surgery for implantation of a cannula in the dPAG, the animals were submitted to the test. To assess the involvement of 5-HT 1A receptors on the effect of tramadol, we combined the 5-HT 1A receptor antagonist, WAY100635 (0.37nmol), microinjected intra-dPAG, 10min prior to the administration of tramadol (32mg/kg, i.p.). WAY100635 did not block the panicolytic-like effect of tramadol. We also associated the non-selective opioid receptor antagonist, naloxone, systemically (1mg/kg, i.p.) or intra-dPAG (0.5nmol) administered 10min prior to tramadol (32mg/kg, i.p.). Naloxone blocked the panicolytic-like effect of tramadol in both routes of administrations, showing that tramadol modulates acute panic defensive behaviours through its interaction with opioid receptors located in the dPAG. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Kappa-opioid receptors mediate the antidepressant-like activity of hesperidin in the mouse forced swimming test.

    Filho, Carlos B; Del Fabbro, Lucian; de Gomes, Marcelo G; Goes, André T R; Souza, Leandro C; Boeira, Silvana P; Jesse, Cristiano R

    2013-01-05

    The opioid system has been implicated as a contributing factor for major depression and is thought to play a role in the mechanism of action of antidepressants. This study investigated the involvement of the opioid system in the antidepressant-like effect of hesperidin in the mouse forced swimming test. Our results demonstrate that hesperidin (0.1, 0.3 and 1 mg/kg; intraperitoneal) decreased the immobility time in the forced swimming test without affecting locomotor activity in the open field test. The antidepressant-like effect of hesperidin (0.3 mg/kg) in the forced swimming test was prevented by pretreating mice with naloxone (1 mg/kg, a nonselective opioid receptor antagonist) and 2-(3,4-dichlorophenyl)-Nmethyl-N-[(1S)-1-(3-isothiocyanatophenyl)-2-(1-pyrrolidinyl)ethyl] acetamide (DIPPA (1 mg/kg), a selective κ-opioid receptor antagonist), but not with naloxone methiodide (1 mg/kg, a peripherally acting opioid receptor antagonist), naltrindole (3 mg/kg, a selective δ-opioid receptor antagonist), clocinnamox (1 mg/kg, a selective μ-opioid receptor antagonist) or caffeine (3 mg/kg, a nonselective adenosine receptor antagonist). In addition, a sub-effective dose of hesperidin (0.01 mg/kg) produced a synergistic antidepressant-like effect in the forced swimming test when combined with a sub-effective dose of morphine (1 mg/kg). The antidepressant-like effect of hesperidin in the forced swimming test on mice was dependent on its interaction with the κ-opioid receptor, but not with the δ-opioid, μ-opioid or adenosinergic receptors. Taken together, these results suggest that hesperidin possesses antidepressant-like properties and may be of interest as a therapeutic agent for the treatment of depressive disorders. Published by Elsevier B.V.

  17. δ-opioid receptor and somatostatin receptor-4 heterodimerization: possible implications in modulation of pain associated signaling.

    Rishi K Somvanshi

    Full Text Available Pain relief is the principal action of opioids. Somatostatin (SST, a growth hormone inhibitory peptide is also known to alleviate pain even in cases when opioids fail. Recent studies have shown that mice are prone to sustained pain and devoid of analgesic effect in the absence of somatostatin receptor 4 (SSTR4. In the present study, using brain slices, cultured neurons and HEK-293 cells, we showed that SSTR4 and δ-Opioid receptor (δOR exist in a heteromeric complex and function in synergistic manner. SSTR4 and δOR co-expressed in cortical/striatal brain regions and spinal cord. Using cultured neuronal cells, we describe the heterogeneous complex formation of SSTR4 and δOR at neuronal cell body and processes. Cotransfected cells display inhibition of cAMP/PKA and co-activation of SSTR4 and δOR oppose receptor trafficking induced by individual receptor activation. Furthermore, downstream signaling pathways either associated with withdrawal or pain relief are modulated synergistically with a predominant role of SSTR4. Inhibition of cAMP/PKA and activation of ERK1/2 are the possible cellular adaptations to prevent withdrawal induced by chronic morphine use. Our results reveal direct intra-membrane interaction between SSTR4 and δOR and provide insights for the molecular mechanism for the anti-nociceptive property of SST in combination with opioids as a potential therapeutic approach to avoid undesirable withdrawal symptoms.

  18. Kappa Opioid Receptors Mediate where Fear Is Expressed Following Extinction Training

    Cole, Sindy; Richardson, Rick; McNally, Gavan P.

    2011-01-01

    Six experiments used a within-subjects renewal design to examine the involvement of kappa opioid receptors (KORs) in regulating the expression and recovery of extinguished fear. Rats were trained to fear a tone conditioned stimulus (CS) via pairings with foot shock in a distinctive context (A). This was followed by extinction training of the CS in…

  19. Molecular Docking, Molecular Dynamics, and Structure-Activity Relationship Explorations of 14-Oxygenated N-Methylmorphinan-6-ones as Potent μ-Opioid Receptor Agonists.

    Noha, Stefan M; Schmidhammer, Helmut; Spetea, Mariana

    2017-06-21

    Among opioids, morphinans are of major importance as the most effective analgesic drugs acting primarily via μ-opioid receptor (μ-OR) activation. Our long-standing efforts in the field of opioid analgesics from the class of morphinans led to N-methylmorphinan-6-ones differently substituted at positions 5 and 14 as μ-OR agonists inducing potent analgesia and fewer undesirable effects. Herein we present the first thorough molecular modeling study and structure-activity relationship (SAR) explorations aided by docking and molecular dynamics (MD) simulations of 14-oxygenated N-methylmorphinan-6-ones to gain insights into their mode of binding to the μ-OR and interaction mechanisms. The structure of activated μ-OR provides an essential model for how ligand/μ-OR binding is encoded within small chemical differences in otherwise structurally similar morphinans. We reveal important molecular interactions that these μ-agonists share and distinguish them. The molecular docking outcomes indicate the crucial role of the relative orientation of the ligand in the μ-OR binding site, influencing the propensity of critical non-covalent interactions that are required to facilitate ligand/μ-OR interactions and receptor activation. The MD simulations point out minor differences in the tendency to form hydrogen bonds by the 4,5α-epoxy group, along with the tendency to affect the 3-7 lock switch. The emerged SARs reveal the subtle interplay between the substituents at positions 5 and 14 in the morphinan scaffold by enabling the identification of key structural elements that determine the distinct pharmacological profiles. This study provides a significant structural basis for understanding ligand binding and μ-OR activation by the 14-oxygenated N-methylmorphinan-6-ones, which should be useful for guiding drug design.

  20. Mu opioid receptor availability in people with psychiatric disorders who died by suicide: a case control study.

    Scarr, Elizabeth; Money, Tammie Terese; Pavey, Geoffrey; Neo, Jaclyn; Dean, Brian

    2012-08-28

    Mu opioid receptors have previously been shown to be altered in people with affective disorders who died as a result of suicide. We wished to determine whether these changes were more widespread and independent of psychiatric diagnoses. Mu receptor levels were determined using [3 H]DAMGO binding in BA24 from 51 control subjects; 38 people with schizophrenia (12 suicides); 20 people with major depressive disorder (15 suicides); 13 people with bipolar disorder (5 suicides) and 9 people who had no history of psychiatric disorders but who died as a result of suicide. Mu receptor levels were further determined in BA9 and caudate-putamen from 38 people with schizophrenia and 20 control subjects using [3 H]DAMGO binding and, in all three regions, using Western blots. Data was analysed using one-way ANOVAs with Bonferroni's Multiple Comparison Test or, where data either didn't approximate to a binomial distribution or the sample size was too small to determine distribution, a Kruskal-Wallis test with Dunn's Multiple Comparison Test. [3 H]DAMGO binding density was lower in people who had died as a result of suicide (pendogenous ligands occupying these receptors.

  1. A photoaffinity ligand for dopamine D2 receptors: azidoclebopride.

    Niznik, H B; Guan, J H; Neumeyer, J L; Seeman, P

    1985-02-01

    In order to label D2 dopamine receptors selectively and covalently by means of a photosensitive compound, azidoclebopride was synthesized directly from clebopride. The dissociation constant (KD) of clebopride for the D2 dopamine receptor (canine brain striatum) was 1.5 nM, while that for azidoclebopride was 21 nM. The affinities of both clebopride and azidoclebopride were markedly reduced in the absence of sodium chloride. In the presence of ultraviolet light, azidoclebopride inactivated D2 dopamine receptors irreversibly, as indicated by the inability of the receptors to bind [3H]spiperone. Maximal photoinactivation of about 60% of the D2 dopamine receptors occurred at 1 microM azidoclebopride; 30% of the receptors were inactivated at 80 nM azidoclebopride (pseudo-IC50). Dopamine agonists selectively protected the D2 receptors from being inactivated by azidoclebopride, the order of potency being (-)-N-n-propylnorapomorphine greater than apomorphine greater than (+/-)-6,7-dihydroxy-2-aminotetralin greater than (+)-N-n-propylnorapomorphine greater than dopamine greater than noradrenaline greater than serotonin. Similarly, dopaminergic antagonists prevented the photoinactivation of D2 receptors by azidoclebopride with the following order of potency: spiperone greater than (+)-butaclamol greater than haloperidol greater than clebopride greater than (-)-sulpiride greater than (-)-butaclamol. The degree of D2 dopamine receptor photoinduced inactivation by azidoclebopride was not significantly affected by scavengers such as p-aminobenzoic acid and dithiothreitol. Furthermore, irradiation of striatal membranes with a concentration of azidoclebopride sufficient to inactivate dopamine D2 receptors by 60% did not significantly reduce dopamine D1, serotonin (S2), benzodiazepine, alpha 1- or beta-noradrenergic receptors. This study describes the use of a novel and selective photoaffinity ligand for brain dopamine D2 receptors. The molecule, in radiolabeled form, may aid in the

  2. Ligand-independent recruitment of steroid receptor coactivators to estrogen receptor by cyclin D1

    Zwijsen, R.M.L.; Buckle, R.S.; Hijmans, E.M.; Loomans, C.J.M.; Bernards, R.A.

    1998-01-01

    The estrogen receptor (ER) is an important regulator of growth and differentiation of breast epithelium. Transactivation by ER depends on a leucine-rich motif, which constitutes a ligand-regulated binding site for steroid receptor coactivators (SRCs). Cyclin D1 is frequently amplified in breast

  3. Non-conventional Frizzled ligands and Wnt receptors.

    Hendrickx, Marijke; Leyns, Luc

    2008-05-01

    The Wnt family of secreted signaling factors plays numerous roles in embryonic development and in stem cell biology. In the adult, Wnt signaling is involved in tissue homeostasis and mutations that lead to the overexpression of Wnt can be linked to cancer. Wnt signaling is transduced intracellularly by the Frizzled (Fzd) family of receptors. In the canonical pathway, accumulation of beta-catenin and the subsequent formation of a complex with T cell factors (TCF) or lymphoid enhancing factors (Lef) lead to target gene activation. The identification of Ryk as an alternative Wnt receptor and the discovery of the novel Fzd ligands Norrie disease protein (NDP) and R-Spondin, changed the traditional view of Wnts binding to Fzd receptors. Mouse R-Spondin cooperates with Wnt signaling and Low density lipoprotein (LDL) receptor related protein (LRP) to activate beta-catenin dependent gene expression and is involved in processes such as limb and placental development in the mouse. NDP is the product of the Norrie disease gene and controls vascular development in the retina, inner ear and in the female reproductive system during pregnancy. In this review a functional overview of the interactions of the different Wnt and non-Wnt ligands with the Fzd receptors is given as well as a survey of Wnts binding to Ryk and we discuss the biological significance of these interactions.

  4. Expression of μ, κ, and δ opioid receptor messenger RNA in the human CNS: a 33P in situ hybridization study

    Peckys, D.; Landwehrmeyer, G.B.

    1999-01-01

    The existence of at least three opioid receptor types, referred to as μ, κ, and δ, is well established. Complementary DNAs corresponding to the pharmacologically defined μ, κ, and δ opioid receptors have been isolated in various species including man. The expression patterns of opioid receptor transcripts in human brain has not been established with a cellular resolution, in part because of the low apparent abundance of opioid receptor messenger RNAs in human brain. To visualize opioid receptor messenger RNAs we developed a sensitive in situ hybridization histochemistry method using 33 P-labelled RNA probes. In the present study we report the regional and cellular expression of μ, κ, and δ opioid receptor messenger RNAs in selected areas of the human brain. Hybridization of the different opioid receptor probes resulted in distinct labelling patterns. For the μ and κ opioid receptor probes, the most intense regional signals were observed in striatum, thalamus, hypothalamus, cerebral cortex, cerebellum and certain brainstem areas as well as the spinal cord. The most intense signals for the δ opioid receptor probe were found in cerebral cortex. Expression of opioid receptor transcripts was restricted to subpopulations of neurons within most regions studied demonstrating differences in the cellular expression patterns of μ, κ, and δ opioid receptor messenger RNAs in numerous brain regions. The messenger RNA distribution patterns for each opioid receptor corresponded in general to the distribution of opioid receptor binding sites as visualized by receptor autoradiography. However, some mismatches, for instance between μ opioid receptor receptor binding and μ opioid receptor messenger RNA expression in the anterior striatum, were observed. A comparison of the distribution patterns of opioid receptor messenger RNAs in the human brain and that reported for the rat suggests a homologous expression pattern in many regions. However, in the human brain, κ

  5. Dynorphin/Kappa Opioid Receptor Signaling in Preclinical Models of Alcohol, Drug, and Food Addiction.

    Karkhanis, Anushree; Holleran, Katherine M; Jones, Sara R

    2017-01-01

    The dynorphin/kappa opioid receptor (KOR) system is implicated in the "dark side" of addiction, in which stress exacerbates maladaptive responses to drug and alcohol exposure. For example, acute stress and acute ethanol exposure result in an elevation in dynorphin, the KOR endogenous ligand. Activation of KORs results in modulation of several neurotransmitters; however, this chapter will focus on its regulatory effects on dopamine in mesolimbic areas. Specifically, KOR activation has an inhibitory effect on dopamine release, thereby influencing reward processing. Repeated stimulation of KORs, for example, via chronic drug and/or stress exposure, results in increased function of the dynorphin/KOR system. This augmentation in KOR function shifts the homeostatic balance in favor of an overall reduction in dopamine signaling via either by reducing dopamine release or by increasing dopamine transporter function. This chapter examines the effects of chronic ethanol exposure on KOR function and the downstream effects on dopamine transmission. Additionally, the impact of chronic cocaine exposure and its effects on KOR function will be explored. Further, KORs may also be involved in driving excessive consumption of food, contributing to the risk of developing obesity. While some studies have shown that KOR agonists reduce drug intake, other studies have shown that antagonists reduce addiction-like behaviors, demonstrating therapeutic potential. For example, KOR inhibition reduces ethanol intake in dependent animals, motivation to self-administer cocaine in chronic stress-exposed animals, and food consumption in obese animals. This chapter will delve into the mechanisms by which modulation of the dynorphin/KOR system may be therapeutic. © 2017 Elsevier Inc. All rights reserved.

  6. Molecular modeling of ligand-receptor interactions in the OR5 olfactory receptor.

    Singer, M S; Shepherd, G M

    1994-06-02

    Olfactory receptors belong to the superfamily of seven transmembrane domain, G protein-coupled receptors. In order to begin analysis of mechanisms of receptor activation, a computer model of the OR5 olfactory receptor has been constructed and compared with other members of this superfamily. We have tested docking of the odor molecule lyral, which is known to activate the OR5 receptor. The results point to specific ligand-binding residues on helices III through VII that form a binding pocket in the receptor. Some of these residues occupy sequence positions identical to ligand-binding residues conserved among other superfamily members. The results provide new insights into possible molecular mechanisms of odor recognition and suggest hypotheses to guide future experimental studies using site-directed mutagenesis.

  7. 5D-QSAR for spirocyclic sigma1 receptor ligands by Quasar receptor surface modeling.

    Oberdorf, Christoph; Schmidt, Thomas J; Wünsch, Bernhard

    2010-07-01

    Based on a contiguous and structurally as well as biologically diverse set of 87 sigma(1) ligands, a 5D-QSAR study was conducted in which a quasi-atomistic receptor surface modeling approach (program package Quasar) was applied. The superposition of the ligands was performed with the tool Pharmacophore Elucidation (MOE-package), which takes all conformations of the ligands into account. This procedure led to four pharmacophoric structural elements with aromatic, hydrophobic, cationic and H-bond acceptor properties. Using the aligned structures a 3D-model of the ligand binding site of the sigma(1) receptor was obtained, whose general features are in good agreement with previous assumptions on the receptor structure, but revealed some novel insights since it represents the receptor surface in more detail. Thus, e.g., our model indicates the presence of an H-bond acceptor moiety in the binding site as counterpart to the ligands' cationic ammonium center, rather than a negatively charged carboxylate group. The presented QSAR model is statistically valid and represents the biological data of all tested compounds, including a test set of 21 ligands not used in the modeling process, with very good to excellent accuracy [q(2) (training set, n=66; leave 1/3 out) = 0.84, p(2) (test set, n=21)=0.64]. Moreover, the binding affinities of 13 further spirocyclic sigma(1) ligands were predicted with reasonable accuracy (mean deviation in pK(i) approximately 0.8). Thus, in addition to novel insights into the requirements for binding of spirocyclic piperidines to the sigma(1) receptor, the presented model can be used successfully in the rational design of new sigma(1) ligands. Copyright (c) 2010 Elsevier Masson SAS. All rights reserved.

  8. Endogenous opioids regulate moment-to-moment neuronal communication and excitability

    Winters, Bryony L.; Gregoriou, Gabrielle C.; Kissiwaa, Sarah A.; Wells, Oliver A.; Medagoda, Danashi I.; Hermes, Sam M.; Burford, Neil T.; Alt, Andrew; Aicher, Sue A.; Bagley, Elena E.

    2017-01-01

    Fear and emotional learning are modulated by endogenous opioids but the cellular basis for this is unknown. The intercalated cells (ITCs) gate amygdala output and thus regulate the fear response. Here we find endogenous opioids are released by synaptic stimulation to act via two distinct mechanisms within the main ITC cluster. Endogenously released opioids inhibit glutamate release through the δ-opioid receptor (DOR), an effect potentiated by a DOR-positive allosteric modulator. Postsynaptically, the opioids activate a potassium conductance through the μ-opioid receptor (MOR), suggesting for the first time that endogenously released opioids directly regulate neuronal excitability. Ultrastructural localization of endogenous ligands support these functional findings. This study demonstrates a new role for endogenously released opioids as neuromodulators engaged by synaptic activity to regulate moment-to-moment neuronal communication and excitability. These distinct actions through MOR and DOR may underlie the opposing effect of these receptor systems on anxiety and fear. PMID:28327612

  9. Steroid receptors and their ligands: Effects on male gamete functions

    Aquila, Saveria; De Amicis, Francesca

    2014-01-01

    In recent years a new picture of human sperm biology is emerging. It is now widely recognized that sperm contain nuclear encoded mRNA, mitochondrial encoded RNA and different transcription factors including steroid receptors, while in the past sperm were considered incapable of transcription and translation. One of the main targets of steroid hormones and their receptors is reproductive function. Expression studies on Progesterone Receptor, estrogen receptor, androgen receptor and their specific ligands, demonstrate the presence of these systems in mature spermatozoa as surface but also as nuclear conventional receptors, suggesting that both systemic and local steroid hormones, through sperm receptors, may influence male reproduction. However, the relationship between the signaling events modulated by steroid hormones and sperm fertilization potential as well as the possible involvement of the specific receptors are still controversial issues. The main line of this review highlights the current research in human sperm biology examining new molecular systems of response to the hormones as well as specific regulatory pathways controlling sperm cell fate and biological functions. Most significant studies regarding the identification of steroid receptors are reported and the mechanistic insights relative to signaling pathways, together with the change in sperm metabolism energy influenced by steroid hormones are discussed.The reviewed evidences suggest important effects of Progesterone, Estrogen and Testosterone and their receptors on spermatozoa and implicate the involvement of both systemic and local steroid action in the regulation of male fertility potential. - Highlights: • One of the main targets of steroid hormones and their receptors is reproductive function. • Pg/PR co-work to stimulate enzymatic activities to sustain a capacitation process. • E2/ERs regulate sperm motility, capacitation and acrosome reaction and act as survival factors. • Androgens

  10. Steroid receptors and their ligands: Effects on male gamete functions

    Aquila, Saveria; De Amicis, Francesca, E-mail: francesca.deamicis@unical.it

    2014-11-01

    In recent years a new picture of human sperm biology is emerging. It is now widely recognized that sperm contain nuclear encoded mRNA, mitochondrial encoded RNA and different transcription factors including steroid receptors, while in the past sperm were considered incapable of transcription and translation. One of the main targets of steroid hormones and their receptors is reproductive function. Expression studies on Progesterone Receptor, estrogen receptor, androgen receptor and their specific ligands, demonstrate the presence of these systems in mature spermatozoa as surface but also as nuclear conventional receptors, suggesting that both systemic and local steroid hormones, through sperm receptors, may influence male reproduction. However, the relationship between the signaling events modulated by steroid hormones and sperm fertilization potential as well as the possible involvement of the specific receptors are still controversial issues. The main line of this review highlights the current research in human sperm biology examining new molecular systems of response to the hormones as well as specific regulatory pathways controlling sperm cell fate and biological functions. Most significant studies regarding the identification of steroid receptors are reported and the mechanistic insights relative to signaling pathways, together with the change in sperm metabolism energy influenced by steroid hormones are discussed.The reviewed evidences suggest important effects of Progesterone, Estrogen and Testosterone and their receptors on spermatozoa and implicate the involvement of both systemic and local steroid action in the regulation of male fertility potential. - Highlights: • One of the main targets of steroid hormones and their receptors is reproductive function. • Pg/PR co-work to stimulate enzymatic activities to sustain a capacitation process. • E2/ERs regulate sperm motility, capacitation and acrosome reaction and act as survival factors. • Androgens

  11. Sex and age-dependent effects of a maternal junk food diet on the mu-opioid receptor in rat offspring.

    Gugusheff, Jessica R; Bae, Sung Eun; Rao, Alexandra; Clarke, Iain J; Poston, Lucilla; Taylor, Paul D; Coen, Clive W; Muhlhausler, Beverly S

    2016-03-15

    Perinatal junk food exposure increases the preference for palatable diets in juvenile and adult rat offspring. Previous studies have implicated reduced sensitivity of the opioid pathway in the programming of food preferences; however it is not known when during development these changes in opioid signalling first emerge. This study aimed to determine the impact of a maternal junk food (JF) diet on mu-opioid receptor (MuR) expression and ligand binding in two key regions of the reward pathway, the nucleus accumbens (NAc) and the ventral tegmental area (VTA) in rats during the early suckling (postnatal day (PND) 1 and 7) and late suckling/early post-weaning (PND 21 and 28) periods. Female rats were fed either a JF or a control diet for two weeks prior to mating and throughout pregnancy and lactation. MuR expression in the VTA was significantly reduced in female JF offspring on PND 21 and 28 (by 32% and 57% respectively, Pjunk food exposure on MuR mRNA expression or binding were detected at these time points in male offspring. These findings provide evidence that the opioid signalling system is a target of developmental programming by the end of the third postnatal week in females, but not in males. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. μ opioid receptor activation hyperpolarizes respiratory-controlling Kölliker-Fuse neurons and suppresses post-inspiratory drive.

    Levitt, Erica S; Abdala, Ana P; Paton, Julian F R; Bissonnette, John M; Williams, John T

    2015-10-01

    In addition to reductions in respiratory rate, opioids also cause aspiration and difficulty swallowing, indicating impairment of the upper airways. The Kölliker-Fuse (KF) maintains upper airway patency and a normal respiratory pattern. In this study, activation of μ opioid receptors in the KF reduced respiratory frequency and tidal volume in anaesthetized rats. Nerve recordings in an in situ preparation showed that activation of μ opioid receptors in the KF eliminated the post-inspiration phase of the respiratory cycle. In brain slices, μ opioid agonists hyperpolarized a distinct population (61%) of KF neurons by activation of an inwardly rectifying potassium conductance. These results suggest that KF neurons that are hyperpolarized by opioids could contribute to opioid-induced respiratory disturbances, particularly the impairment of upper airways. Opioid-induced respiratory effects include aspiration and difficulty swallowing, suggesting impairment of the upper airways. The pontine Kölliker-Fuse nucleus (KF) controls upper airway patency and regulates respiration, in particular the inspiratory/expiratory phase transition. Given the importance of the KF in coordinating respiratory pattern, the mechanisms of μ opioid receptor activation in this nucleus were investigated at the systems and cellular level. In anaesthetized, vagi-intact rats, injection of opioid agonists DAMGO or [Met(5) ]enkephalin (ME) into the KF reduced respiratory frequency and amplitude. The μ opioid agonist DAMGO applied directly into the KF of the in situ arterially perfused working heart-brainstem preparation of rat resulted in robust apneusis (lengthened low amplitude inspiration due to loss of post-inspiratory drive) that was rapidly reversed by the opioid antagonist naloxone. In brain slice preparations, activation of μ opioid receptors on KF neurons hyperpolarized a distinct population (61%) of neurons. As expected, the opioid-induced hyperpolarization reduced the excitability of

  13. Human Mu Opioid Receptor (OPRM1A118G) polymorphism is associated with brain mu- opioid receptor binding potential in smokers

    Ray, R.; Logan, J.; Ray, R.; Ruparel, K.; Newberg, A.; Wileyto, E.P.; Loughead, J.W.; Divgi, C.; Blendy, J.A.; Logan, J.; Zubieta, J.-K.; Lerman, C.

    2011-04-15

    Evidence points to the endogenous opioid system, and the mu-opioid receptor (MOR) in particular, in mediating the rewarding effects of drugs of abuse, including nicotine. A single nucleotide polymorphism (SNP) in the human MOR gene (OPRM1 A118G) has been shown to alter receptor protein level in preclinical models and smoking behavior in humans. To clarify the underlying mechanisms for these associations, we conducted an in vivo investigation of the effects of OPRM1 A118G genotype on MOR binding potential (BP{sub ND} or receptor availability). Twenty-two smokers prescreened for genotype (12 A/A, 10 */G) completed two [{sup 11}C] carfentanil positron emission tomography (PET) imaging sessions following overnight abstinence and exposure to a nicotine-containing cigarette and a denicotinized cigarette. Independent of session, smokers homozygous for the wild-type OPRM1 A allele exhibited significantly higher levels of MOR BP{sub ND} than smokers carrying the G allele in bilateral amygdala, left thalamus, and left anterior cingulate cortex. Among G allele carriers, the extent of subjective reward difference (denicotinized versus nicotine cigarette) was associated significantly with MOR BP{sub ND} difference in right amygdala, caudate, anterior cingulate cortex, and thalamus. Future translational investigations can elucidate the role of MORs in nicotine addiction, which may lead to development of novel therapeutics.

  14. Human Mu Opioid Receptor (OPRM1A118G) polymorphism is associated with brain mu- opioid receptor binding potential in smokers

    Ray, R.; Logan, J.; Ruparel, K.; Newberg, A.; Wileyto, E.P.; Loughead, J.W.; Divgi, C.; Blendy, J.A.; Logan, J.; Zubieta, J.-K.; Lerman, C.

    2011-01-01

    Evidence points to the endogenous opioid system, and the mu-opioid receptor (MOR) in particular, in mediating the rewarding effects of drugs of abuse, including nicotine. A single nucleotide polymorphism (SNP) in the human MOR gene (OPRM1 A118G) has been shown to alter receptor protein level in preclinical models and smoking behavior in humans. To clarify the underlying mechanisms for these associations, we conducted an in vivo investigation of the effects of OPRM1 A118G genotype on MOR binding potential (BP ND or receptor availability). Twenty-two smokers prescreened for genotype (12 A/A, 10 */G) completed two [ 11 C] carfentanil positron emission tomography (PET) imaging sessions following overnight abstinence and exposure to a nicotine-containing cigarette and a denicotinized cigarette. Independent of session, smokers homozygous for the wild-type OPRM1 A allele exhibited significantly higher levels of MOR BP ND than smokers carrying the G allele in bilateral amygdala, left thalamus, and left anterior cingulate cortex. Among G allele carriers, the extent of subjective reward difference (denicotinized versus nicotine cigarette) was associated significantly with MOR BP ND difference in right amygdala, caudate, anterior cingulate cortex, and thalamus. Future translational investigations can elucidate the role of MORs in nicotine addiction, which may lead to development of novel therapeutics.

  15. Distribution of mu, delta, and kappa opioid receptor binding sites in the brain of the one-day-old domestic chick (Gallus domesticus): An in vitro quantitative autoradiographic study

    Csillag, A.; Bourne, R.C.; Stewart, M.G. (Open Univ., Milton Keynes (England))

    1990-12-15

    Three highly specific opioid ligands--(D-Ala2,Gly-ol)-enkephalin (DAGO) for mu (mu) receptor sites, (D-Pen2,D-Pen5)-enkephalin (DPDPE) for delta (delta) sites, and U-69593 for kappa (kappa) sites--were used to determine the regional distribution of the three major subtypes of opioid receptor binding sites in the brains of 1-day-old domestic chicks by the technique of quantitative receptor autoradiography. While there was a degree of heterogeneity in the binding levels of each of the ligands, some notable similarities existed in the binding of the mu and kappa ligands in several forebrain regions, and in the optic tectum of the midbrain where mu and delta binding was very high. In the forebrain there was a high level of binding of mu and kappa ligands in the hyperstriatum, and for the mu ligand there was a very distinct lamination of binding sites in hyperstriatum accessorium, intercalatum supremum, dorsale and ventrale. Levels of binding of the mu and kappa ligands were also high in nucleus basalis, and (for mu only) in the neostriatum. The distribution of binding of the delta specific ligand in the forebrain showed marked differences to that of mu and kappa, being particularly low in the hyperstriatum and neostriatum. Very high levels of labelling of delta binding sites were, however, found in the nucleus rotundus. Binding of the three ligands was generally low or absent in the cerebellum and medulla, apart from a distinct labelling of the granule cell layer by the mu-ligand. A kinetic analysis was made of the binding of the three ligands to whole forebrain sections using scintillation counting methods.

  16. Interacting cannabinoid and opioid receptors in the nucleus accumbens core control adolescent social play

    Antonia Manduca

    2016-11-01

    Full Text Available Social play behavior is a highly rewarding, developmentally important form of social interaction in young mammals. However, its neurobiological underpinnings remain incompletely understood. Previous work has suggested that opioid and endocannabinoid neurotransmission interact in the modulation of social play. Therefore, we combined behavioral, pharmacological, electrophysiological and genetic approaches to elucidate the role of the endocannabinoid 2-arachidonoylglycerol (2-AG in social play, and how cannabinoid and opioid neurotransmission interact to control social behavior in adolescent rodents. Systemic administration of the 2-AG hydrolysis inhibitor JZL184 or the opioid receptor agonist morphine increased social play behavior in adolescent rats. These effects were blocked by systemic pretreatment with either CB1 cannabinoid receptor (CB1R or mu-opioid receptor (MOR antagonists. The social play-enhancing effects of systemic morphine or JZL184 treatment were also prevented by direct infusion of the CB1R antagonist SR141716 and the MOR antagonist naloxone into the nucleus accumbens core (NAcC. Searching for synaptic correlates of these effects in adolescent NAcC excitatory synapses, we observed that CB1R antagonism blocked the effect of the MOR agonist DAMGO and, conversely, that naloxone reduced the effect of a cannabinoid agonist. These results were recapitulated in mice, and completely abolished in CB1R and MOR knockout mice, suggesting that the functional interaction between CB1R and MOR in the NAcC in the modulation of mediates social behavior is widespread in rodents. The data shed new light on the mechanism by which endocannabinoid lipids and opioid peptides interact to orchestrate rodent socioemotional behaviors.

  17. Ghrelin interacts with neuropeptide Y Y1 and opioid receptors to increase food reward.

    Skibicka, Karolina P; Shirazi, Rozita H; Hansson, Caroline; Dickson, Suzanne L

    2012-03-01

    Ghrelin, a stomach-derived hormone, is an orexigenic peptide that was recently shown to potently increase food reward behavior. The neurochemical circuitry that links ghrelin to the mesolimbic system and food reward behavior remains unclear. Here we examined the contribution of neuropeptide Y (NPY) and opioids to ghrelin's effects on food motivation and intake. Both systems have well-established links to the mesolimbic ventral tegmental area (VTA) and reward/motivation control. NPY mediates the effect of ghrelin on food intake via activation of NPY-Y1 receptor (NPY-Y1R); their connection with respect to motivated behavior is unexplored. The role of opioids in any aspect of ghrelin's action on food-oriented behaviors is unknown. Rats were trained in a progressive ratio sucrose-induced operant schedule to measure food reward/motivation behavior. Chow intake was measured immediately after the operant test. In separate experiments, we explored the suppressive effects of a selective NPY-Y1R antagonist or opioid receptor antagonist naltrexone, injected either intracerebroventricularly or intra-VTA, on ghrelin-induced food reward behavior. The ventricular ghrelin-induced increase in sucrose-motivated behavior and chow intake were completely blocked by intracerebroventricular pretreatment with either an NPY-Y1R antagonist or naltrexone. The intra-VTA ghrelin-induced sucrose-motivated behavior was blocked only by intra-VTA naltrexone. In contrast, the intra-VTA ghrelin-stimulated chow intake was attenuated only by intra-VTA NPY-Y1 blockade. Finally, ghrelin infusion was associated with an elevated VTA μ-opioid receptor expression. Thus, we identify central NPY and opioid signaling as the necessary mediators of food intake and reward effects of ghrelin and localize these interactions to the mesolimbic VTA.

  18. The effect of opioid receptor blockade on the neural processing of thermal stimuli.

    Eszter D Schoell

    Full Text Available The endogenous opioid system represents one of the principal systems in the modulation of pain. This has been demonstrated in studies of placebo analgesia and stress-induced analgesia, where anti-nociceptive activity triggered by pain itself or by cognitive states is blocked by opioid antagonists. The aim of this study was to characterize the effect of opioid receptor blockade on the physiological processing of painful thermal stimulation in the absence of cognitive manipulation. We therefore measured BOLD (blood oxygen level dependent signal responses and intensity ratings to non-painful and painful thermal stimuli in a double-blind, cross-over design using the opioid receptor antagonist naloxone. On the behavioral level, we observed an increase in intensity ratings under naloxone due mainly to a difference in the non-painful stimuli. On the neural level, painful thermal stimulation was associated with a negative BOLD signal within the pregenual anterior cingulate cortex, and this deactivation was abolished by naloxone.

  19. Analgesia produced by exposure to 2450-MHz radiofrequency radiation (RFR) is mediated by brain mu- and kappa-opioid receptors

    Salomon, G.; Park, E.J.; Quock, R.M. (Univ. of Illinois, Rockford (United States))

    1992-02-26

    This study was conducted to identify the opioid receptor subtype(s) responsible for RFR-induced analgesia. Male Swiss Webster mice, 20-25 g, were exposed to 20 mW/cm{sup 2} RFR in a 2,450-MHz waveguide system for 10 min, then tested 15 min later in the abdominal constriction paradigm which detects {mu}- and {kappa}-opioid activity. Immediately following RFR exposure, different groups of mice were pretreated intracerebroventricularly with different opioid receptor blockers with selectivity for {mu}- or {kappa}-opioid receptors. Results show that RFR-induced analgesia was attenuated by higher but not lower doses of the non-selective antagonist naloxone, but the selective {mu}-opioid antagonist {beta}-funaltrexamine and by the selective {kappa}-opioid antagonist norbinaltorphimine. RFR-induced analgesia was also reduced by subcutaneous pretreatment with 5.0 mg/kg of the {mu}-/{kappa}-opioid antagonist({minus})-5,9-diethyl-{alpha}-5,9-dialkyl-2{prime}-hydroxy-6,7-benzomorphan(MR-2266). These findings suggest that RFR-induced analgesia may be mediated by both {mu}- and {kappa}-opioid mechanisms.

  20. Cell-Autonomous Regulation of Mu-Opioid Receptor Recycling by Substance P

    Shanna L. Bowman

    2015-03-01

    Full Text Available How neurons coordinate and reprogram multiple neurotransmitter signals is an area of broad interest. Here, we show that substance P (SP, a neuropeptide associated with inflammatory pain, reprograms opioid receptor recycling and signaling. SP, through activation of the neurokinin 1 (NK1R receptor, increases the post-endocytic recycling of the mu-opioid receptor (MOR in trigeminal ganglion (TG neurons in an agonist-selective manner. SP-mediated protein kinase C (PKC activation is both required and sufficient for increasing recycling of exogenous and endogenous MOR in TG neurons. The target of this cross-regulation is MOR itself, given that mutation of either of two PKC phosphorylation sites on MOR abolishes the SP-induced increase in recycling and resensitization. Furthermore, SP enhances the resensitization of fentanyl-induced, but not morphine-induced, antinociception in mice. Our results define a physiological pathway that cross-regulates opioid receptor recycling via direct modification of MOR and suggest a mode of homeostatic interaction between the pain and analgesic systems.

  1. Differential expression of VEGF ligands and receptors in prostate cancer.

    Woollard, David J; Opeskin, Kenneth; Coso, Sanja; Wu, Di; Baldwin, Megan E; Williams, Elizabeth D

    2013-05-01

    Prostate cancer disseminates to regional lymph nodes, however the molecular mechanisms responsible for lymph node metastasis are poorly understood. The vascular endothelial growth factor (VEGF) ligand and receptor family have been implicated in the growth and spread of prostate cancer via activation of the blood vasculature and lymphatic systems. The purpose of this study was to comprehensively examine the expression pattern of VEGF ligands and receptors in the glandular epithelium, stroma, lymphatic vasculature and blood vessels in prostate cancer. The localization of VEGF-A, VEGF-C, VEGF-D, VEGF receptor (VEGFR)-1, VEGFR-2, and VEGFR-3 was examined in cancerous and adjacent benign prostate tissue from 52 subjects representing various grades of prostate cancer. Except for VEGFR-2, extensive staining was observed for all ligands and receptors in the prostate specimens. In epithelial cells, VEGF-A and VEGFR-1 expression was higher in tumor tissue compared to benign tissue. VEGF-D and VEGFR-3 expression was significantly higher in benign tissue compared to tumor in the stroma and the endothelium of lymphatic and blood vessels. In addition, the frequency of lymphatic vessels, but not blood vessels, was lower in tumor tissue compared with benign tissue. These results suggest that activation of VEGFR-1 by VEGF-A within the carcinoma, and activation of lymphatic endothelial cell VEGFR-3 by VEGF-D within the adjacent benign stroma may be important signaling mechanisms involved in the progression and subsequent metastatic spread of prostate cancer. Thus inhibition of these pathways may contribute to therapeutic strategies for the management of prostate cancer. Copyright © 2012 Wiley Periodicals, Inc.

  2. Feeding Releases Endogenous Opioids in Humans.

    Tuulari, Jetro J; Tuominen, Lauri; de Boer, Femke E; Hirvonen, Jussi; Helin, Semi; Nuutila, Pirjo; Nummenmaa, Lauri

    2017-08-23

    The endogenous opioid system supports a multitude of functions related to appetitive behavior in humans and animals, and it has been proposed to govern hedonic aspects of feeding thus contributing to the development of obesity. Here we used positron emission tomography to investigate whether feeding results in hedonia-dependent endogenous opioid release in humans. Ten healthy males were recruited for the study. They were scanned with the μ-opioid-specific ligand [ 11 C]carfentanil three times, as follows: after a palatable meal, a nonpalatable meal, and after an overnight fast. Subjective mood, satiety, and circulating hormone levels were measured. Feeding induced significant endogenous opioid release throughout the brain. This response was more pronounced following a nonpalatable meal versus a palatable meal, and independent of the subjective hedonic responses to feeding. We conclude that feeding consistently triggers cerebral opioid release even in the absence of subjective pleasure associated with feeding, suggesting that metabolic and homeostatic rather than exclusively hedonic responses play a role in the feeding-triggered cerebral opioid release. SIGNIFICANCE STATEMENT The endogenous opioid system supports both hedonic and homeostatic functions. It has been proposed that overeating and concomitant opioid release could downregulate opioid receptors and promote the development of obesity. However, it remains unresolved whether feeding leads to endogenous opioid release in humans. We used in vivo positron emission tomography to test whether feeding triggers cerebral opioid release and whether this response is associated with pleasurable sensations. We scanned volunteers using the μ-opioid receptor-specific radioligand [ 11 C]carfentanil three times, as follows: after an overnight fast, after consuming a palatable meal, and after consuming a nonpalatable meal. Feeding led to significant endogenous opioid release, and this occurred also in the absence of feeding

  3. Opioid receptors in human neuroblastoma SH-SY5Y cells: evidence for distinct morphine (. mu. ) and enkephalin (delta) binding sites

    Kazmi, S.M.I.; Mishra, R.K.

    1986-06-13

    Human neuroblastoma SH-SY5Y cells exhibited a heterogeneous population of ..mu.. and delta types of opioid binding sites. These specific binding sites displayed the characteristic saturability, stereospecificity and reversibility, expected of a receptor. Scatchard analysis of (/sup 3/H)-D-Ala/sup 2/-D-Leu/sup 5/-enkephalin (DADLE) in the presence of 10/sup -5/M D-Pro/sup 4/-morphiceptin (to block the ..mu.. receptors) and the competitive displacement by various highly selective ligands yielded the binding parameters of delta sites which closely resemble those of the delta receptors in brain and mouse neuroblastoma clones. Similarly, the high affinity binding of (/sup 3/H)-dihydromorphine, together with the higher potency of morphine analogues to displace (/sup 3/H)-naloxone binding established the presence of ..mu.. sites. Guanine nucleotides and NaCl significantly inhibited the association and increased the dissociation of (/sup 3/H)-DADLE binding.

  4. Modelling the PKPD of oxycodone in experimental pain - impact of opioid receptor polymorphisms

    Olsen, Rasmus; Foster, David J R; Upton, Richard N

    2016-01-01

    BACKGROUND: Polymorphisms in the opioid receptor genes may affect the pharmacodynamics (PD) of oxycodone and be part of the reason behind the diversity in clinical response. The aim of the analysis was to model the exposure-response profile of oxycodone for three different pain variables and search...... for genetic covariates. Model simulations were used to predict how population and effect-size impact the power to detect clinical significant SNPs. METHOD: The population pharmacokinetic-pharmacodynamic (PKPD) model of oral single-dosed oxycodone was based on pooled data from three published studies...... in healthy volunteers. Pain tolerance data from muscle pressure (n=36), visceral pressure (n=54) and skin pinch (n=34) were included. Genetic associations with 18 opioid-receptor SNPs were explored using a stepwise covariate approach. Model simulations were performed using the estimated model parameters...

  5. 2012 David W. Robertson Award for Excellence in Medicinal Chemistry: Neoclerodanes as Atypical Opioid Receptor Ligands⊥

    Prisinzano, Thomas E.

    2013-01-01

    The neoclerodane diterpene salvinorin A is the major active component of the hallucinogenic mint plant Salvia divinorum Epling & Játiva (Lamiaceae). Since the finding that salvinorin A exerts its potent psychotropic actions through the activation of opioid receptors, the site of action of morphine and related analogues, there has been much interest in elucidating the underlying mechanisms behind its effects. These effects are particularly remarkable, because (1) salvinorin A is the first reported non-nitrogenous opioid receptor agonist, and (2) its effects are not mediated through the previously investigated targets of psychotomimetics. This perspective outlines our research program, illustrating a new direction to the development of tools to further elucidate the biological mechanisms of drug tolerance and dependence. The information gained from these efforts is expected to facilitate the design of novel agents to treat pain, drug abuse, and other CNS disorders. PMID:23548164

  6. Salvinorin A, a kappa-opioid receptor (KOP-r agonist hallucinogen: Pharmacology and potential template for novel pharmacotherapeutic agents in neuropsychiatric disorders

    Eduardo eButelman

    2015-09-01

    Full Text Available Salvinorin A is a potent hallucinogen, isolated from the ethnomedical plant Salvia divinorum. Salvinorin A is a selective high efficacy kappa-opioid receptor (KOPr agonist, and thus implicates the KOPr system and its endogenous agonist ligands (the dynorphins in higher functions, including cognition, and perceptual effects. Salvinorin A is the only selective KOPr ligand to be widely available outside research or medical settings, and salvinorin A- containing products have undergone frequent non-medical use. KOPr/dynorphin systems in the brain are known to be powerful counter-modulatory mechanisms to dopaminergic function, which is important in mood and reward engendered by natural and drug reinforcers (including drugs of abuse. KOPr activation (including by salvinorin A can thus cause aversion and anhedonia in preclinical models. Salvinorin A is also a completely new scaffold for medicinal chemistry approaches, since it is a non-nitrogenous neoclerodane, unlike all other known opioid ligands. Ongoing efforts have the goal of discovering novel semi-synthetic salvinorin analogs with potential KOPr-mediated pharmacotherapeutic effects (including partial agonist or biased agonist effects, with a reduced burden of undesirable effects associated with salvinorin A.

  7. Ivy and neurogliaform interneurons are a major target of μ opioid receptor modulation

    Krook-Magnuson, Esther; Luu, Lillian; Lee, Sang-Hun; Varga, Csaba; Soltesz, Ivan

    2011-01-01

    Mu opioid receptors (μORs) are selectively expressed on interneurons in area CA1 of the hippocampus. Fast-spiking, parvalbumin expressing, basket cells express μORs, but circumstantial evidence suggests that another major, unidentified, GABAergic cell class must also be modulated by μORs. Here we report that the abundant, dendritically targeting, neurogliaform family of cells (Ivy and neurogliaform cells) is a previously unrecognized target of direct modulation by μORs. Ivy and neurogliaform ...

  8. Atypical Opioid Mechanisms of Control of Injury-Induced Cutaneous Pain by Delta Receptors

    2017-07-01

    i.e. mu opioid receptor agonists such as morphine) cause unacceptable side effects including addiction . Injuries suffered most frequently by active...slides. The slides were then processed for fluorescent in situ hybridization with RNAscope technology (ACD Biosystems) to detect Oprd1 mRNA, as...tissue as done in Bardoni et al., Neuron, 2014) and negative controls (no probe). Controls indicated that the technology and reagents work as expected

  9. Toll-like receptor 9 is required for opioid-induced microglia apoptosis.

    Lei He

    2011-04-01

    Full Text Available Opioids have been widely applied in clinics as one of the most potent pain relievers for centuries, but their abuse has deleterious physiological effects beyond addiction. However, the underlying mechanism by which microglia in response to opioids remains largely unknown. Here we show that morphine induces the expression of Toll-like receptor 9 (TLR9, a key mediator of innate immunity and inflammation. Interestingly, TLR9 deficiency significantly inhibited morphine-induced apoptosis in microglia. Similar results were obtained when endogenous TLR9 expression was suppressed by the TLR9 inhibitor CpGODN. Inhibition of p38 MAPK by its specific inhibitor SB203580 attenuated morphine-induced microglia apoptosis in wild type microglia. Morphine caused a dramatic decrease in Bcl-2 level but increase in Bax level in wild type microglia, but not in TLR9 deficient microglia. In addition, morphine treatment failed to induce an increased levels of phosphorylated p38 MAPK and MAP kinase kinase 3/6 (MKK3/6, the upstream MAPK kinase of p38 MAPK, in either TLR9 deficient or µ-opioid receptor (µOR deficient primary microglia, suggesting an involvement of MAPK and µOR in morphine-mediated TLR9 signaling. Moreover, morphine-induced TLR9 expression and microglia apoptosis appears to require μOR. Collectively, these results reveal that opioids prime microglia to undergo apoptosis through TLR9 and µOR as well. Taken together, our data suggest that inhibition of TLR9 and/or blockage of µOR is capable of preventing opioid-induced brain damage.

  10. Acute inflammation induces segmental, bilateral, supraspinally mediated opioid release in the rat spinal cord, as measured by μ-opioid receptor internalization

    Chen, Wenling; Marvizón, Juan Carlos G.

    2009-01-01

    The objective of this study was to measure opioid release in the spinal cord during acute and long-term inflammation using μ-opioid receptor (MOR) internalization. In particular, we determined whether opioid release occurs in the segments receiving the noxious signals or in the entire spinal cord, and whether it involves supraspinal signals. Internalization of neurokinin 1 receptors (NK1Rs) was measured to track the intensity of the noxious stimulus. Rats received peptidase inhibitors intrathecally to protect opioids from degradation. Acute inflammation of the hindpaw with formalin induced moderate MOR internalization in the L5 segment bilaterally, whereas NK1R internalization occurred only ipsilaterally. MOR internalization was restricted to the lumbar spinal cord, regardless of whether the peptidase inhibitors were injected in a lumbar or thoracic site. Formalin-induced MOR internalization was substantially reduced by isoflurane anesthesia. It was also markedly reduced by a lidocaine block of the cervical-thoracic spinal cord (which did not affect the evoked NK1R internalization) indicating that spinal opioid release is mediated supraspinally. In the absence of peptidase inhibitors, formalin and hindpaw clamp induced a small amount of MOR internalization, which was significantly higher than in controls. To study spinal opioid release during chronic inflammation, we injected Complete Freund's Adjuvant (CFA) in the hindpaw and peptidase inhibitors intrathecally. Two days later, no MOR or NK1R internalization was detected. Furthermore, CFA inflammation decreased MOR internalization induced by clamping the inflamed hindpaw. These results show that acute inflammation, but not chronic inflammation, induce segmental opioid release in the spinal cord that involves supraspinal signals. PMID:19298846

  11. Acute inflammation induces segmental, bilateral, supraspinally mediated opioid release in the rat spinal cord, as measured by mu-opioid receptor internalization.

    Chen, W; Marvizón, J C G

    2009-06-16

    The objective of this study was to measure opioid release in the spinal cord during acute and long-term inflammation using mu-opioid receptor (MOR) internalization. In particular, we determined whether opioid release occurs in the segments receiving the noxious signals or in the entire spinal cord, and whether it involves supraspinal signals. Internalization of neurokinin 1 receptors (NK1Rs) was measured to track the intensity of the noxious stimulus. Rats received peptidase inhibitors intrathecally to protect opioids from degradation. Acute inflammation of the hind paw with formalin induced moderate MOR internalization in the L5 segment bilaterally, whereas NK1R internalization occurred only ipsilaterally. MOR internalization was restricted to the lumbar spinal cord, regardless of whether the peptidase inhibitors were injected in a lumbar or thoracic site. Formalin-induced MOR internalization was substantially reduced by isoflurane anesthesia. It was also markedly reduced by a lidocaine block of the cervical-thoracic spinal cord (which did not affect the evoked NK1R internalization) indicating that spinal opioid release is mediated supraspinally. In the absence of peptidase inhibitors, formalin and hind paw clamp induced a small amount of MOR internalization, which was significantly higher than in controls. To study spinal opioid release during chronic inflammation, we injected complete Freund's adjuvant (CFA) in the hind paw and peptidase inhibitors intrathecally. Two days later, no MOR or NK1R internalization was detected. Furthermore, CFA inflammation decreased MOR internalization induced by clamping the inflamed hind paw. These results show that acute inflammation, but not chronic inflammation, induces segmental opioid release in the spinal cord that involves supraspinal signals.

  12. Opioid receptor subtypes mediating the noise-induced decreases in high-affinity choline uptake in the rat brain.

    Lai, H; Carino, M A

    1992-07-01

    Acute (20 min) exposure to 100-dB white noise elicits a naltrexone-sensitive decrease in sodium-dependent high-affinity choline uptake in the frontal cortex and hippocampus of the rat. In the present study, the subtypes of opioid receptors involved were investigated by pretreating rats with microinjection of specific opioid-receptor antagonists into the lateral cerebroventricle before noise exposure. We found that the noise-induced decrease in high-affinity choline uptake in the hippocampus was blocked by pretreatment with either mu-, delta-, or kappa-opioid-receptor antagonists, whereas the effect of noise on frontal cortical high-affinity choline uptake was blocked by a mu- and delta- but not by a kappa-antagonist. These data further confirm the role of endogenous opioids in mediating the effects of noise on central cholinergic activity and indicate that different neural mechanisms are involved in the effects of noise on the frontal cortical and hippocampal cholinergic systems.

  13. Progress on the application of ligand receptor binding assays in radiopharmaceuticals

    Zhou Xue; Qian Jinping; Kong Aiying; Zhu Lin

    2010-01-01

    Receptor binding assay is an important drug screening method, which can quickly and inexpensively study the interactions between the targeted receptor and the potential ligands in vitro and provide the information of the relative binding affinity of ligand-receptor. The imaging of many radiopharmaceuticals is based on highly selective radioligand-receptor binding. The technique plays an important role in the design and screening of receptor-targeting radiopharmaceuticals. (authors)

  14. The effect of benfotiamine on mu-opioid receptor mediated antinociception in experimental diabetes.

    Nacitarhan, C; Minareci, E; Sadan, G

    2014-03-01

    Diabetic neuropathy is a prevalent, disabling disorder. Currently, the only treatments available to patients with diabetic neuropathy are glucose control and pain management. B vitamin present neuroprotective effects, which are suggested to be related to their analgesic action in various models of neuropathic pain. According to our literature knowledge there is no report about antinociceptive effects of thiamine as benfotiamine and opioids together in diabetic mice. The purpose of this study was to determine the effects of benfotiamine on the antinociception produced by mu-opioid receptor agonist fentanyl in diabetic mice. The effects of benfotiamine on antinociception produced by fentanyl in diabetic mice were studied in 4 groups. Antinociceptive effect was determined with tail flick, hot plate and formalin test. Our results showed that, mu-opioid agonist fentanyl in benfotiamine applied diabetic group caused more potent antinociceptive effect than in diabetic group without benfotiamine treatment. In brief benfotiamine supplement in diet did not bring out antinociceptive effect itself, but during development of STZ diabetes, benfotiamine replacement increased the antinociceptive effect of fentanyl in mice tail-flick test. This effect is probably due to the replacement of benfotiamine efficiency occurring in diabetes mellitus. Finally, we suppose that oral benfotiamine replacement therapy may be useful to ameliorate analgesic effect of mu-opioid agonists on neuropathic pain in diabetic case. © J. A. Barth Verlag in Georg Thieme Verlag KG Stuttgart · New York.

  15. Desensitization and Tolerance of Mu Opioid Receptors on Pontine Kölliker-Fuse Neurons.

    Levitt, Erica S; Williams, John T

    2018-01-01

    Acute desensitization of mu opioid receptors is thought to be an initial step in the development of tolerance to opioids. Given the resistance of the respiratory system to develop tolerance, desensitization of neurons in the Kölliker-Fuse (KF), a key area in the respiratory circuit, was examined. The activation of G protein-coupled inwardly rectifying potassium current was measured using whole-cell voltage-clamp recordings from KF and locus coeruleus (LC) neurons contained in acute rat brain slices. A saturating concentration of the opioid agonist [Met 5 ]-enkephalin (ME) caused significantly less desensitization in KF neurons compared with LC neurons. In contrast to LC, desensitization in KF neurons was not enhanced by activation of protein kinase C or in slices from morphine-treated rats. Cellular tolerance to ME and morphine was also lacking in KF neurons from morphine-treated rats. The lack of cellular tolerance in KF neurons correlates with the relative lack of tolerance to the respiratory depressant effect of opioids. Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.

  16. Kappa opioid receptors in rat spinal cord vary across the estrous cycle.

    Chang, P C; Aicher, S A; Drake, C T

    2000-04-07

    Kappa opioid receptors (KORs) were immunocytochemically localized in the lumbosacral spinal cord of female rats in different stages of the estrous cycle to examine the influence of hormonal status on receptor density. KOR labeling was primarily in fine processes and a few neuronal cell bodies in the superficial dorsal horn and the dorsolateral funiculus. Quantitative light microscopic densitometry of the superficial dorsal horn revealed that rats in diestrus had significantly lower KOR densities than those in proestrus or estrus. This suggests that female reproductive hormones regulate spinal KOR levels, which may contribute to variations in analgesic effectiveness of KOR agonists across the estrous cycle.

  17. Site-specific effects of the nonsteroidal anti-inflammatory drug lysine clonixinate on rat brain opioid receptors.

    Ortí, E; Coirini, H; Pico, J C

    1999-04-01

    In addition to effects in the periphery through inhibition of prostaglandin synthesis, several lines of evidence suggest that nonsteroidal anti-inflammatory drugs (NSAIDs) act in the central nervous system. The possibility that the central action of NSAIDs involves regulation of opioid receptors was investigated by quantitative autoradiography of mu, delta, and kappa sites in rat brain slices. Increased (p lysine clonixinate. Labeling of delta receptors was lower in the lateral septum, and kappa sites decreased in thalamic nuclei. These effects were not mediated through direct interaction with opioid-binding sites, since receptor-binding assays using rat brain membranes confirmed that clonixinate up to 1 x 10(-4) mol/l does not inhibit mu, delta, and kappa receptor specific binding. Central effects of NSAIDs might, therefore, involve interaction with the opioid receptor system through indirect mechanisms.

  18. MIPs are ancestral ligands for the sex peptide receptor.

    Kim, Young-Joon; Bartalska, Katarina; Audsley, Neil; Yamanaka, Naoki; Yapici, Nilay; Lee, Ju-Youn; Kim, Yong-Chul; Markovic, Milica; Isaac, Elwyn; Tanaka, Yoshiaki; Dickson, Barry J

    2010-04-06

    Upon mating, females of many animal species undergo dramatic changes in their behavior. In Drosophila melanogaster, postmating behaviors are triggered by sex peptide (SP), which is produced in the male seminal fluid and transferred to female during copulation. SP modulates female behaviors via sex peptide receptor (SPR) located in a small subset of internal sensory neurons that innervate the female uterus and project to the CNS. Although required for postmating responses only in these female sensory neurons, SPR is expressed broadly in the CNS of both sexes. Moreover, SPR is also encoded in the genomes of insects that lack obvious SP orthologs. These observations suggest that SPR may have additional ligands and functions. Here, we identify myoinhibitory peptides (MIPs) as a second family of SPR ligands that is conserved across a wide range of invertebrate species. MIPs are potent agonists for Drosophila, Aedes, and Aplysia SPRs in vitro, yet are unable to trigger postmating responses in vivo. In contrast to SP, MIPs are not produced in male reproductive organs, and are not required for postmating behaviors in Drosophila females. We conclude that MIPs are evolutionarily conserved ligands for SPR, which are likely to mediate functions other than the regulation of female reproductive behaviors.

  19. Population pharmacokinetics of nalmefene in healthy subjects and its relation to μ-opioid receptor occupancy.

    Kyhl, Lars-Erik Broksoe; Li, Shen; Faerch, Kirstine Ullitz; Soegaard, Birgitte; Larsen, Frank; Areberg, Johan

    2016-02-01

    The aims of this study were to develop a population pharmacokinetic (PK) model to describe the PK of nalmefene in healthy subjects and to relate the exposure of nalmefene to the μ-opioid receptor occupancy by simulations in the target population. Data from nine phase I studies (243 subjects) with extensive blood sampling were pooled and used for the population PK model building. Data from four other phase I studies (85 subjects) were pooled and used as an external validation dataset. Eight subjects from an imaging study contributed occupancy data and the pharmacokinetic/pharmacodynamic (PK/PD) relationship was modelled. Combining the population PK model and the PK/PD relationship enabled simulations to predict μ-opioid occupancy. A two compartment model with first order absorption best described the nalmefene PK data. The typical subject in the population was estimated to have a systemic clearance of 60.4 l h(-1) and a central volume of distribution of 266 l. Absolute oral bioavailability was estimated to 41% without food intake and with food about 53%. Simulation of the μ-opioid receptor occupancy shows that the 95% confidence bound is within or above 60-90% occupancy for up to 22-24 h after a single dose of 20 mg nalmefene. A robust population PK model for nalmefene was developed. Based on the concentration-occupancy model the μ-opioid receptor occupancy after a single 20 mg dose of nalmefene is predicted to be above the target therapeutic occupancy for about 24 h in about 95% of the target population. © 2015 The British Pharmacological Society.

  20. Naloxone-sensitive, haloperidol-sensitive, [3H](+)SKF-10047-binding protein partially purified from rat liver and rat brain membranes: an opioid/sigma receptor?

    Tsao, L I; Su, T P

    1997-02-01

    A naloxone-sensitive, haloperidol-sensitive, [3H](+)SKF-10047-binding protein was partially purified from rat liver and rat brain membranes in an affinity chromatography originally designed to purify sigma receptors. Detergent-solubilized extracts from membranes were adsorbed to Sephadex G-25 resin containing an affinity ligand for sigma receptors: N-(2- 3,4-dichlorophenyl]ethyl)-N-(6-aminohexyl)-(2-[1-pyrrolidinyl]) ethylamine (DAPE). After eluting the resin with haloperidol, a protein that bound [3H](+)SKF-10047 was detected in the eluates. However, the protein was not the sigma receptor. [3H](+)SKF-10047 binding to the protein was inhibited by the following compounds in the order of decreasing potency: (+)pentazocine > (-) pentazocine > (+/-)cyclazocine > (-)morphine > (-)naloxone > haloperidol > (+)SKF-10047 > DADLE > (-)SKF-10047. Further, the prototypic sigma receptor ligands, such as 1,3-di-o-tolylguanidine (DTG), (+)3-PPP, and progesterone, bound poorly to the protein. Tryptic digestion and heat treatment of the affinity-purified protein abolished radioligand binding. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) of the partially-purified protein from the liver revealed a major diffuse band with a molecular mass of 31 kDa, a polypeptide of 65 kDa, and another polypeptide of > 97 kDa. This study demonstrates the existence of a novel protein in the rat liver and rat brain which binds opioids, benzomorphans, and haloperidol with namomolar affinity. The protein resembles the opioid/sigma receptor originally proposed by Martin et al. [(1976): J. Pharmacol. Exp. Ther., 197:517-532.]. A high degree of purification of this protein has been achieved in the present study.

  1. Deletion of the δ opioid receptor gene impairs place conditioning but preserves morphine reinforcement.

    Le Merrer, Julie; Plaza-Zabala, Ainhoa; Del Boca, Carolina; Matifas, Audrey; Maldonado, Rafael; Kieffer, Brigitte L

    2011-04-01

    Converging experimental data indicate that δ opioid receptors contribute to mediate drug reinforcement processes. Whether their contribution reflects a role in the modulation of drug reward or an implication in conditioned learning, however, has not been explored. In the present study, we investigated the impact of δ receptor gene knockout on reinforced conditioned learning under several experimental paradigms. We assessed the ability of δ receptor knockout mice to form drug-context associations with either morphine (appetitive)- or lithium (aversive)-induced Pavlovian place conditioning. We also examined the efficiency of morphine to serve as a positive reinforcer in these mice and their motivation to gain drug injections, with operant intravenous self-administration under fixed and progressive ratio schedules and at two different doses. Mutant mice showed impaired place conditioning in both appetitive and aversive conditions, indicating disrupted context-drug association. In contrast, mutant animals displayed intact acquisition of morphine self-administration and reached breaking-points comparable to control subjects. Thus, reinforcing effects of morphine and motivation to obtain the drug were maintained. Collectively, the data suggest that δ receptor activity is not involved in morphine reinforcement but facilitates place conditioning. This study reveals a novel aspect of δ opioid receptor function in addiction-related behaviors. Copyright © 2011 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

  2. The Mu opioid receptor promotes opioid and growth factor-induced proliferation, migration and Epithelial Mesenchymal Transition (EMT in human lung cancer.

    Frances E Lennon

    Full Text Available Recent epidemiologic studies implying differences in cancer recurrence based on anesthetic regimens raise the possibility that the mu opioid receptor (MOR can influence cancer progression. Based on our previous observations that overexpression of MOR in human non-small cell lung cancer (NSCLC cells increased tumor growth and metastasis, this study examined whether MOR regulates growth factor receptor signaling and epithelial mesenchymal transition (EMT in human NSCLC cells. We utilized specific siRNA, shRNA, chemical inhibitors and overexpression vectors in human H358 NSCLC cells that were either untreated or treated with various concentrations of DAMGO, morphine, fentanyl, EGF or IGF. Cell function assays, immunoblot and immunoprecipitation assays were then performed. Our results indicate MOR regulates opioid and growth factor-induced EGF receptor signaling (Src, Gab-1, PI3K, Akt and STAT3 activation which is crucial for consequent human NSCLC cell proliferation and migration. In addition, human NSCLC cells treated with opioids, growth factors or MOR overexpression exhibited an increase in snail, slug and vimentin and decrease ZO-1 and claudin-1 protein levels, results consistent with an EMT phenotype. Further, these effects were reversed with silencing (shRNA or chemical inhibition of MOR, Src, Gab-1, PI3K, Akt and STAT3 (p<0.05. Our data suggest a possible direct effect of MOR on opioid and growth factor-signaling and consequent proliferation, migration and EMT transition during lung cancer progression. Such an effect provides a plausible explanation for the epidemiologic findings.

  3. Killer Cell Immunoglobulin-like Receptors and their Ligands

    Tajik N.

    2010-09-01

    Full Text Available The Natural killer (NK cells are a subset of lymphocytes comprising around 10% of total lymphocytes in peripheral blood. Due to their role in the innate response, NK cells provide a ‘first line of defense’ against infectious agents and cancer and are also thought to play a role in autoimmunity. The killer cell immunoglobulin-like receptors (KIR are regulatory surface molecules, found on NK cells and on a subset of T lymphocytes. The genes for KIR are present on chromosome 19 in the leukocyte receptor complex and show a major difference for both the type and number of KIR genes present among different ethnic groups. They have been divided into two groups of 2D or 3D, depending on the number of external immunoglobulin domains. The presence of a long cytoplasmic tail with two immune tyrosine-based inhibitory motifs (ITIM allows the transduction of inhibitory signals and characterizes the inhibitory KIRs (2DL and 3DL, whereas the presence of short cytoplasmic tails corresponds to the activating KIR receptors (2DS and 3DS.These polymorphic receptors interact with specific motifs on human leukocyte antigen (HLA class I molecules, modulate NK cytolytic activity. Some KIRs are known to interact with HLA-C molecules of target cells, HLA-Bw4 molecules and HLA-A3/11. For some KIRs the corresponding ligands are still unknown.

  4. The μ-opioid receptor gene and smoking initiation and nicotine dependence

    Kendler Kenneth S

    2006-08-01

    Full Text Available Abstract The gene encoding the mu-opioid receptor (OPRM1 is reported to be associated with a range of substance dependence. Experiments in knockout mice indicate that the mu-opioid receptor may mediate reinforcing effects of nicotine. In humans, opioid antagonist naltrexone may reduce the reinforcing effects of tobacco smoking. Additionally, the OPRM1 gene is located in a region showing linkage to nicotine dependence. The OPRM1 is thus a plausible candidate gene for smoking behavior. To investigate whether OPRM1 contributes to the susceptibility of smoking initiation and nicotine dependence, we genotyped 11 SNPs in the gene for 688 Caucasian subjects of lifetime smokers and nonsmokers. Three SNPs showed nominal significance for smoking initiation and one reached significance for nicotine dependence. The global test for three-marker (rs9479757-rs2075572-rs10485057 haplotypes was significant for smoking initiation (p = 0.0022. The same three-marker haplotype test was marginal (p = 0.0514 for nicotine dependence. These results suggest that OPRM1 may be involved in smoking initiation and nicotine dependence.

  5. Involvement of direct inhibition of NMDA receptors in the effects of sigma-receptor ligands on glutamate neurotoxicity in vitro.

    Nishikawa, H; Hashino, A; Kume, T; Katsuki, H; Kaneko, S; Akaike, A

    2000-09-15

    This study was performed to examine the roles of the N-methyl-D-aspartate (NMDA) receptor/phencyclidine (PCP) channel complex in the protective effects of sigma-receptor ligands against glutamate neurotoxicity in cultured cortical neurons derived from fetal rats. A 1-h exposure of cultures to glutamate caused a marked loss of viability, as determined by Trypan blue exclusion. This acute neurotoxicity of glutamate was prevented by NMDA receptor antagonists. Expression of sigma(1) receptor mRNA in cortical cultures was confirmed by reverse transcription polymerase chain reaction (RT-PCR). sigma Receptor ligands with affinity for NMDA receptor channels including the PCP site, such as (+)-N-allylnormetazocine ((+)-SKF10,047), haloperidol, and R(-)-N-(3-phenyl-1-propyl)-1-phenyl-2-aminopropane ((-)-PPAP), prevented glutamate neurotoxicity in a concentration-dependent manner. In contrast, other sigma-receptor ligands without affinity for NMDA receptors, such as carbetapentane and R(+)-3-(3-hydroxyphenyl)-N-propylpiperidine ((+)-3-PPP), did not show neuroprotective effects. Putative endogenous sigma receptor ligands such as pregnenolone, progesterone, and dehydroepiandrosterone did not affect glutamate neurotoxicity. The protective effects of (+)-SKF10,047, haloperidol, and (-)-PPAP were not affected by the sigma(1) receptor antagonist rimcazole. These results suggested that a direct interaction with NMDA receptors but not with sigma receptors plays a crucial role in the neuroprotective effects of sigma receptor ligands with affinity for NMDA receptors.

  6. Somatostatin and opioid receptors do not regulate proliferation or apoptosis of the human multiple myeloma U266 cells

    Allouche Stéphane

    2009-06-01

    Full Text Available Abstract Background opioid and somatostatin receptors (SSTRs that can assemble as heterodimer were individually reported to modulate malignant cell proliferation and to favour apoptosis. Materials and methods: SSTRs and opioid receptors expression were examined by RT-PCR, western-blot and binding assays, cell proliferation was studied by XTT assay and propidium iodide (PI staining and apoptosis by annexin V-PI labelling. Results almost all human malignant haematological cell lines studied here expressed the five SSTRs. Further experiments were conducted on the human U266 multiple myeloma cells, which express also μ-opioid receptors (MOP-R. XTT assays and cell cycle studies provide no evidence for a significant effect upon opioid or somatostatin receptors stimulation. Furthermore, neither direct effect nor potentiation of the Fas-receptor pathway was detected on apoptosis after these treatments. Conclusion these data suggest that SSTRs or opioid receptors expression is not a guaranty for an anti-tumoral action in U266 cell line.

  7. Effects of the kappa opioid receptor antagonist MR-2266-BS on the acquisition of ethanol preference

    Sandi, C.; Borrell, J.; Guaza, C. (Cajal Institute, Madrid (Spain))

    1990-01-01

    Using a paradigm by which rats forced to drink a weak ethanol solution develop ethanol preference in consecutive retention testing days, the effects of the administration of the kappa opioid antagonist MR-2266-BS, prior to or after the forced ethanol session, were studied. Pre-conditioning subcutaneous (s.c.) administration of 1 mg/kg of MR-2266-BS induced a decrease in subsequent ethanol consumption without significantly modifying the acquisition of ethanol preference. Post-conditioning administration of MR-2266-BS induced both a dose-dependent reduction in ethanol consumption and in preference throughout the three following days. The results of the present study provide further support of the involvement of kappa-type opioids on drinking behavior, and suggest that kappa receptors may be involved in the consumption and development of preference to ethanol.

  8. Effect of Iboga alkaloids on µ-opioid receptor-coupled G protein activation.

    Tamara Antonio

    Full Text Available The iboga alkaloids are a class of small molecules defined structurally on the basis of a common ibogamine skeleton, some of which modify opioid withdrawal and drug self-administration in humans and preclinical models. These compounds may represent an innovative approach to neurobiological investigation and development of addiction pharmacotherapy. In particular, the use of the prototypic iboga alkaloid ibogaine for opioid detoxification in humans raises the question of whether its effect is mediated by an opioid agonist action, or if it represents alternative and possibly novel mechanism of action. The aim of this study was to independently replicate and extend evidence regarding the activation of μ-opioid receptor (MOR-related G proteins by iboga alkaloids.Ibogaine, its major metabolite noribogaine, and 18-methoxycoronaridine (18-MC, a synthetic congener, were evaluated by agonist-stimulated guanosine-5´-O-(γ-thio-triphosphate ([(35S]GTPγS binding in cells overexpressing the recombinant MOR, in rat thalamic membranes, and autoradiography in rat brain slices.In rat thalamic membranes ibogaine, noribogaine and 18-MC were MOR antagonists with functional Ke values ranging from 3 uM (ibogaine to 13 uM (noribogaine and 18MC. Noribogaine and 18-MC did not stimulate [(35S]GTPγS binding in Chinese hamster ovary cells expressing human or rat MORs, and had only limited partial agonist effects in human embryonic kidney cells expressing mouse MORs. Ibogaine did not did not stimulate [(35S]GTPγS binding in any MOR expressing cells. Noribogaine did not stimulate [(35S]GTPγS binding in brain slices using autoradiography. An MOR agonist action does not appear to account for the effect of these iboga alkaloids on opioid withdrawal. Taken together with existing evidence that their mechanism of action also differs from that of other non-opioids with clinical effects on opioid tolerance and withdrawal, these findings suggest a novel mechanism of action, and

  9. Characterization of melanocortin receptor ligands on cloned brain melanocortin receptors and on grooming behavior in the rat

    Gispen, W.H.; Adan, R.A.H.; Szklarczyk, A.W.; Oosterom, J.; Brakkee, J.H.; Nijenhuis, W.A.; Schaaper, W.M.; Meloen, R.H.

    1999-01-01

    Since the melanocortin MC3 and melanocortin MC4 receptors are the main melanocortin receptor subtypes expressed in rat brain, we characterized the activity and affinity of nine melanocortin receptor ligands using these receptors in vitro, as well as their activity in a well-defined

  10. Ligands specify estrogen receptor alpha nuclear localization and degradation

    Caze-Subra Stéphanie

    2010-12-01

    Full Text Available Abstract Background The estrogen receptor alpha (ERα is found predominately in the nucleus, both in hormone stimulated and untreated cells. Intracellular distribution of the ERα changes in the presence of agonists but the impact of different antiestrogens on the fate of ERα is a matter of debate. Results A MCF-7 cell line stably expressing GFP-tagged human ERα (SK19 cell line was created to examine the localization of ligand-bound GFP-ERα. We combined digitonin-based cell fractionation analyses with fluorescence and immuno-electron microscopy to determine the intracellular distribution of ligand-bound ERα and/or GFP-ERα. Using fluorescence- and electron microscopy we demonstrate that both endogenous ERα and GFP-ERα form numerous nuclear focal accumulations upon addition of agonist, 17β-estradiol (E2, and pure antagonists (selective estrogen regulator disruptor; SERD, ICI 182,780 or RU58,668, while in the presence of partial antagonists (selective estrogen regulator modulator; SERM, 4-hydroxytamoxifen (OHT or RU39,411, diffuse nuclear staining persisted. Digitonin based cell fractionation analyses confirmed that endogenous ERα and GFP-ERα predominantly reside in the nuclear fraction. Overall ERα protein levels were reduced after estradiol treatment. In the presence of SERMs ERα was stabilized in the nuclear soluble fraction, while in the presence of SERDs protein levels decreased drastically and the remaining ERα was largely found in a nuclear insoluble fraction. mRNA levels of ESR1 were reduced compared to untreated cells in the presence of all ligands tested, including E2. E2 and SERDs induced ERα degradation occurred in distinct nuclear foci composed of ERα and the proteasome providing a simple explanation for ERα sequestration in the nucleus. Conclusions Our results indicate that chemical structure of ligands directly affect the nuclear fate and protein turnover of the estrogen receptor alpha independently of their impact on

  11. Cell-Autonomous Regulation of Mu-Opioid Receptor Recycling by Substance P

    Bowman, Shanna L.; Soohoo, Amanda L.; Shiwarski, Daniel J.; Schulz, Stefan; Pradhan, Amynah A.; Puthenveedu, Manojkumar A.

    2015-01-01

    SUMMARY How neurons coordinate and reprogram multiple neurotransmitter signals is an area of broad interest. Here, we show that substance P (SP), a neuropep-tide associated with inflammatory pain, reprograms opioid receptor recycling and signaling. SP, through activation of the neurokinin 1 (NK1R) receptor, increases the post-endocytic recycling of the muopioid receptor (MOR) in trigeminal ganglion (TG) neurons in an agonist-selective manner. SP-mediated protein kinase C (PKC) activation is both required and sufficient for increasing recycling of exogenous and endogenous MOR in TG neurons. The target of this cross-regulation is MOR itself, given that mutation of either of two PKC phosphorylation sites on MOR abolishes the SP-induced increase in recycling and resensitization. Furthermore, SP enhances the resensitization of fentanyl-induced, but not morphine-induced, antinociception in mice. Our results define a physiological pathway that cross-regulates opioid receptor recycling via direct modification of MOR and suggest a mode of homeo-static interaction between the pain and analgesic systems. PMID:25801029

  12. Reduced emotional and corticosterone responses to stress in μ-opioid receptor knockout mice

    Ide, Soichiro; Sora, Ichiro; Ikeda, Kazutaka; Minami, Masabumi; Uhl, George R.; Ishihara, Kumatoshi

    2014-01-01

    The detailed mechanisms of emotional modulation in the nervous system by opioids remain to be elucidated, although the opioid system is well known to play important roles in the mechanisms of analgesia and drug dependence. In the present study, we conducted behavioral tests of anxiety and depression and measured corticosterone concentrations in both male and female μ-opioid receptor knockout (MOP-KO) mice to reveal the involvement of μ-opioid receptors in stress-induced emotional responses. MOP-KO mice entered more and spent more time in the open arms of the elevated plus maze compared with wild-type mice. MOP-KO mice also displayed significantly decreased immobility in a 15 min tail-suspension test compared with wild-type mice. Similarly, MOP-KO mice exhibited significantly decreased immobility on days 2, 3, and 4 in a 6 min forced swim test conducted for 5 consecutive days. The increase in plasma corticosterone concentration induced by tail-suspension, repeated forced swim, or restraint stress was reduced in MOP-KO mice compared with wild-type mice. Corticosterone levels were not different between wild-type and MOP-KO mice before stress exposure. In contrast, although female mice tended to exhibit fewer anxiety-like responses in the tail-suspension test in both genotypes, no significant gender differences were observed in stress-induced emotional responses. These results suggest that MOPs play an important facilitatory role in emotional responses to stress, including anxiety- and depression-like behavior and corticosterone levels. PMID:19596019

  13. alpha-Adrenoceptor and opioid receptor modulation of clonidine-induced antinociception.

    Sierralta, F; Naquira, D; Pinardi, G; Miranda, H F

    1996-10-01

    1. The antinociceptive action of clonidine (Clon) and the interactions with alpha 1, alpha 2 adrenoceptor and opioid receptor antagonists was evaluated in mice by use of chemical algesiometric test (acetic acid writhing test). 2. Clon produced a dose-dependent antinociceptive action and the ED50 for intracerebroventricular (i.c.v.) was lower than for intraperitoneal (i.p.) administration (1 ng kg-1 vs 300 ng kg-1). The parallelism of the dose-response curves indicates activation of a common receptor subtype. 3. Systemic administration of prazosin and terazosin displayed antinociceptive activity. Pretreatment with prazosin produced a dual action: i.c.v. Clon effect did not change, and i.p. Clon effect was enhanced. Yohimbine i.c.v. or i.p. did not induce antinonciception, but antagonized Clon-induced activity. These results suggest that alpha 1- and alpha 2-adrenoceptors, either located at the pre- and/or post-synaptic level, are involved in the control of spinal antinociception. 4. Naloxone (NX) and naltrexone (NTX) induced antinociceptive effects at low doses (microgram kg-1 range) and a lower antinociceptive effect at higher doses (mg kg-1 range). Low doses of NX or NTX antagonized Clon antinociception, possibly in relation to a preferential mu opioid receptor antagonism. In contrast, high doses of NX or NTX increased the antinociceptive activity of Clon, which could be due to an enhanced inhibition of the release of substance P. 5. The results obtained in the present work suggest the involvement of alpha 1-, alpha 2-adrenoceptor and opioid receptors in the modulation of the antinociceptive activity of clonidine, which seems to be exerted either at spinal and/or supraspinal level.

  14. Concomitant duplications of opioid peptide and receptor genes before the origin of jawed vertebrates.

    Görel Sundström

    Full Text Available BACKGROUND: The opioid system is involved in reward and pain mechanisms and consists in mammals of four receptors and several peptides. The peptides are derived from four prepropeptide genes, PENK, PDYN, PNOC and POMC, encoding enkephalins, dynorphins, orphanin/nociceptin and beta-endorphin, respectively. Previously we have described how two rounds of genome doubling (2R before the origin of jawed vertebrates formed the receptor family. METHODOLOGY/PRINCIPAL FINDINGS: Opioid peptide gene family members were investigated using a combination of sequence-based phylogeny and chromosomal locations of the peptide genes in various vertebrates. Several adjacent gene families were investigated similarly. The results show that the ancestral peptide gene gave rise to two additional copies in the genome doublings. The fourth member was generated by a local gene duplication, as the genes encoding POMC and PNOC are located on the same chromosome in the chicken genome and all three teleost genomes that we have studied. A translocation has disrupted this synteny in mammals. The PDYN gene seems to have been lost in chicken, but not in zebra finch. Duplicates of some peptide genes have arisen in the teleost fishes. Within the prepropeptide precursors, peptides have been lost or gained in different lineages. CONCLUSIONS/SIGNIFICANCE: The ancestral peptide and receptor genes were located on the same chromosome and were thus duplicated concomitantly. However, subsequently genetic linkage has been lost. In conclusion, the system of opioid peptides and receptors was largely formed by the genome doublings that took place early in vertebrate evolution.

  15. Flow Cytometry-Based Bead-Binding Assay for Measuring Receptor Ligand Specificity

    Sprokholt, Joris K.; Hertoghs, Nina; Geijtenbeek, Teunis B. H.

    2016-01-01

    In this chapter we describe a fluorescent bead-binding assay, which is an efficient and feasible method to measure interaction between ligands and receptors on cells. In principle, any ligand can be coated on fluorescent beads either directly or via antibodies. Binding between ligand-coated beads

  16. The future of type 1 cannabinoid receptor allosteric ligands.

    Alaverdashvili, Mariam; Laprairie, Robert B

    2018-02-01

    Allosteric modulation of the type 1 cannabinoid receptor (CB1R) holds great therapeutic potential. This is because allosteric modulators do not possess intrinsic efficacy, but instead augment (positive allosteric modulation) or diminish (negative allosteric modulation) the receptor's response to endogenous ligand. Consequently, CB1R allosteric modulators have an effect ceiling which allows for the tempering of CB1R signaling without the desensitization, tolerance, dependence, and psychoactivity associated with orthosteric compounds. Pain, movement disorders, epilepsy, obesity are all potential therapeutic targets for CB1R allosteric modulation. Several challenges exist for the development of CB1R allosteric modulators, such as receptor subtype specificity, translation to in vivo systems, and mixed allosteric/agonist/inverse agonist activity. Despite these challenges, elucidation of crystal structures of CB1R and compound design based on structure-activity relationships will advance the field. In this review, we will cover recent progress for CB1R allosteric modulators and discuss the future promise of this research.

  17. Identification of VDR Antagonists among Nuclear Receptor Ligands Using Virtual Screening

    Kelly Teske

    2014-04-01

    Full Text Available Herein, we described the development of two virtual screens to identify new vitamin D receptor (VDR antagonists among nuclear receptor (NR ligands. Therefore, a database of 14330 nuclear receptor ligands and their NR affinities was assembled using the online available “Binding Database.” Two different virtual screens were carried out in conjunction with a reported VDR crystal structure applying a stringent and less stringent pharmacophore model to filter docked NR ligand conformations. The pharmacophore models were based on the spatial orientation of the hydroxyl functionalities of VDR's natural ligands 1,25(OH2D3 and 25(OH2D3. The first virtual screen identified 32 NR ligands with a calculated free energy of VDR binding of more than -6.0 kJ/mol. All but nordihydroguaiaretic acid (NDGA are VDR ligands, which inhibited the interaction between VDR and coactivator peptide SRC2-3 with an IC50 value of 15.8 μM. The second screen identified 162 NR ligands with a calculated free energy of VDR binding of more than -6.0 kJ/mol. More than half of these ligands were developed to bind VDR followed by ERα/β ligands (26%, TRα/β ligands (7%, and LxRα/β ligands (7%. The binding between VDR and ERα ligand H6036 as well as TRα/β ligand triiodothyronine and a homoserine analog thereof was confirmed by fluorescence polarization.

  18. Novel photoaffinity ligands for the GA-receptor

    Suttle, J.C.; Hultstrand, J.F.; Tanaka, F.S.

    1990-01-01

    Previous studies from this laboratory have shown that certain N-substituted phthalimides (NSPs) exhibit GA-like activity in a range of specific bioassays and that bioactive NSPs compete with [ 3 H]-GA 4 for soluble binding sites in cucumber homogenates. As such, these compounds may prove useful in the purification and characterization of GA receptor proteins. To this end, five azido-NSPs have been synthesized and are currently being screened for biological activity and photochemical stability. Three azido-NSPs elicit α-amylase production in barley half-seeds and stimulate tissue elongation in d 5 maize, lettuce, sunflower, and soybean. Further evaluations are in progress and these data as well as the utility of these compounds as photo-affinity ligands will be discussed

  19. β-Arrestin-2 knockout prevents development of cellular μ-opioid receptor tolerance but does not affect opioid-withdrawal-related adaptations in single PAG neurons.

    Connor, M; Bagley, E E; Chieng, B C; Christie, M J

    2015-01-01

    Tolerance to the behavioural effects of morphine is blunted in β-arrestin-2 knockout mice, but opioid withdrawal is largely unaffected. The cellular mechanisms of tolerance have been studied in some neurons from β-arrestin-2 knockouts, but tolerance and withdrawal mechanisms have not been examined at the cellular level in periaqueductal grey (PAG) neurons, which are crucial for central tolerance and withdrawal phenomena. μ-Opioid receptor (MOPr) inhibition of voltage-gated calcium channel currents (ICa ) was examined by patch-clamp recordings from acutely dissociated PAG neurons from wild-type and β-arrestin-2 knockout mice treated chronically with morphine (CMT) or vehicle. Opioid withdrawal-induced activation of GABA transporter type 1 (GAT-1) currents was determined using perforated patch recordings from PAG neurons in brain slices. MOPr inhibition of ICa in PAG neurons was unaffected by β-arrestin-2 deletion. CMT impaired coupling of MOPrs to ICa in PAG neurons from wild-type mice, but this cellular tolerance was not observed in neurons from CMT β-arrestin-2 knockouts. However, β-arrestin-2 knockouts displayed similar opioid-withdrawal-induced activation of GAT-1 currents as wild-type PAG neurons. In β-arrestin-2 knockout mice, the central neurons involved in the anti-nociceptive actions of opioids also fail to develop cellular tolerance to opioids following chronic morphine. The results also provide the first cellular physiological evidence that opioid withdrawal is not disrupted by β-arrestin-2 deletion. However, the unaffected basal sensitivity to opioids in PAG neurons provides further evidence that changes in basal MOPr sensitivity cannot account for the enhanced acute nociceptive response to morphine reported in β-arrestin-2 knockouts. This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2. © 2014 The British

  20. Imaging of a glioma using peripheral benzodiazepine receptor ligands

    Starosta-Rubinstein, S.; Ciliax, B.J.; Penney, J.B.; McKeever, P.; Young, A.B.

    1987-02-01

    Two types of benzodiazepine receptors have been demonstrated in mammalian tissues, one which is localized on neuronal elements in brain and the other, on glial cells and in peripheral tissues such as kidney. In vivo administration of /sup 3/H-labeled PK 11195 (1-(2-chlorophenyl-N-methyl-N-(1-methylpropyl)-3-isoquinoline carboxamide) or (/sup 3/H)flunitrazepam with 5 mg of clonazepam per kg to rats with intracranial C6 gliomas resulted in high levels of tritiated-drug binding to the tumor as shown by quantitative autoradiography. Pharmacological studies indicated that the bound drugs labeled the peripheral benzodiazepine binding site. Binding to the peripheral benzodiazepine site was confirmed primarily to malignant cells with little binding to adjacent normal brain tissue or to necrotic tissue. Tumor cell binding was completely inhibited by preadministration of the peripheral benzodiazepine blocking agent PK 11195 at 5 mg/kg. The centrally selective benzodiazepine ligand clonazepam had no effect on PK 11195 binding to the tumor cells. When binding to other tumor cell lines grown in nude mice and nude athymic rats was evaluated, little or no peripheral benzodiazepine binding was detected on human pheochromocytoma (RN1) and neuroblastoma (SK-N-MC, SK-N-SH) tumor cells, respectively. However, high densities of peripheral benzodiazepine binding sites were observed on tumors derived from a human glioma cell line (ATCC HTB 14, U-87 MG). The presence of high concentrations of specific peripheral benzodiazepine receptors on glial tumors suggests that human primary central nervous system tumors could be imaged and diagnosed using peripheral benzodiazepine ligands labeled with positron- or gamma-emitting isotopes.

  1. Development of radioiodinated receptor ligands for cerebral single photon emission tomography

    Knapp, F.F. Jr.; McPherson, D.W.

    1992-01-01

    In the last decade the use of radiolabeled ligands for the imaging of cerebral receptors by emission computed tomography (ECT) has seen rapid growth. The opportunity to routinely perform cerebral single photon emission tomography (SPET) with iodine-123-labeled ligands depends on the availability of receptor ligands into which iodine can be introduced without decreasing the required high target receptor specificity. The use of iodine-123-labeled receptor-specific ligands also depends on the availability of high purity iodine-123 at reasonable costs and the necessary imaging instrumentation. In this paper, the development and current stage of evaluation of various iodine-123-labeled ligands for SPET imaging of dopaminergic, serotonergic and muscarinic acetylcholinergic receptor classes are discussed

  2. Morphine withdrawal enhances constitutive μ-opioid receptor activity in the ventral tegmental area.

    Meye, Frank J; van Zessen, Ruud; Smidt, Marten P; Adan, Roger A H; Ramakers, Geert M J

    2012-11-14

    μ-Opioid receptors (MORs) in the ventral tegmental area (VTA) are pivotally involved in addictive behavior. While MORs are typically activated by opioids, they can also become constitutively active in the absence of any agonist. In the current study, we present evidence that MOR constitutive activity is highly relevant in the mouse VTA, as it regulates GABAergic input to dopamine neurons. Specifically, suppression of MOR constitutive activity with the inverse agonist KC-2-009 enhanced GABAergic neurotransmission onto VTA dopamine neurons. This inverse agonistic effect was fully blocked by the specific MOR neutral antagonist CTOP, which had no effect on GABAergic transmission itself. We next show that withdrawal from chronic morphine further increases the magnitude of inverse agonistic effects at the MOR, suggesting enhanced MOR constitutive activity. We demonstrate that this increase can be an adaptive response to the detrimental elevation in cAMP levels known to occur during morphine withdrawal. These findings offer important insights in the physiological occurrence and function of MOR constitutive activity, and have important implications for therapeutic strategies aimed at normalizing MOR signaling during addiction and opioid overdose.

  3. Expression and purification of functional human mu opioid receptor from E.coli.

    Yanbin Ma

    Full Text Available N-terminally his-tagged human mu opioid receptor, a G protein-coupled receptor was produced in E.coli employing synthetic codon-usage optimized constructs. The receptor was expressed in inclusion bodies and membrane-inserted in different E.coli strains. By optimizing the expression conditions the expression level for the membrane-integrated receptor was raised to 0.3-0.5 mg per liter of culture. Milligram quantities of receptor could be enriched by affinity chromatography from IPTG induced cultures grown at 18°C. By size exclusion chromatography the protein fraction with the fraction of alpha-helical secondary structure expected for a 7-TM receptor was isolated, by CD-spectroscopy an alpha-helical content of ca. 45% was found for protein solubilised in the detergent Fos-12. Receptor in Fos-12 micelles was shown to bind endomorphin-1 with a K(D of 61 nM. A final yield of 0.17 mg functional protein per liter of culture was obtained.

  4. Ligand Receptor-Mediated Regulation of Growth in Plants.

    Haruta, Miyoshi; Sussman, Michael R

    2017-01-01

    Growth and development of multicellular organisms are coordinately regulated by various signaling pathways involving the communication of inter- and intracellular components. To form the appropriate body patterns, cellular growth and development are modulated by either stimulating or inhibiting these pathways. Hormones and second messengers help to mediate the initiation and/or interaction of the various signaling pathways in all complex multicellular eukaryotes. In plants, hormones include small organic molecules, as well as larger peptides and small proteins, which, as in animals, act as ligands and interact with receptor proteins to trigger rapid biochemical changes and induce the intracellular transcriptional and long-term physiological responses. During the past two decades, the availability of genetic and genomic resources in the model plant species, Arabidopsis thaliana, has greatly helped in the discovery of plant hormone receptors and the components of signal transduction pathways and mechanisms used by these immobile but highly complex organisms. Recently, it has been shown that two of the most important plant hormones, auxin and abscisic acid (ABA), act through signaling pathways that have not yet been recognized in animals. For example, auxins stimulate cell elongation by bringing negatively acting transcriptional repressor proteins to the proteasome to be degraded, thus unleashing the gene expression program required for increasing cell size. The "dormancy" inducing hormone, ABA, binds to soluble receptor proteins and inhibits a specific class of protein phosphatases (PP2C), which activates phosphorylation signaling leading to transcriptional changes needed for the desiccation of the seeds prior to entering dormancy. While these two hormone receptors have no known animal counterparts, there are also many similarities between animal and plant signaling pathways. For example, in plants, the largest single gene family in the genome is the protein kinase

  5. Muscarinic acetylcholine receptors: location of the ligand binding site

    Hulme, E.; Wheatley, M.; Curtis, C.; Birdsall, N.

    1987-01-01

    The key to understanding the pharmacological specificity of muscarinic acetylcholine receptors (mAChR's) is the location within the receptor sequence of the amino acid residues responsible for ligand binding. To approach this problem, they have purified mAChR's from rat brain to homogeneity by sequential ion-exchange chromatography, affinity chromatography and molecular weight fractionation. Following labelling of the binding site with an alkylating affinity label, 3 H-propylbenzilycholine mustard aziridinium ion ( 3 H-PrBCM), the mAChR was digested with a lysine-specific endoproteinase, and a ladder of peptides of increasing molecular weight, each containing the glycosylated N-terminus, isolated by chromatography on wheat-germ agglutinin sepharose. The pattern of labelling showed that a residue in the peptides containing transmembrane helices 2 and/or 3 of the mAChR was alkylated. The linkage was cleaved by 1 M hydroxylamine, showing that 3 H-PrBCM was attached to an acidic residue, whose properties strongly suggested it to be embedded in a hydrophobic intramembrane region of the mAChR. Examination of the cloned sequence of the mAChR reveals several candidate residues, the most likely of which is homologous to an aspartic acid residue thought to protonate the retinal Schiff's base in the congeneric protein rhodopsin

  6. Acetylcholine receptors and cholinergic ligands: biochemical and genetic aspects in Torpedo californica and Drosophila melanogaster

    Rosenthal, L.S.

    1987-01-01

    This study evaluates the biochemical and genetic aspects of the acetylcholine receptor proteins and cholinergic ligands in Drosophila melanogaster and Torpedo californica. Included are (1) a comparative study of nicotinic ligand-induced cation release from acetylcholine receptors isolated from Torpedo californica and from Drosophila melanogaster, (2) solution studies of the cholinergic ligands, nikethamide and ethamivan, aimed at measuring internal molecular rotational barriers in solvents of different polarity; and (3) the isolation and characterization of the gene(s) for the acetylcholine receptor in Drosophila melasogaster. Acetylcholine receptor proteins isolated from Drosphila melanogaster heads were found to behave kinetically similar (with regards to cholinergic ligand-induced 155 Eu: 3+ displacement from prelabeled proteins) to receptor proteins isolated from Torpedo californica electric tissue, providing additional biochemical evidence for the existence of a Drosophila acetylcholine receptor

  7. Engineering of Olfactory Receptor OlfCc1 for Directed Ligand Sensitivity

    Berke, Allison Paige

    2013-01-01

    Abstract Engineering of Olfactory Receptor OlfCc1 for Directed Ligand Sensitivityby Allison Paige Berke Joint Doctor of Philosophywith the University of California San FranciscoUniversity of California, Berkeley Professor Song Li, ChairDue to structural similarity, OlfCc1and its mammalian analogue V2R2 are hypothesized to respond to amino acid ligands in a calcium-mediated fashion. By analyzing receptor structure and making targeted mutations, the specificity and sensitivity of the receptor s...

  8. Lateralized kappa opioid receptor signaling from the amygdala central nucleus promotes stress-induced functional pain.

    Nation, Kelsey M; De Felice, Milena; Hernandez, Pablo I; Dodick, David W; Neugebauer, Volker; Navratilova, Edita; Porreca, Frank

    2018-05-01

    The response of diffuse noxious inhibitory controls (DNIC) is often decreased, or lost, in stress-related functional pain syndromes. Because the dynorphin/kappa opioid receptor (KOR) pathway is activated by stress, we determined its role in DNIC using a model of stress-induced functional pain. Male, Sprague-Dawley rats were primed for 7 days with systemic morphine resulting in opioid-induced hyperalgesia. Fourteen days after priming, when hyperalgesia was resolved, rats were exposed to environmental stress and DNIC was evaluated by measuring hind paw response threshold to noxious pressure (test stimulus) after capsaicin injection in the forepaw (conditioning stimulus). Morphine priming without stress did not alter DNIC. However, stress produced a loss of DNIC in morphine-primed rats in both hind paws that was abolished by systemic administration of the KOR antagonist, nor-binaltorphimine (nor-BNI). Microinjection of nor-BNI into the right, but not left, central nucleus of the amygdala (CeA) prevented the loss of DNIC in morphine-primed rats. Diffuse noxious inhibitory controls were not modulated by bilateral nor-BNI in the rostral ventromedial medulla. Stress increased dynorphin content in both the left and right CeA of primed rats, reaching significance only in the right CeA; no change was observed in the rostral ventromedial medulla or hypothalamus. Although morphine priming alone is not sufficient to influence DNIC, it establishes a state of latent sensitization that amplifies the consequences of stress. After priming, stress-induced dynorphin/KOR signaling from the right CeA inhibits DNIC in both hind paws, likely reflecting enhanced descending facilitation that masks descending inhibition. Kappa opioid receptor antagonists may provide a new therapeutic strategy for stress-related functional pain disorders.

  9. Potential ligand-binding residues in rat olfactory receptors identified by correlated mutation analysis

    Singer, M. S.; Oliveira, L.; Vriend, G.; Shepherd, G. M.

    1995-01-01

    A family of G-protein-coupled receptors is believed to mediate the recognition of odor molecules. In order to identify potential ligand-binding residues, we have applied correlated mutation analysis to receptor sequences from the rat. This method identifies pairs of sequence positions where residues remain conserved or mutate in tandem, thereby suggesting structural or functional importance. The analysis supported molecular modeling studies in suggesting several residues in positions that were consistent with ligand-binding function. Two of these positions, dominated by histidine residues, may play important roles in ligand binding and could confer broad specificity to mammalian odor receptors. The presence of positive (overdominant) selection at some of the identified positions provides additional evidence for roles in ligand binding. Higher-order groups of correlated residues were also observed. Each group may interact with an individual ligand determinant, and combinations of these groups may provide a multi-dimensional mechanism for receptor diversity.

  10. Glucagon-like peptide-1 receptor ligand interactions: structural cross talk between ligands and the extracellular domain.

    Graham M West

    Full Text Available Activation of the glucagon-like peptide-1 receptor (GLP-1R in pancreatic β-cells potentiates insulin production and is a current therapeutic target for the treatment of type 2 diabetes mellitus (T2DM. Like other class B G protein-coupled receptors (GPCRs, the GLP-1R contains an N-terminal extracellular ligand binding domain. N-terminal truncations on the peptide agonist generate antagonists capable of binding to the extracellular domain, but not capable of activating full length receptor. The main objective of this study was to use Hydrogen/deuterium exchange (HDX to identify how the amide hydrogen bonding network of peptide ligands and the extracellular domain of GLP-1R (nGLP-1R were altered by binding interactions and to then use this platform to validate direct binding events for putative GLP-1R small molecule ligands. The HDX studies presented here for two glucagon-like peptide-1 receptor (GLP-1R peptide ligands indicates that the antagonist exendin-4[9-39] is significantly destabilized in the presence of nonionic detergents as compared to the agonist exendin-4. Furthermore, HDX can detect stabilization of exendin-4 and exendin-4[9-39] hydrogen bonding networks at the N-terminal helix [Val19 to Lys27] upon binding to the N-terminal extracellular domain of GLP-1R (nGLP-1R. In addition we show hydrogen bonding network stabilization on nGLP-1R in response to ligand binding, and validate direct binding events with the extracellular domain of the receptor for putative GLP-1R small molecule ligands.

  11. Divergent Ah Receptor Ligand Selectivity during Hominin Evolution.

    Hubbard, Troy D; Murray, Iain A; Bisson, William H; Sullivan, Alexis P; Sebastian, Aswathy; Perry, George H; Jablonski, Nina G; Perdew, Gary H

    2016-10-01

    We have identified a fixed nonsynonymous sequence difference between humans (Val381; derived variant) and Neandertals (Ala381; ancestral variant) in the ligand-binding domain of the aryl hydrocarbon receptor (AHR) gene. In an exome sequence analysis of four Neandertal and Denisovan individuals compared with nine modern humans, there are only 90 total nucleotide sites genome-wide for which archaic hominins are fixed for the ancestral nonsynonymous variant and the modern humans are fixed for the derived variant. Of those sites, only 27, including Val381 in the AHR, also have no reported variability in the human dbSNP database, further suggesting that this highly conserved functional variant is a rare event. Functional analysis of the amino acid variant Ala381 within the AHR carried by Neandertals and nonhuman primates indicate enhanced polycyclic aromatic hydrocarbon (PAH) binding, DNA binding capacity, and AHR mediated transcriptional activity compared with the human AHR. Also relative to human AHR, the Neandertal AHR exhibited 150-1000 times greater sensitivity to induction of Cyp1a1 and Cyp1b1 expression by PAHs (e.g., benzo(a)pyrene). The resulting CYP1A1/CYP1B1 enzymes are responsible for PAH first pass metabolism, which can result in the generation of toxic intermediates and perhaps AHR-associated toxicities. In contrast, the human AHR retains the ancestral sensitivity observed in primates to nontoxic endogenous AHR ligands (e.g., indole, indoxyl sulfate). Our findings reveal that a functionally significant change in the AHR occurred uniquely in humans, relative to other primates, that would attenuate the response to many environmental pollutants, including chemicals present in smoke from fire use during cooking. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  12. (D-Pen2,4 prime -125I-Phe4,D-Pen5)enkephalin: A selective high affinity radioligand for delta opioid receptors with exceptional specific activity

    Knapp, R.J.; Sharma, S.D.; Toth, G.; Duong, M.T.; Fang, L.; Bogert, C.L.; Weber, S.J.; Hunt, M.; Davis, T.P.; Wamsley, J.K. (Department of Pharmacology, University of Arizona, College of Medicine, Tucson (United States))

    1991-09-01

    (D-Pen2,4{prime}-125I-Phe4,D-Pen5)enkephalin ((125I)DPDPE) is a highly selective radioligand for the delta opioid receptor with a specific activity (2200 Ci/mmol) that is over 50-fold greater than that of tritium-labeled DPDPE analogs. (125I)DPDPE binds to a single site in rat brain membranes with an equilibrium dissociation constant (Kd) value of 421 {plus minus} 67 pM and a receptor density (Bmax) value of 36.4 {plus minus} 2.7 fmol/mg protein. The high affinity of this site for delta opioid receptor ligands and its low affinity for mu or kappa receptor-selective ligands are consistent with its being a delta opioid receptor. The distribution of these sites in rat brain, observed by receptor autoradiography, is also consistent with that of delta opioid receptors. Association and dissociation binding kinetics of 1.0 nM (125I) DPDPE are monophasic at 25 degrees C. The association rate (k + 1 = 5.80 {plus minus} 0.88 {times} 10(7) M-1 min-1) is about 20- and 7-fold greater than that measured for 1.0 nM (3H) DPDPE and 0.8 nM (3H) (D-Pen2,4{prime}-Cl-Phe4, D-Pen5)enkephalin, respectively. The dissociation rate of (125I)DPDPE (0.917 {plus minus} 0.117 {times} 10(-2) min-1) measured at 1.0 nM is about 3-fold faster than is observed for either of the other DPDPE analogs. The rapid binding kinetics of (125I)DPDPE is advantageous because binding equilibrium is achieved with much shorter incubation times than are required for other cyclic enkephalin analogs. This, in addition to its much higher specific activity, makes (125I)DPDPE a valuable new radioligand for studies of delta opioid receptors.

  13. Activation of Peripheral κ-Opioid Receptors Normalizes Caffeine Effects Modified in Nicotine-Dependent Rats during Nicotine Withdrawal.

    Sudakov, S K; Bogdanova, N G

    2016-10-01

    The study examined the effect of peripheral (intragastric) ICI-204,448, an agonist of gastric κ-opioid receptors, on the psychostimulating and anxiolytic effects of caffeine in nicotinedependent rats at the stage of nicotine withdrawal. In these rats, the effects of caffeine (10 mg/kg) were perverted. In nicotine-dependent rats, caffeine produced an anxiolytic effect accompanied by pronounced stimulation of motor activity, in contrast to anxiogenic effect induced by caffeine in intact rats without nicotine dependence. During nicotine withdrawal, nicotine-dependent rats demonstrated enhanced sensitivity to nicotine. Intragastric administration of κ-opioid receptor agonist ICI-204,448 normalized the effect of caffeine in nicotinedependent rats. We have previously demonstrated that activation of peripheral κ-opioid receptors inhibited central κ-opioid activity and eliminated manifestations of nicotine withdrawal syndrome in nicotine-dependent rats, e.g. metabolism activation, stimulation of motor activity, and enhancement of food consumption. In its turn, inhibition of central κ-opioid structures activates the brain adenosine system, which can attenuate the caffeine-induced effects in nicotine-dependent rats.

  14. Cell surface receptors for signal transduction and ligand transport: a design principles study.

    Harish Shankaran

    2007-06-01

    Full Text Available Receptors constitute the interface of cells to their external environment. These molecules bind specific ligands involved in multiple processes, such as signal transduction and nutrient transport. Although a variety of cell surface receptors undergo endocytosis, the systems-level design principles that govern the evolution of receptor trafficking dynamics are far from fully understood. We have constructed a generalized mathematical model of receptor-ligand binding and internalization to understand how receptor internalization dynamics encodes receptor function and regulation. A given signaling or transport receptor system represents a particular implementation of this module with a specific set of kinetic parameters. Parametric analysis of the response of receptor systems to ligand inputs reveals that receptor systems can be characterized as being: i avidity-controlled where the response control depends primarily on the extracellular ligand capture efficiency, ii consumption-controlled where the ability to internalize surface-bound ligand is the primary control parameter, and iii dual-sensitivity where both the avidity and consumption parameters are important. We show that the transferrin and low-density lipoprotein receptors are avidity-controlled, the vitellogenin receptor is consumption-controlled, and the epidermal growth factor receptor is a dual-sensitivity receptor. Significantly, we show that ligand-induced endocytosis is a mechanism to enhance the accuracy of signaling receptors rather than merely serving to attenuate signaling. Our analysis reveals that the location of a receptor system in the avidity-consumption parameter space can be used to understand both its function and its regulation.

  15. Mechanism Governing Human Kappa-Opioid Receptor Expression under Desferrioxamine-Induced Hypoxic Mimic Condition in Neuronal NMB Cells

    Jennifer Babcock

    2017-01-01

    Full Text Available Cellular adaptation to hypoxia is a protective mechanism for neurons and relevant to cancer. Treatment with desferrioxamine (DFO to induce hypoxia reduced the viability of human neuronal NMB cells. Surviving/attached cells exhibited profound increases of expression of the human kappa-opioid receptor (hKOR and hypoxia inducible factor-1α (HIF-1α. The functional relationship between hKOR and HIF-1α was investigated using RT-PCR, Western blot, luciferase reporter, mutagenesis, siRNA and receptor-ligand binding assays. In surviving neurons, DFO increased HIF-1α expression and its amount in the nucleus. DFO also dramatically increased hKOR expression. Two (designated as HIFC and D out of four potential HIF response elements of the hKOR gene (HIFA–D synergistically mediated the DFO response. Mutation of both elements completely abolished the DFO-induced effect. The CD11 plasmid (containing HIFC and D with an 11 bp spacing produced greater augmentation than that of the CD17 plasmid (HIFC and D with a 17 bp-spacing, suggesting that a proper topological interaction of these elements synergistically enhanced the promoter activity. HIF-1α siRNA knocked down the increase of endogenous HIF-1α messages and diminished the DFO-induced increase of hKOR expression. Increased hKOR expression resulted in the up-regulation of hKOR protein. In conclusion, the adaptation of neuronal hKOR under hypoxia was governed by HIF-1, revealing a new mechanism of hKOR regulation.

  16. Cutaneous nociceptors lack sensitisation, but reveal μ-opioid receptor-mediated reduction in excitability to mechanical stimulation in neuropathy

    Schmidt Yvonne

    2012-11-01

    Full Text Available Abstract Background Peripheral nerve injuries often trigger a hypersensitivity to tactile stimulation. Behavioural studies demonstrated efficient and side effect-free analgesia mediated by opioid receptors on peripheral sensory neurons. However, mechanistic approaches addressing such opioid properties in painful neuropathies are lacking. Here we investigated whether opioids can directly inhibit primary afferent neuron transmission of mechanical stimuli in neuropathy. We analysed the mechanical thresholds, the firing rates and response latencies of sensory fibres to mechanical stimulation of their cutaneous receptive fields. Results Two weeks following a chronic constriction injury of the saphenous nerve, mice developed a profound mechanical hypersensitivity in the paw innervated by the damaged nerve. Using an in vitro skin-nerve preparation we found no changes in the mechanical thresholds and latencies of sensory fibres from injured nerves. The firing rates to mechanical stimulation were unchanged or reduced following injury. Importantly, μ-opioid receptor agonist [D-Ala2,N-Me-Phe4,Gly5]-ol-enkephalin (DAMGO significantly elevated the mechanical thresholds of nociceptive Aδ and C fibres. Furthermore, DAMGO substantially diminished the mechanically evoked discharges of C nociceptors in injured nerves. These effects were blocked by DAMGO washout and pre-treatment with the selective μ-opioid receptor antagonist Cys2-Tyr3-Orn5-Pen7-amide. DAMGO did not alter the responses of sensory fibres in uninjured nerves. Conclusions Our findings suggest that behaviourally manifested neuropathy-induced mechanosensitivity does not require a sensitised state of cutaneous nociceptors in damaged nerves. Yet, nerve injury renders nociceptors sensitive to opioids. Prevention of action potential generation or propagation in nociceptors might represent a cellular mechanism underlying peripheral opioid-mediated alleviation of mechanical hypersensitivity in neuropathy.

  17. Heterologous activation of protein kinase C stimulates phosphorylation of delta-opioid receptor at serine 344, resulting in beta-arrestin- and clathrin-mediated receptor internalization

    Xiang, B; Yu, G H; Guo, J

    2001-01-01

    The purpose of the current study is to investigate the effect of opioid-independent, heterologous activation of protein kinase C (PKC) on the responsiveness of opioid receptor and the underlying molecular mechanisms. Our result showed that removing the C terminus of delta opioid receptor (DOR......) containing six Ser/Thr residues abolished both DPDPE- and phorbol 12-myristate 13-acetate (PMA)-induced DOR phosphorylation. The phosphorylation levels of DOR mutants T352A, T353A, and T358A/T361A/S363S were comparable to that of the wild-type DOR, whereas S344G substitution blocked PMA-induced receptor......, and ionomycin resulted in DOR internalization that required phosphorylation of Ser-344. Expression of dominant negative beta-arrestin and hypertonic sucrose treatment blocked PMA-induced DOR internalization, suggesting that PKC mediates DOR internalization via a beta-arrestin- and clathrin-dependent mechanism...

  18. The potent opioid agonist, (+)-cis-3-methylfentanyl binds pseudoirreversibly to the opioid receptor complex in vitro and in vivo: Evidence for a novel mechanism of action

    Band, L.; Xu, Heng; Bykov, V.; Rothman, R.B.; Kim, Chongho; Newman, A.; Jacobson, A.E.; Rice, K.C. (NIDDK, Bethesda, MD (USA)); Greig, N. (NIA, Bethesda, MD (USA))

    1990-01-01

    The present study demonstrates that pretreatment of rat brain membranes with (+)-cis-3-methylfentanyl ((+)-cis-MF), followed by extensive washing of the membranes, produces a wash-resistant decreasing in the binding of ({sup 3}H)-(D-ala{sup 2}, D-leu{sup 5})enkephalin to the d binding site of the opioid receptor complex ({delta}{sub cx} binding site). Intravenous administration of (+)-cis-MF (50 {mu}g/kg) to rats produced a pronounced catalepsy and also produced a wash-resistant masking of {delta}{sub cx} and {mu} binding sites in membranes prepared 120 min post-injection. Administration of 1 mg/kg i.v. of the opioid antagonist, 6-desoxy-6{beta}-fluoronaltrexone (cycloFOXY), 100 min after the injection of (+)-cis-MF (20 min prior to the preparation of membranes) completely reversed the catatonia and restored masked {delta}{sub cx} binding sites to control levels. This was not observed with (+)-cycloFOXY. The implications of these and other findings for the mechanism of action of (+)-cis-MF and models of the opioid receptors are discussed.

  19. A response calculus for immobilized T cell receptor ligands

    Andersen, P S; Menné, C; Mariuzza, R A

    2001-01-01

    determine the level of T cell activation. When fitted to T cell responses against purified ligands immobilized on plastic surfaces, the 2D-affinity model adequately simulated changes in cellular activation as a result of varying ligand affinity and ligand density. These observations further demonstrated...

  20. Design and Synthesis of an 18F-Labeled Version of Phenylethyl Orvinol ([18F]FE-PEO for PET-Imaging of Opioid Receptors

    Gjermund Henriksen

    2012-09-01

    Full Text Available The semisynthetic oripavine derivative phenethyl orvinol (PEO, a full agonist at opioid receptors (OR, is an attractive structural motif for developing 18F-labeled PET tracers with a high degree of sensitivity for competition between endogenous and exogenous OR-ligands. The target cold reference compound 6-O-(2-fluoroethyl-6-O-desmethylphenylethyl orvinol (FE-PEO was obtained via two separate reaction routes. A three-step synthesis was developed for the preparation of a tosyloxyethyl precursor (TE-TDPEO, the key precursor for a direct, nucleophilic radiofluorination to yield [18F]FE-PEO. The developed radiosynthesis provides the target compound in relevantly high yield and purity, and is adaptable to routine production.

  1. 1,2,3-triazolyl amino acids as AMPA receptor ligands

    Stanley, Nathan J.; Pedersen, Daniel Sejer; Nielsen, Birgitte

    2010-01-01

    The central nervous system glutamate receptors are an important target for drug discovery. Herein we report initial investigations into the synthesis and glutamate receptor activity of 1,2,3-triazolyl amino acids. Two compounds were found to be selective AMPA receptor ligands, which warrant further...

  2. Structural determinants for selective recognition of peptide ligands for endothelin receptor subtypes ETA and ETB.

    Lättig, Jens; Oksche, Alexander; Beyermann, Michael; Rosenthal, Walter; Krause, Gerd

    2009-07-01

    The molecular basis for recognition of peptide ligands endothelin-1, -2 and -3 in endothelin receptors is poorly understood. Especially the origin of ligand selectivity for ET(A) or ET(B) is not clearly resolved. We derived sequence-structure-function relationships of peptides and receptors from mutational data and homology modeling. Our major findings are the dissection of peptide ligands into four epitopes and the delineation of four complementary structural portions on receptor side explaining ligand recognition in both endothelin receptor subtypes. In addition, structural determinants for ligand selectivity could be described. As a result, we could improve the selectivity of BQ3020 about 10-fold by a single amino acid substitution, validating our hypothesis for ligand selectivity caused by different entrances to the receptors' transmembrane binding sites. A narrow tunnel shape in ET(A) is restrictive for a selected group of peptide ligands' N-termini, whereas a broad funnel-shaped entrance in ET(B) accepts a variety of different shapes and properties of ligands.

  3. Effects of opioid drugs on dopamine mediated locomotor activity in rats

    Leathern, L L

    1986-01-01

    Opioid drugs influence various behavioural parameters including locomotor activity in experimental animals. The interaction between the opioid and dopaminergic systems is one possible explanation for the effect of opioid drugs on locomotor activity. In this study behavioural and biochemical assays were done to investigate the interaction between the opioid and dopaminergic systems. Behavioural studies were done by measurement of locomotor activity (LA) of rats after acute or chronic pretreatment with opioid andor dopaminergic drugs. Biochemical studies were in the form of radioligand binding assays, the effect on the number (Bmax) and affinity (K/sub D/) of receptors was measured after chronic pretreatment with opioid andor dopaminergic drugs. The opioid drugs used are morphine, nalbuphine and naloxone. Dopaminergic drugs used included: agonists-apomorphine and piribedil; antagonists-pimozide, haloperidol, chlorpromazine. In the acute situation increased LA was obtained with morphine and the DA agonists. A correlation between the behavioural and biochemical assays was found. Chronic pretreatment with morphine enhanced apomorphine induced LA, this supersensitivity was also measured as an increased receptor density (Bmax) of D2 receptors in the striatum. Chronic morphine pretreatment caused a decrease in morphine induced LA, while this subsensitivity was not apparent in the ligand binding assays - where no change in receptor number was observed. Chronic naloxone pretreatment enhanced morphine induced LA, as well as increased the Bmax of opioid receptors in the whole brain. It is concluded that an interaction between the opioid and dopaminergic systems does exist, and may account for the mechanism of action of the opioids.

  4. Monitoring ligand-receptor interactions by photonic force microscopy

    Jeney, Sylvia [M E Mueller Institute for Structural Biology, Biozentrum, University of Basel, Klingelbergstrasse 70, Basel, 4056 (Switzerland); Mor, Flavio; Forro, Laszlo [Laboratory of Complex Matter Physics (LPMC), Ecole Polytechnique Federale de Lausanne (EPFL), CH-1015 Lausanne (Switzerland); Koszali, Roland [Institute for Information and Communication Technologies (IICT), University of Applied Sciences of Western Switzerland (HEIG-VD), Rue Galilee 15, CH 1401 Yverdon-les-bains (Switzerland); Moy, Vincent T, E-mail: sylvia.jeney@unibas.ch, E-mail: vmoy@miami.edu [Department of Physiology and Biophysics, University of Miami Miller School of Medicine, 1600 NW 10th Avenue, Miami, FL 33136 (United States)

    2010-06-25

    We introduce a method for the acquisition of single molecule force measurements of ligand-receptor interactions using the photonic force microscope (PFM). Biotin-functionalized beads, manipulated with an optical trap, and a streptavidin-functionalized coverslip were used to measure the effect of different pulling forces on the lifetime of individual streptavidin-biotin complexes. By optimizing the design of the optical trap and selection of the appropriate bead size, pulling forces in excess of 50 pN were achieved. Based on the amplitude of three-dimensional (3D) thermal position fluctuations of the attached bead, we were able to select for a bead-coverslip interaction that was mediated by a single streptavidin-biotin complex. Moreover, the developed experimental system was greatly accelerated by automation of data acquisition and analysis. In force-dependent kinetic measurements carried out between streptavidin and biotin, we observed that the streptavidin-biotin complex exhibited properties of a catch bond, with the lifetime increasing tenfold when the pulling force increased from 10 to 20 pN. We also show that silica beads were more appropriate than polystyrene beads for the force measurements, as tethers, longer than 200 nm, could be extracted from polystyrene beads.

  5. Mutual inactivation of Notch receptors and ligands facilitates developmental patterning.

    David Sprinzak

    2011-06-01

    Full Text Available Developmental patterning requires juxtacrine signaling in order to tightly coordinate the fates of neighboring cells. Recent work has shown that Notch and Delta, the canonical metazoan juxtacrine signaling receptor and ligand, mutually inactivate each other in the same cell. This cis-interaction generates mutually exclusive sending and receiving states in individual cells. It generally remains unclear, however, how this mutual inactivation and the resulting switching behavior can impact developmental patterning circuits. Here we address this question using mathematical modeling in the context of two canonical pattern formation processes: boundary formation and lateral inhibition. For boundary formation, in a model motivated by Drosophila wing vein patterning, we find that mutual inactivation allows sharp boundary formation across a broader range of parameters than models lacking mutual inactivation. This model with mutual inactivation also exhibits robustness to correlated gene expression perturbations. For lateral inhibition, we find that mutual inactivation speeds up patterning dynamics, relieves the need for cooperative regulatory interactions, and expands the range of parameter values that permit pattern formation, compared to canonical models. Furthermore, mutual inactivation enables a simple lateral inhibition circuit architecture which requires only a single downstream regulatory step. Both model systems show how mutual inactivation can facilitate robust fine-grained patterning processes that would be difficult to implement without it, by encoding a difference-promoting feedback within the signaling system itself. Together, these results provide a framework for analysis of more complex Notch-dependent developmental systems.

  6. Monitoring ligand-receptor interactions by photonic force microscopy

    Jeney, Sylvia; Mor, Flavio; Forro, Laszlo; Koszali, Roland; Moy, Vincent T

    2010-01-01

    We introduce a method for the acquisition of single molecule force measurements of ligand-receptor interactions using the photonic force microscope (PFM). Biotin-functionalized beads, manipulated with an optical trap, and a streptavidin-functionalized coverslip were used to measure the effect of different pulling forces on the lifetime of individual streptavidin-biotin complexes. By optimizing the design of the optical trap and selection of the appropriate bead size, pulling forces in excess of 50 pN were achieved. Based on the amplitude of three-dimensional (3D) thermal position fluctuations of the attached bead, we were able to select for a bead-coverslip interaction that was mediated by a single streptavidin-biotin complex. Moreover, the developed experimental system was greatly accelerated by automation of data acquisition and analysis. In force-dependent kinetic measurements carried out between streptavidin and biotin, we observed that the streptavidin-biotin complex exhibited properties of a catch bond, with the lifetime increasing tenfold when the pulling force increased from 10 to 20 pN. We also show that silica beads were more appropriate than polystyrene beads for the force measurements, as tethers, longer than 200 nm, could be extracted from polystyrene beads.

  7. Do orphan G-protein-coupled receptors have ligand-independent functions? New insights from receptor heterodimers

    Levoye, Angélique; Dam, Julie; Ayoub, Mohammed A; Guillaume, Jean-Luc; Jockers, Ralf

    2006-01-01

    G-protein-coupled receptors (GPCRs) are important drug targets and are involved in virtually every biological process. However, there are still more than 140 orphan GPCRs, and deciphering their function remains a priority for fundamental and clinical research. Research on orphan GPCRs has concentrated mainly on the identification of their natural ligands, whereas recent data suggest additional ligand-independent functions for these receptors. This emerging concept is connected with the observ...

  8. Sodium modulates opioid receptors through a membrane component different from G-proteins. Demonstration by target size analysis

    Ott, S.; Costa, T.; Herz, A.

    1988-01-01

    The target size for opioid receptor binding was studied after manipulations known to affect the interactions between receptor and GTP-binding regulatory proteins (G-proteins). Addition of GTP or its analogs to the binding reaction, exposure of intact cells to pertussis toxin prior to irradiation, or treatment of irradiated membranes with N-ethylmaleimide did not change the target size (approximately equal to 100 kDa) for opioid receptors in NG 108-15 cells and rat brain. These data suggest that the 100-kDa species does not include an active subunit of a G-protein or alternatively that GTP does not promote the dissociation of the receptor-G-protein complex. The presence of Na+ (100 mM) in the radioligand binding assay induced a biphasic decay curve for agonist binding and a flattening of the monoexponential decay curve for a partial agonist. In both cases the effect was explained by an irradiation-induced loss of the low affinity state of the opioid receptor produced by the addition of Na+. This suggests that an allosteric inhibitor that mediates the effect of sodium on the receptor is destroyed at low doses of irradiation, leaving receptors which are no longer regulated by sodium. The effect of Na+ on target size was slightly increased by the simultaneous addition of GTP but was not altered by pertussis toxin treatment. Thus, the sodium unit is distinct from G-proteins and may represent a new component of the opioid receptor complex. Assuming a simple bimolecular model of one Na+ unit/receptor, the size of this inhibitor can be measured as 168 kDa

  9. Transcriptional activation by the thyroid hormone receptor through ligand-dependent receptor recruitment and chromatin remodelling.

    Grøntved, Lars; Waterfall, Joshua J; Kim, Dong Wook; Baek, Songjoon; Sung, Myong-Hee; Zhao, Li; Park, Jeong Won; Nielsen, Ronni; Walker, Robert L; Zhu, Yuelin J; Meltzer, Paul S; Hager, Gordon L; Cheng, Sheue-yann

    2015-04-28

    A bimodal switch model is widely used to describe transcriptional regulation by the thyroid hormone receptor (TR). In this model, the unliganded TR forms stable, chromatin-bound complexes with transcriptional co-repressors to repress transcription. Binding of hormone dissociates co-repressors and facilitates recruitment of co-activators to activate transcription. Here we show that in addition to hormone-independent TR occupancy, ChIP-seq against endogenous TR in mouse liver tissue demonstrates considerable hormone-induced TR recruitment to chromatin associated with chromatin remodelling and activated gene transcription. Genome-wide footprinting analysis using DNase-seq provides little evidence for TR footprints both in the absence and presence of hormone, suggesting that unliganded TR engagement with repressive complexes on chromatin is, similar to activating receptor complexes, a highly dynamic process. This dynamic and ligand-dependent interaction with chromatin is likely shared by all steroid hormone receptors regardless of their capacity to repress transcription in the absence of ligand.

  10. Multiple pathways of sigma(1) receptor ligand uptakes into primary cultured neuronal cells.

    Yamamoto, H; Karasawa, J; Sagi, N; Takahashi, S; Horikomi, K; Okuyama, S; Nukada, T; Sora, I; Yamamoto, T

    2001-08-03

    Although many antipsychotics have affinities for sigma receptors, the transportation pathway of exogenous sigma(1) receptor ligands to intracellular type-1 sigma receptors are not fully understood. In this study, sigma(1) receptor ligand uptakes were studied using primary cultured neuronal cells. [(3)H](+)-pentazocine and [(3)H](R)-(+)-1-(4-chlorophenyl)-3-[4-(2-methoxyethyl)piperazin-1-yl]methyl-2-pyrrolidinone L-tartrate (MS-377), used as a selective sigma(1) receptor ligands, were taken up in a time-, energy- and temperature-dependent manner, suggesting that active transport mechanisms were involved in their uptakes. sigma(1) receptor ligands taken up into primary cultured neuronal cells were not restricted to agonists, but also concerned antagonists. The uptakes of these ligands were mainly Na(+)-independent. Kinetic analysis of [(3)H](+)-pentazocine and [(3)H]MS-377 uptake showed K(m) values (microM) of 0.27 and 0.32, and V(max) values (pmol/mg protein/min) of 17.4 and 9.4, respectively. Although both ligands were incorporated, the pharmacological properties of these two ligands were different. Uptake of [(3)H](+)-pentazocine was inhibited in the range 0.4-7.1 microM by all the sigma(1) receptor ligands used, including N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]ethylamine monohydrochloride (NE-100), a selective sigma(1) receptor ligand. In contrast, the inhibition of [(3)H]MS-377 uptake was potently inhibited by haloperidol, characterized by supersensitivity (IC(50), approximately 2 nM) and was inhibited by NE-100 with low sensitivity (IC(50), 4.5 microM). Moreover, kinetic analysis revealed that NE-100 inhibited [(3)H]MS-377 uptake in a noncompetitive manner, suggesting that NE-100 acted at a site different from the uptake sites of [(3)H]MS-377. These findings suggest that there are at least two uptake pathways for sigma(1) receptor ligands in primary cultured neuronal cells (i.e. a haloperidol-sensitive pathway and another, unclear, pathway). In

  11. Modulation of melanocortin- induced changes in spinal nociception by µ-opioid receptor agonist and antagonist in neuropathic rats

    Gispen, W.H.; Starowitcz, K.; Przewlocki, R.; Przewlocka, B.

    2002-01-01

    Co-localization of opioid and melanocortin receptor expression, especially at the spinal cord level in the dorsal horn and in the gray matter surrounding the central canal led to the suggestion that melanocortins might play a role in nociceptive processes. In the present studies, we aimed to

  12. Immunohistochemical observations of methionine-enkephalin and delta opioid receptor in the digestive system of Octopus ocellatus.

    Sha, Ailong; Sun, Hushan; Wang, Yiyan

    2013-02-01

    The study was designed to determine whether methionine-enkephalin (met-Enk) or delta opioid receptor was present in the digestive system of Octopus ocellatus. The results showed that they were both in the bulbus oris, esophagus, crop, stomach, gastric cecum, intestine, posterior salivary glands of O. ocellatus, one of them, met-Enk in the rectum, anterior salivary glands, digestive gland. And the distributions were extensive in the digestive system. Strong or general met-Enk immunoreactivity was observed in the inner epithelial cells of the bulbus oris, esophagus, stomach, gastric cecum, intestine, anterior salivary glands and the adventitia of the intestine and rectum, and so was the delta opioid receptor immunoreactivity in the inner epithelial cells of the bulbus oris, esophagus, and crop, however, they were weak in other parts. Combining with delta opioid receptor, met-Enk may be involved in the regulations of food intake, absorption, movement of gastrointestinal smooth muscle and secretion of digestive gland. The different densities of met-Enk and delta opioid receptor may be related to the different functions in the digestive system of O. ocellatus. Copyright © 2012 Elsevier Ltd. All rights reserved.

  13. Preserved cardiac mitochondrial function and reduced ischaemia/reperfusion injury afforded by chronic continuous hypoxia: Role of opioid receptors

    Maslov, L. N.; Naryzhnaya, N. V.; Prokudina, E. S.; Kolář, František; Gorbunov, A. S.; Zhang, Y.; Wang, H.; Tsibulnikov, S.Yu.; Portnichenko, A. G.; Lasukova, T. V.; Lishmanov, Yu. B.

    2015-01-01

    Roč. 42, č. 5 (2015), s. 496-501 ISSN 1440-1681 R&D Projects: GA ČR(CZ) GAP303/12/1162 Institutional support: RVO:67985823 Keywords : cardioprotection * chronic hypoxia * ischaemia/reperfusion * mitochondrial function * opioid receptors Subject RIV: ED - Physiology Impact factor: 2.004, year: 2015

  14. Challenges predicting ligand-receptor interactions of promiscuous proteins: the nuclear receptor PXR.

    Sean Ekins

    2009-12-01

    Full Text Available Transcriptional regulation of some genes involved in xenobiotic detoxification and apoptosis is performed via the human pregnane X receptor (PXR which in turn is activated by structurally diverse agonists including steroid hormones. Activation of PXR has the potential to initiate adverse effects, altering drug pharmacokinetics or perturbing physiological processes. Reliable computational prediction of PXR agonists would be valuable for pharmaceutical and toxicological research. There has been limited success with structure-based modeling approaches to predict human PXR activators. Slightly better success has been achieved with ligand-based modeling methods including quantitative structure-activity relationship (QSAR analysis, pharmacophore modeling and machine learning. In this study, we present a comprehensive analysis focused on prediction of 115 steroids for ligand binding activity towards human PXR. Six crystal structures were used as templates for docking and ligand-based modeling approaches (two-, three-, four- and five-dimensional analyses. The best success at external prediction was achieved with 5D-QSAR. Bayesian models with FCFP_6 descriptors were validated after leaving a large percentage of the dataset out and using an external test set. Docking of ligands to the PXR structure co-crystallized with hyperforin had the best statistics for this method. Sulfated steroids (which are activators were consistently predicted as non-activators while, poorly predicted steroids were docked in a reverse mode compared to 5alpha-androstan-3beta-ol. Modeling of human PXR represents a complex challenge by virtue of the large, flexible ligand-binding cavity. This study emphasizes this aspect, illustrating modest success using the largest quantitative data set to date and multiple modeling approaches.

  15. 5-Hydroxytryptamine 1A/7 and 4alpha receptors differentially prevent opioid-induced inhibition of brain stem cardiorespiratory function.

    Wang, Xin; Dergacheva, Olga; Kamendi, Harriet; Gorini, Christopher; Mendelowitz, David

    2007-08-01

    Opioids evoke respiratory depression, bradycardia, and reduced respiratory sinus arrhythmia, whereas serotonin (5-HT) agonists stimulate respiration and cardiorespiratory interactions. This study tested whether serotonin agonists can prevent the inhibitory effects of opioids on cardiorespiratory function. Spontaneous and rhythmic inspiratory-related activity and gamma-aminobutyric acid (GABA) neurotransmission to premotor parasympathetic cardioinhibitory neurons in the nucleus ambiguus were recorded simultaneously in an in vitro thick slice preparation. The mu-opioid agonist fentanyl inhibited respiratory frequency. The 5-hydroxytryptamine 1A/7 receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin increased respiratory frequency by itself and also prevented the fentanyl-induced respiratory depression. The 5-hydroxytryptamine 4alpha agonist BIMU-8 did not by itself change inspiratory activity but prevented the mu-opioid-mediated respiratory depression. Both spontaneous and inspiratory-evoked GABAergic neurotransmission to cardiac vagal neurons were inhibited by fentanyl. 8-Hydroxy-2-(di-n-propylamino)tetralin inhibited spontaneous but not inspiratory-evoked GABAergic activity to parasympathetic cardiac neurons. However, 8-hydroxy-2-(di-n-propylamino)tetralin differentially altered the opioid-mediated depression of inspiratory-evoked GABAergic activity but did not change the opioid-induced reduction in spontaneous GABAergic neurotransmission. In contrast, BIMU-8 did not alter GABAergic neurotransmission to cardiac vagal neurons by itself but prevented the fentanyl depression of both spontaneous and inspiratory-elicited GABAergic neurotransmission to cardiac vagal neurons. In the presence of tetrodotoxin, the inhibition of GABAergic inhibitory postsynaptic currents with fentanyl is prevented by coapplication of BIMU-8, indicating that BIMU-8 acts at presynaptic GABAergic terminals to prevent fentanyl-induced depression. These results suggest that activation of 5

  16. Ligands, cell-based models, and readouts required for Toll-like receptor action.

    Dellacasagrande, Jerome

    2012-02-01

    This chapter details the tools that are available to study Toll-like receptor (TLR) biology in vitro. This includes ligands, host cells, and readouts. The use of modified TLRs to circumvent some technical problems is also discussed.

  17. Is the isolated ligand binding domain a good model of the domain in the native receptor?

    Deming, Dustin; Cheng, Qing; Jayaraman, Vasanthi

    2003-05-16

    Numerous studies have used the atomic level structure of the isolated ligand binding domain of the glutamate receptor to elucidate the agonist-induced activation and desensitization processes in this group of proteins. However, no study has demonstrated the structural equivalence of the isolated ligand binding fragments and the protein in the native receptor. In this report, using visible absorption spectroscopy we show that the electronic environment of the antagonist 6-cyano-7-nitro-2,3-dihydroxyquinoxaline is identical for the isolated protein and the native glutamate receptors expressed in cells. Our results hence establish that the local structure of the ligand binding site is the same in the two proteins and validate the detailed structure-function relationships that have been developed based on a comparison of the structure of the isolated ligand binding domain and electrophysiological consequences in the native receptor.

  18. Reduced basal ganglia μ-opioid receptor availability in trigeminal neuropathic pain: A pilot study

    DosSantos Marcos

    2012-09-01

    Full Text Available Abstract Background Although neuroimaging techniques have provided insights into the function of brain regions involved in Trigeminal Neuropathic Pain (TNP in humans, there is little understanding of the molecular mechanisms affected during the course of this disorder. Understanding these processes is crucial to determine the systems involved in the development and persistence of TNP. Findings In this study, we examined the regional μ-opioid receptor (μOR availability in vivo (non-displaceable binding potential BPND of TNP patients with positron emission tomography (PET using the μOR selective radioligand [11C]carfentanil. Four TNP patients and eight gender and age-matched healthy controls were examined with PET. Patients with TNP showed reduced μOR BPND in the left nucleus accumbens (NAc, an area known to be involved in pain modulation and reward/aversive behaviors. In addition, the μOR BPND in the NAc was negatively correlated with the McGill sensory and total pain ratings in the TNP patients. Conclusions Our findings give preliminary evidence that the clinical pain in TNP patients can be related to alterations in the endogenous μ-opioid system, rather than only to the peripheral pathology. The decreased availability of μORs found in TNP patients, and its inverse relationship to clinical pain levels, provide insights into the central mechanisms related to this condition. The results also expand our understanding about the impact of chronic pain on the limbic system.

  19. Functional role of peripheral opioid receptors in the regulation of cardiac spinal afferent nerve activity during myocardial ischemia

    Longhurst, John C.

    2013-01-01

    Thinly myelinated Aδ-fiber and unmyelinated C-fiber cardiac sympathetic (spinal) sensory nerve fibers are activated during myocardial ischemia to transmit the sensation of angina pectoris. Although recent observations showed that myocardial ischemia increases the concentrations of opioid peptides and that the stimulation of peripheral opioid receptors inhibits chemically induced visceral and somatic nociception, the role of opioids in cardiac spinal afferent signaling during myocardial ischemia has not been studied. The present study tested the hypothesis that peripheral opioid receptors modulate cardiac spinal afferent nerve activity during myocardial ischemia by suppressing the responses of cardiac afferent nerve to ischemic mediators like bradykinin and extracellular ATP. The nerve activity of single unit cardiac afferents was recorded from the left sympathetic chain (T2–T5) in anesthetized cats. Forty-three ischemically sensitive afferent nerves (conduction velocity: 0.32–3.90 m/s) with receptive fields in the left and right ventricles were identified. The responses of these afferent nerves to repeat ischemia or ischemic mediators were further studied in the following protocols. First, epicardial administration of naloxone (8 μmol), a nonselective opioid receptor antagonist, enhanced the responses of eight cardiac afferent nerves to recurrent myocardial ischemia by 62%, whereas epicardial application of vehicle (PBS) did not alter the responses of seven other cardiac afferent nerves to ischemia. Second, naloxone applied to the epicardial surface facilitated the responses of seven cardiac afferent nerves to epicardial ATP by 76%. Third, administration of naloxone enhanced the responses of seven other afferent nerves to bradykinin by 85%. In contrast, in the absence of naloxone, cardiac afferent nerves consistently responded to repeated application of ATP (n = 7) or bradykinin (n = 7). These data suggest that peripheral opioid peptides suppress the

  20. Embryonic expression of the transforming growth factor beta ligand and receptor genes in chicken.

    Cooley, James R; Yatskievych, Tatiana A; Antin, Parker B

    2014-03-01

    Transforming growth factor-beta (TGFβ) signaling regulates a myriad of biological processes during embryogenesis, in the adult, and during the manifestation of disease. TGFβ signaling is propagated through one of three TGFβ ligands interacting with Type I and Type II receptors, and Type III co-receptors. Although TGFβ signaling is regulated partly by the combinatorial expression patterns of TGFβ receptors and ligands, a comprehensive gene expression analysis has not been published. Here we report the embryonic mRNA expression patterns in chicken embryos of the canonical TGFβ ligands (TGFB1, TGFB2, and TGFB3) and receptors (TGFBR1, TGFBR2, TGFBR3), plus the Activin A receptor, type 1 (ACVR1) and co receptor Endoglin (ENG) that also transduce TGFβ signaling. TGFB ligands and receptors show dynamic and frequently overlapping expression patterns in numerous embryonic cell layers and structures. Integrating expression information identifies combinations of ligands and receptors that are involved in specific developmental processes including somitogenesis, cardiogenesis and vasculogenesis. Copyright © 2013 Wiley Periodicals, Inc.

  1. Expression and Purification of Functional Ligand-binding Domains of T1R3 Taste Receptors

    Nie,Y.; Hobbs, J.; Vigues, S.; Olson, W.; Conn, G.; Munger, S.

    2006-01-01

    Chemosensory receptors, including odor, taste, and vomeronasal receptors, comprise the largest group of G protein-coupled receptors (GPCRs) in the mammalian genome. However, little is known about the molecular determinants that are critical for the detection and discrimination of ligands by most of these receptors. This dearth of understanding is due in part to difficulties in preparing functional receptors suitable for biochemical and biophysical analyses. Here we describe in detail two strategies for the expression and purification of the ligand-binding domain of T1R taste receptors, which are constituents of the sweet and umami taste receptors. These class C GPCRs contain a large extracellular N-terminal domain (NTD) that is the site of interaction with most ligands and that is amenable to expression as a separate polypeptide in heterologous cells. The NTD of mouse T1R3 was expressed as two distinct fusion proteins in Escherichia coli and purified by column chromatography. Spectroscopic analysis of the purified NTD proteins shows them to be properly folded and capable of binding ligands. This methodology should not only facilitate the characterization of T1R ligand interactions but may also be useful for dissecting the function of other class C GPCRs such as the large family of orphan V2R vomeronasal receptors.

  2. Differential regulation of. mu. , delta, kappa opioid receptors by Mn/sup + +/

    Szuecs, M.; Oetting, G.M.; Coscia, C.J.

    1986-03-05

    Differential effects of Mn/sup + +/ on three opioid receptor subtypes of rat brain membranes were evaluated. Concentration dependency studies performed with 0.05-20 mM Mn/sup + +/ revealed that only the delta receptors are stimulated at any concentration. The binding of 1 nM /sup 3/H-DAGO was not stimulated by low concentrations (< 1mM) of Mn/sup + +/, and was significantly inhibited at higher concentrations (40% at 20 mM). 1 nM /sup 3/H-EKC (+100nM DAGO and 100nM DADLE) binding was inhibited by Mn/sup + +/ in the entire concentration range. While regulation of ..mu.. receptor binding did not change during postnatal development, delta and kappa binding displayed a pronounced developmental time-dependency. Kappa sites were hardly affected by Mn/sup + +/ at day 5, and adult levels of inhibition were reached only after the third week postnatal. In contrast, 1 nM /sup 3/H-DADLE (+10nM DAGO) binding was most sensitive to Mn/sup + +/ on day 5 after birth (100% stimulation with 5-20 mM). The ED/sub 50/ of Mn/sup + +/ stimulation was unchanged during maturation. These immature delta sites displayed a similar extent of Mn/sup + +/ reversal of Gpp(NH)p inhibition as seen in microsomes, which represent a good model of N/sub i/-uncoupled receptors. These data suggest that ..mu.., delta and kappa receptors are differently coupled to N/sub i/. Moreover, a second divalent cation binding site, in addition to that on N/sub i/ might exist for delta receptors.

  3. A ligand channel through the G protein coupled receptor opsin.

    Peter W Hildebrand

    Full Text Available The G protein coupled receptor rhodopsin contains a pocket within its seven-transmembrane helix (TM structure, which bears the inactivating 11-cis-retinal bound by a protonated Schiff-base to Lys296 in TM7. Light-induced 11-cis-/all-trans-isomerization leads to the Schiff-base deprotonated active Meta II intermediate. With Meta II decay, the Schiff-base bond is hydrolyzed, all-trans-retinal is released from the pocket, and the apoprotein opsin reloaded with new 11-cis-retinal. The crystal structure of opsin in its active Ops* conformation provides the basis for computational modeling of retinal release and uptake. The ligand-free 7TM bundle of opsin opens into the hydrophobic membrane layer through openings A (between TM1 and 7, and B (between TM5 and 6, respectively. Using skeleton search and molecular docking, we find a continuous channel through the protein that connects these two openings and comprises in its central part the retinal binding pocket. The channel traverses the receptor over a distance of ca. 70 A and is between 11.6 and 3.2 A wide. Both openings are lined with aromatic residues, while the central part is highly polar. Four constrictions within the channel are so narrow that they must stretch to allow passage of the retinal beta-ionone-ring. Constrictions are at openings A and B, respectively, and at Trp265 and Lys296 within the retinal pocket. The lysine enforces a 90 degrees elbow-like kink in the channel which limits retinal passage. With a favorable Lys side chain conformation, 11-cis-retinal can take the turn, whereas passage of the all-trans isomer would require more global conformational changes. We discuss possible scenarios for the uptake of 11-cis- and release of all-trans-retinal. If the uptake gate of 11-cis-retinal is assigned to opening B, all-trans is likely to leave through the same gate. The unidirectional passage proposed previously requires uptake of 11-cis-retinal through A and release of photolyzed all

  4. Comparative tissue distribution of conformationally restricted radioiodinated vesamicol receptor ligands

    Efange, S M.N.; Khare, A B; Langason, R B

    1995-05-01

    Three conformationally restricted analogs of vesamicol, 1'-[1-(3-iodobenzyl)-4-hydroxypiperidin-3-yl]-spirol[1H-indene-1,4'- piperidine] (5), 1'-[1-(3-iodobenzyl)-4-hydroxypiperidin-3-yl]-3,4-dihydrospiro[indene-1,4'- piperidine] (6) and 1'-[1-(3-iodobenzyl)-4-hydroxypiperidin-3-yl)]-3,4-dihydrospiro[naphthalene- 1(2H),4'-piperidine] (7), were labelled with iodine-125 and evaluated as potential radioligands for mapping vesamicol receptor (VR) density and cholinergic function in vivo. All compounds showed similar kinetics in most tissues. However, differences were observed in the brain. Although comparable levels of each corresponding enantiomeric pair were obtained initially in the brain, the levels of the dextrorotatory enantiomers (+)-5, (+)-6 and (+)-7 were found to decrease by 72-82% over a period of 3 h. In contrast, the brain levels of the corresponding levorotatory isomers were maintained throughout the duration of the experiment. Among the dextrorotatory isomers, (+)-6 showed the highest brain extraction, while (+)-7 showed the lowest. In tissue dissection experiments, the levels of (+)-5, (+)-6 and (+)-7 were highest in the striatum and moderate to low in the cortex and cerebellum. Co-administration of haloperidol with (+)-6 decreased the levels of the latter in the striatum by 27%, while the levels in the cortex and cerebellum were each reduced by 60%. In addition, haloperidol failed to affect the regional distribution of (+)-7 in the brain. However, both haloperidol and spiperone increased the striatal levels of (+)-5 by 67 and 76%, respectively, suggesting that the binding of this radioligand is related to cholinergic function. Furthermore, haloperidol reduced the concentration of (+)-5 in the cortex and cerebellum by 25 and 33%, respectively, thereby implicating the sigma site as a secondary target for this ligand in the cortex.

  5. Comparative tissue distribution of conformationally restricted radioiodinated vesamicol receptor ligands

    Efange, S.M.N.; Khare, A.B.; Langason, R.B.

    1995-01-01

    Three conformationally restricted analogs of vesamicol, 1'-[1-(3-iodobenzyl)-4-hydroxypiperidin-3-yl]-spirol[1H-indene-1,4'- piperidine] (5), 1'-[1-(3-iodobenzyl)-4-hydroxypiperidin-3-yl]-3,4-dihydrospiro[indene-1,4'- piperidine] (6) and 1'-[1-(3-iodobenzyl)-4-hydroxypiperidin-3-yl)-3,4-dihydrospiro[naphthalene- 1(2H),4'-piperidine] (7), were labelled with iodine-125 and evaluated as potential radioligands for mapping vesamicol receptor (VR) density and cholinergic function in vivo. All compounds showed similar kinetics in most tissues. However, differences were observed in the brain. Although comparable levels of each corresponding enantiomeric pair were obtained initially in the brain, the levels of the dextrorotatory enantiomers (+)-5, (+)-6 and (+)-7 were found to decrease by 72-82% over a period of 3 h. In contrast, the brain levels of the corresponding levorotatory isomers were maintained throughout the duration of the experiment. Among the dextrorotatory isomers, (+)-6 showed the highest brain extraction, while (+)-7 showed the lowest. In tissue dissection experiments, the levels of (+)-5, (+)-6 and (+)-7 were highest in the striatum and moderate to low in the cortex and cerebellum. Co-administration of haloperidol with (+)-6 decreased the levels of the latter in the striatum by 27%, while the levels in the cortex and cerebellum were each reduced by 60%. In addition, haloperidol failed to affect the regional distribution of (+)-7 in the brain. However, both haloperidol and spiperone increased the striatal levels of (+)-5 by 67 and 76%, respectively, suggesting that the binding of this radioligand is related to cholinergic function. Furthermore, haloperidol reduced the concentration of (+)-5 in the cortex and cerebellum by 25 and 33%, respectively, thereby implicating the sigma site as a secondary target for this ligand in the cortex

  6. Internalization mechanisms of the epidermal growth factor receptor after activation with different ligands.

    Lasse Henriksen

    Full Text Available The epidermal growth factor receptor (EGFR regulates normal growth and differentiation, but dysregulation of the receptor or one of the EGFR ligands is involved in the pathogenesis of many cancers. There are eight ligands for EGFR, however most of the research into trafficking of the receptor after ligand activation focuses on the effect of epidermal growth factor (EGF and transforming growth factor-α (TGF-α. For a long time it was believed that clathrin-mediated endocytosis was the major pathway for internalization of the receptor, but recent work suggests that different pathways exist. Here we show that clathrin ablation completely inhibits internalization of EGF- and TGF-α-stimulated receptor, however the inhibition of receptor internalization in cells treated with heparin-binding EGF-like growth factor (HB-EGF or betacellulin (BTC was only partial. In contrast, clathrin knockdown fully inhibits EGFR degradation after all ligands tested. Furthermore, inhibition of dynamin function blocked EGFR internalization after stimulation with all ligands. Knocking out a number of clathrin-independent dynamin-dependent pathways of internalization had no effect on the ligand-induced endocytosis of the EGFR. We suggest that EGF and TGF-α lead to EGFR endocytosis mainly via the clathrin-mediated pathway. Furthermore, we suggest that HB-EGF and BTC also lead to EGFR endocytosis via a clathrin-mediated pathway, but can additionally use an unidentified internalization pathway or better recruit the small amount of clathrin remaining after clathrin knockdown.

  7. Internalization Mechanisms of the Epidermal Growth Factor Receptor after Activation with Different Ligands

    Henriksen, Lasse; Grandal, Michael Vibo; Knudsen, Stine Louise Jeppe; van Deurs, Bo; Grøvdal, Lene Melsæther

    2013-01-01

    The epidermal growth factor receptor (EGFR) regulates normal growth and differentiation, but dysregulation of the receptor or one of the EGFR ligands is involved in the pathogenesis of many cancers. There are eight ligands for EGFR, however most of the research into trafficking of the receptor after ligand activation focuses on the effect of epidermal growth factor (EGF) and transforming growth factor-α (TGF-α). For a long time it was believed that clathrin-mediated endocytosis was the major pathway for internalization of the receptor, but recent work suggests that different pathways exist. Here we show that clathrin ablation completely inhibits internalization of EGF- and TGF-α-stimulated receptor, however the inhibition of receptor internalization in cells treated with heparin-binding EGF-like growth factor (HB-EGF) or betacellulin (BTC) was only partial. In contrast, clathrin knockdown fully inhibits EGFR degradation after all ligands tested. Furthermore, inhibition of dynamin function blocked EGFR internalization after stimulation with all ligands. Knocking out a number of clathrin-independent dynamin-dependent pathways of internalization had no effect on the ligand-induced endocytosis of the EGFR. We suggest that EGF and TGF-α lead to EGFR endocytosis mainly via the clathrin-mediated pathway. Furthermore, we suggest that HB-EGF and BTC also lead to EGFR endocytosis via a clathrin-mediated pathway, but can additionally use an unidentified internalization pathway or better recruit the small amount of clathrin remaining after clathrin knockdown. PMID:23472148

  8. Satiety and the role of μ-opioid receptors in the portal vein.

    De Vadder, Filipe; Gautier-Stein, Amandine; Mithieux, Gilles

    2013-12-01

    Mu-opioid receptors (MORs) are known to influence food intake at the brain level, through their involvement in the food reward system. MOR agonists stimulate food intake. On the other hand, MOR antagonists suppress food intake. MORs are also active in peripheral organs, especially in the small intestine where they control the gut motility. Recently, an indirect role in the control of food intake was ascribed to MORs in the extrinsic gastrointestinal neural system. MORs present in the neurons of the portal vein walls sense blood peptides released from the digestion of dietary protein. These peptides behave as MOR antagonists. Their MOR antagonist action initiates a gut-brain circuitry resulting in the induction of intestinal gluconeogenesis, a function controlling food intake. Thus, periportal MORs are a key mechanistic link in the satiety effect of protein-enriched diets. Copyright © 2013 Elsevier Ltd. All rights reserved.

  9. μ Opioid Receptor Expression after Morphine Administration Is Regulated by miR-212/132 Cluster.

    Adrian Garcia-Concejo

    Full Text Available Since their discovery, miRNAs have emerged as a promising therapeutical approach in the treatment of several diseases, as demonstrated by miR-212 and its relation to addiction. Here we prove that the miR-212/132 cluster can be regulated by morphine, through the activation of mu opioid receptor (Oprm1. The molecular pathways triggered after morphine administration also induce changes in the levels of expression of oprm1. In addition, miR-212/132 cluster is actively repressing the expression of mu opioid receptor by targeting a sequence in the 3' UTR of its mRNA. These findings suggest that this cluster is closely related to opioid signaling, and function as a post-transcriptional regulator, modulating morphine response in a dose dependent manner. The regulation of miR-212/132 cluster expression is mediated by MAP kinase pathway, CaMKII-CaMKIV and PKA, through the phosphorylation of CREB. Moreover, the regulation of both oprm1 and of the cluster promoter is mediated by MeCP2, acting as a transcriptional repressor on methylated DNA after prolonged morphine administration. This mechanism explains the molecular signaling triggered by morphine as well as the regulation of the expression of the mu opioid receptor mediated by morphine and the implication of miR-212/132 in these processes.

  10. Dynamic Regulation of Delta-Opioid Receptor in Rat Trigeminal Ganglion Neurons by Lipopolysaccharide-induced Acute Pulpitis.

    Huang, Jin; Lv, Yiheng; Fu, Yunjie; Ren, Lili; Wang, Pan; Liu, Baozhu; Huang, Keqiang; Bi, Jing

    2015-12-01

    Delta-opioid receptor (DOR) and its endogenous ligands distribute in trigeminal system and play a very important role in modulating peripheral inflammatory pain. DOR activation can trigger p44/42 mitogen-activated protein kinase (ERK1/2) and Akt signaling pathways, which participate in anti-inflammatory and neuroprotective effects. In this study, our purpose was to determine the dynamic changes of DOR in trigeminal ganglion (TG) neurons during the process of acute dental pulp inflammation and elucidate its possible mechanism. Forty rats were used to generate lipopolysaccharide-induced acute pulpitis animal models at 6, 12, and 24 hours and sham-operated groups. Acute pulpitis was confirmed by hematoxylin-eosin staining, and TG neuron activation was determined by anti-c-Fos immunohistochemistry. DOR protein and gene expression in TG was investigated by immunohistochemistry, Western blotting, and real-time polymerase chain reaction, and DOR expression in trigeminal nerves and dental pulp was also determined by immunohistochemistry. To further investigate the mechanism of DOR modulating acute inflammation, the change of pErk1/2 and pAkt in TG was examined by immunohistochemistry. Lipopolysaccharide could successfully induce acute pulpitis and activated TG neurons. Acute pulpitis could dynamically increase DOR protein and gene expression at 6, 12, and 24 hours in TG, and DOR dimerization was significantly increased at 12 and 24 hours. Acute pulpitis also induced the dynamic change of DOR protein in trigeminal nerve and dental pulp. Furthermore, ERK1/2 and Akt signaling pathways were inhibited in TG after acute pulpitis. Increased DOR expression and dimerization may play important roles in peripheral acute inflammatory pain. Copyright © 2015 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

  11. Ligand Activation of TAM Family Receptors-Implications for Tumor Biology and Therapeutic Response.

    Davra, Viralkumar; Kimani, Stanley G; Calianese, David; Birge, Raymond B

    2016-11-29

    The TAM family of receptors (i.e., Tyro3, Axl, and Mertk), and their ligands Growth arrest specific factor 6 (Gas6) and Protein S (Pros1) contribute to several oncogenic processes, such as cell survival, invasion, migration, chemo-resistance, and metastasis, whereby expression often correlates with poor clinical outcomes. In recent years, there has been great interest in the study of TAM receptors in cancer, stemming both from their roles as oncogenic signaling receptors, as well as their roles in tumor immunology. As a result, several classes of TAM inhibitors that include small molecule tyrosine kinase inhibitors, monoclonal antibodies, decoy receptors, as well as novel strategies to target TAM ligands are being developed. This paper will review the biology of TAM receptors and their ligands with a focus on cancer, as well as evidence-based data for the continued pursuit of TAM/Gas6 inhibitors in clinical practice.

  12. Structural Determinants for the Binding of Morphinan Agonists to the μ-Opioid Receptor.

    Xiaojing Cong

    Full Text Available Atomistic descriptions of the μ-opioid receptor (μOR noncovalently binding with two of its prototypical morphinan agonists, morphine (MOP and hydromorphone (HMP, are investigated using molecular dynamics (MD simulations. Subtle differences between the binding modes and hydration properties of MOP and HMP emerge from the calculations. Alchemical free energy perturbation calculations show qualitative agreement with in vitro experiments performed in this work: indeed, the binding free energy difference between MOP and HMP computed by forward and backward alchemical transformation is 1.2±1.1 and 0.8±0.8 kcal/mol, respectively, to be compared with 0.4±0.3 kcal/mol from experiment. Comparison with an MD simulation of μOR covalently bound with the antagonist β-funaltrexamine hints to agonist-induced conformational changes associated with an early event of the receptor's activation: a shift of the transmembrane helix 6 relative to the transmembrane helix 3 and a consequent loss of the key R165-T279 interhelical hydrogen bond. This finding is consistent with a previous proposal suggesting that the R165-T279 hydrogen bond between these two helices indicates an inactive receptor conformation.

  13. Endogenous ligands for C-type lectin receptors: the true regulators of immune homeostasis.

    García-Vallejo, Juan J; van Kooyk, Yvette

    2009-07-01

    C-type lectin receptors (CLRs) have long been known as pattern-recognition receptors implicated in the recognition of pathogens by the innate immune system. However, evidence is accumulating that many CLRs are also able to recognize endogenous 'self' ligands and that this recognition event often plays an important role in immune homeostasis. In the present review, we focus on the human and mouse CLRs for which endogenous ligands have been described. Special attention is given to the signaling events initiated upon recognition of the self ligand and the regulation of glycosylation as a switch modulating CLR recognition, and therefore, immune homeostasis.

  14. Differential effects of EGFR ligands on endocytic sorting of the receptor

    Roepstorff, Kirstine; Grandal, Michael Vibo; Henriksen, Lasse

    2009-01-01

    signalling and is a more potent mitogen than EGF. In addition to EGF and TGF-alpha, five EGFR ligands have been identified. Although many of these ligands are upregulated in cancers, very little is known about their effect on EGFR trafficking. We have compared the effect of six different ligands on endocytic...... trafficking of EGFR. We find that, whereas they all stimulate receptor internalization, they have very diverse effects on endocytic sorting. Heparin-binding EGF-like growth factor and Betacellulin target all EGFRs for lysosomal degradation. In contrast, TGF-alpha and epiregulin lead to complete receptor...

  15. A highly sensitive quantitative cytosensor technique for the identification of receptor ligands in tissue extracts.

    Lenkei, Z; Beaudet, A; Chartrel, N; De Mota, N; Irinopoulou, T; Braun, B; Vaudry, H; Llorens-Cortes, C

    2000-11-01

    Because G-protein-coupled receptors (GPCRs) constitute excellent putative therapeutic targets, functional characterization of orphan GPCRs through identification of their endogenous ligands has great potential for drug discovery. We propose here a novel single cell-based assay for identification of these ligands. This assay involves (a) fluorescent tagging of the GPCR, (b) expression of the tagged receptor in a heterologous expression system, (c) incubation of the transfected cells with fractions purified from tissue extracts, and (d) imaging of ligand-induced receptor internalization by confocal microscopy coupled to digital image quantification. We tested this approach in CHO cells stably expressing the NT1 neurotensin receptor fused to EGFP (enhanced green fluorescent protein), in which neurotensin promoted internalization of the NT1-EGFP receptor in a dose-dependent fashion (EC(50) = 0.98 nM). Similarly, four of 120 consecutive reversed-phase HPLC fractions of frog brain extracts promoted internalization of the NT1-EGFP receptor. The same four fractions selectively contained neurotensin, an endogenous ligand of the NT1 receptor, as detected by radioimmunoassay and inositol phosphate production. The present internalization assay provides a highly specific quantitative cytosensor technique with sensitivity in the nanomolar range that should prove useful for the identification of putative natural and synthetic ligands for GPCRs.

  16. Inhibition of GABAergic Neurotransmission by HIV-1 Tat and Opioid Treatment in the Striatum Involves ?-Opioid Receptors

    Xu, Changqing; Fitting, Sylvia

    2016-01-01

    Due to combined antiretroviral therapy (cART), human immunodeficiency virus type 1 (HIV-1) is considered a chronic disease with high prevalence of mild forms of neurocognitive impairments, also referred to as HIV-associated neurocognitive disorders (HAND). Although opiate drug use can exacerbate HIV-1 Tat-induced neuronal damage, it remains unknown how and to what extent opioids interact with Tat on the GABAergic system. We conducted whole-cell recordings in mouse striatal slices and examined...

  17. Single-molecule photobleaching reveals increased MET receptor dimerization upon ligand binding in intact cells

    Dietz, Marina S; Haße, Daniel; Ferraris, Davide M; Göhler, Antonia; Niemann, Hartmut H; Heilemann, Mike

    2013-01-01

    The human receptor tyrosine kinase MET and its ligand hepatocyte growth factor/scatter factor are essential during embryonic development and play an important role during cancer metastasis and tissue regeneration. In addition, it was found that MET is also relevant for infectious diseases and is the target of different bacteria, amongst them Listeria monocytogenes that induces bacterial uptake through the surface protein internalin B. Binding of ligand to the MET receptor is proposed to lead to receptor dimerization. However, it is also discussed whether preformed MET dimers exist on the cell membrane. To address these issues we used single-molecule fluorescence microscopy techniques. Our photobleaching experiments show that MET exists in dimers on the membrane of cells in the absence of ligand and that the proportion of MET dimers increases significantly upon ligand binding. Our results indicate that partially preformed MET dimers may play a role in ligand binding or MET signaling. The addition of the bacterial ligand internalin B leads to an increase of MET dimers which is in agreement with the model of ligand-induced dimerization of receptor tyrosine kinases.

  18. Role of the T cell receptor ligand affinity in T cell activation by bacterial superantigens

    Andersen, P S; Geisler, C; Buus, S

    2001-01-01

    Similar to native peptide/MHC ligands, bacterial superantigens have been found to bind with low affinity to the T cell receptor (TCR). It has been hypothesized that low ligand affinity is required to allow optimal TCR signaling. To test this, we generated variants of Staphylococcus enterotoxin C3...... (SEC3) with up to a 150-fold increase in TCR affinity. By stimulating T cells with SEC3 molecules immobilized onto plastic surfaces, we demonstrate that increasing the affinity of the SEC3/TCR interaction caused a proportional increase in the ability of SEC3 to activate T cells. Thus, the potency...... correlation between ligand affinity and ligand potency indicating that it is the density of receptor-ligand complexes in the T cell contact area that determines TCR signaling strength....

  19. /sup 3/H)-(H-D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2) ((/sup 3/H)CTOP), a potent and highly selective peptide for mu opioid receptors in rat brain

    Hawkins, K.N.; Knapp, R.J.; Lui, G.K.; Gulya, K.; Kazmierski, W.; Wan, Y.P.; Pelton, J.T.; Hruby, V.J.; Yamamura, H.I.

    1989-01-01

    The cyclic, conformationally restricted octapeptide (3H)-(H-D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2) ((3H)CTOP) was synthesized and its binding to mu opioid receptors was characterized in rat brain membrane preparations. Association rates (k+1) of 1.25 x 10(8) M-1 min-1 and 2.49 x 10(8) M-1 min-1 at 25 and 37 degrees C, respectively, were obtained, whereas dissociation rates (k-1) at the same temperatures were 1.93 x 10(-2) min-1 and 1.03 x 10(-1) min-1 at 25 and 37 degrees C, respectively. Saturation isotherms of (3H)CTOP binding to rat brain membranes gave apparent Kd values of 0.16 and 0.41 nM at 25 and 37 degrees C, respectively. Maximal number of binding sites in rat brain membranes were found to be 94 and 81 fmol/mg of protein at 25 and 37 degrees C, respectively. (3H)CTOP binding over a concentration range of 0.1 to 10 nM was best fit by a one site model consistent with binding to a single site. The general effect of different metal ions and guanyl-5'-yl-imidodiphosphate on (3H)CTOP binding was to reduce its affinity. High concentrations (100 mM) of sodium also produced a reduction of the apparent mu receptor density. Utilizing the delta opioid receptor specific peptide (3H)-(D-Pen2,D-Pen5)enkephalin, CTOP appeared to be about 2000-fold more specific for mu vs. delta opioid receptor than naloxone. Specific (3H)CTOP binding was inhibited by a large number of opioid or opiate ligands.

  20. Internalization mechanisms of the epidermal growth factor receptor after activation with different ligands

    Henriksen, Lasse; Grandal, Michael Vibo; Knudsen, Stine Louise Jeppe

    2013-01-01

    after ligand activation focuses on the effect of epidermal growth factor (EGF) and transforming growth factor-α (TGF-α). For a long time it was believed that clathrin-mediated endocytosis was the major pathway for internalization of the receptor, but recent work suggests that different pathways exist....... Here we show that clathrin ablation completely inhibits internalization of EGF- and TGF-α-stimulated receptor, however the inhibition of receptor internalization in cells treated with heparin-binding EGF-like growth factor (HB-EGF) or betacellulin (BTC) was only partial. In contrast, clathrin knockdown...... fully inhibits EGFR degradation after all ligands tested. Furthermore, inhibition of dynamin function blocked EGFR internalization after stimulation with all ligands. Knocking out a number of clathrin-independent dynamin-dependent pathways of internalization had no effect on the ligand...

  1. Revealing a steroid receptor ligand as a unique PPAR[gamma] agonist

    Lin, Shengchen; Han, Ying; Shi, Yuzhe; Rong, Hui; Zheng, Songyang; Jin, Shikan; Lin, Shu-Yong; Lin, Sheng-Cai; Li, Yong (Pitt); (Xiamen)

    2012-06-28

    Peroxisome proliferator-activated receptor gamma (PPAR{gamma}) regulates metabolic homeostasis and is a molecular target for anti-diabetic drugs. We report here the identification of a steroid receptor ligand, RU-486, as an unexpected PPAR{gamma} agonist, thereby uncovering a novel signaling route for this steroid drug. Similar to rosiglitazone, RU-486 modulates the expression of key PPAR{gamma} target genes and promotes adipocyte differentiation, but with a lower adipogenic activity. Structural and functional studies of receptor-ligand interactions reveal the molecular basis for a unique binding mode for RU-486 in the PPAR{gamma} ligand-binding pocket with distinctive properties and epitopes, providing the molecular mechanisms for the discrimination of RU-486 from thiazolidinediones (TZDs) drugs. Our findings together indicate that steroid compounds may represent an alternative approach for designing non-TZD PPAR{gamma} ligands in the treatment of insulin resistance.

  2. Free energy calculations offer insights into the influence of receptor flexibility on ligand-receptor binding affinities.

    Dolenc, Jožica; Riniker, Sereina; Gaspari, Roberto; Daura, Xavier; van Gunsteren, Wilfred F

    2011-08-01

    Docking algorithms for computer-aided drug discovery and design often ignore or restrain the flexibility of the receptor, which may lead to a loss of accuracy of the relative free enthalpies of binding. In order to evaluate the contribution of receptor flexibility to relative binding free enthalpies, two host-guest systems have been examined: inclusion complexes of α-cyclodextrin (αCD) with 1-chlorobenzene (ClBn), 1-bromobenzene (BrBn) and toluene (MeBn), and complexes of DNA with the minor-groove binding ligands netropsin (Net) and distamycin (Dist). Molecular dynamics simulations and free energy calculations reveal that restraining of the flexibility of the receptor can have a significant influence on the estimated relative ligand-receptor binding affinities as well as on the predicted structures of the biomolecular complexes. The influence is particularly pronounced in the case of flexible receptors such as DNA, where a 50% contribution of DNA flexibility towards the relative ligand-DNA binding affinities is observed. The differences in the free enthalpy of binding do not arise only from the changes in ligand-DNA interactions but also from changes in ligand-solvent interactions as well as from the loss of DNA configurational entropy upon restraining.

  3. Synthesis and evaluation of peptide and nucleic acid based Toll-like receptor ligands

    Weterings, Josephus Johannes

    2008-01-01

    Toll-like receptors (TLRs) are receptors that continuously scour their direct surroundings for pathogen associated molecular patterns (PAMPs) of bacterial, viral or fungal origin. TLRs can be found at cells that play a role in the immune system. Binding of the TLR with its corresponding ligand

  4. Ligand-mediated negative regulation of a chimeric transmembrane receptor tyrosine phosphatase

    Desai, D M; Sap, J; Schlessinger, J

    1993-01-01

    CD45, a transmembrane protein tyrosine phosphatase (PTPase), is required for TCR signaling. Multiple CD45 isoforms, differing in the extracellular domain, are expressed in a tissue- and activation-specific manner, suggesting an important function for this domain. We report that a chimeric protein...... that ligand-mediated regulation of receptor-PTPases may have mechanistic similarities with receptor tyrosine kinases....

  5. PET and Hormone Receptor Ligands in Breast Cancer

    Gemignani, Mary

    2006-01-01

    .... To investigate this further, this project's objectives are: To evaluate the use of estrogen-like ligands labeled with positron emitters in preoperatively determining the ER status of breast cancer using PET...

  6. Dynamical Binding Modes Determine Agonistic and Antagonistic Ligand Effects in the Prostate-Specific G-Protein Coupled Receptor (PSGR).

    Wolf, Steffen; Jovancevic, Nikolina; Gelis, Lian; Pietsch, Sebastian; Hatt, Hanns; Gerwert, Klaus

    2017-11-22

    We analysed the ligand-based activation mechanism of the prostate-specific G-protein coupled receptor (PSGR), which is an olfactory receptor that mediates cellular growth in prostate cancer cells. Furthermore, it is an olfactory receptor with a known chemically near identic antagonist/agonist pair, α- and β-ionone. Using a combined theoretical and experimental approach, we propose that this receptor is activated by a ligand-induced rearrangement of a protein-internal hydrogen bond network. Surprisingly, this rearrangement is not induced by interaction of the ligand with the network, but by dynamic van der Waals contacts of the ligand with the involved amino acid side chains, altering their conformations and intraprotein connectivity. Ligand recognition in this GPCR is therefore highly stereo selective, but seemingly lacks any ligand recognition via polar contacts. A putative olfactory receptor-based drug design scheme will have to take this unique mode of protein/ligand action into account.

  7. Endomorphin-2: a biased agonist at the μ-opioid receptor.

    Rivero, Guadalupe; Llorente, Javier; McPherson, Jamie; Cooke, Alex; Mundell, Stuart J; McArdle, Craig A; Rosethorne, Elizabeth M; Charlton, Steven J; Krasel, Cornelius; Bailey, Christopher P; Henderson, Graeme; Kelly, Eamonn

    2012-08-01

    Previously we correlated the efficacy for G protein activation with that for arrestin recruitment for a number of agonists at the μ-opioid receptor (MOPr) stably expressed in HEK293 cells. We suggested that the endomorphins (endomorphin-1 and -2) might be biased toward arrestin recruitment. In the present study, we investigated this phenomenon in more detail for endomorphin-2, using endogenous MOPr in rat brain as well as MOPr stably expressed in HEK293 cells. For MOPr in neurons in brainstem locus ceruleus slices, the peptide agonists [d-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO) and endomorphin-2 activated inwardly rectifying K(+) current in a concentration-dependent manner. Analysis of these responses with the operational model of pharmacological agonism confirmed that endomorphin-2 had a much lower operational efficacy for G protein-mediated responses than did DAMGO at native MOPr in mature neurons. However, endomorphin-2 induced faster desensitization of the K(+) current than did DAMGO. In addition, in HEK293 cells stably expressing MOPr, the ability of endomorphin-2 to induce phosphorylation of Ser375 in the COOH terminus of the receptor, to induce association of arrestin with the receptor, and to induce cell surface loss of receptors was much more efficient than would be predicted from its efficacy for G protein-mediated signaling. Together, these results indicate that endomorphin-2 is an arrestin-biased agonist at MOPr and the reason for this is likely to be the ability of endomorphin-2 to induce greater phosphorylation of MOPr than would be expected from its ability to activate MOPr and to induce activation of G proteins.

  8. Tachykinin NK₁ receptor antagonist co-administration attenuates opioid withdrawal-mediated spinal microglia and astrocyte activation.

    Tumati, Suneeta; Largent-Milnes, Tally M; Keresztes, Attila I; Yamamoto, Takashi; Vanderah, Todd W; Roeske, William R; Hruby, Victor J; Varga, Eva V

    2012-06-05

    Prolonged morphine treatment increases pain sensitivity in many patients. Enhanced spinal Substance P release is one of the adaptive changes associated with sustained opioid exposure. In addition to pain transmitting second order neurons, spinal microglia and astrocytes also express functionally active Tachykinin NK₁ (Substance P) receptors. In the present work we investigated the role of glial Tachykinin NK₁ receptors in morphine withdrawal-mediated spinal microglia and astrocyte activation. Our data indicate that intrathecal co-administration (6 days, twice daily) of a selective Tachykinin NK₁ receptor antagonist (N-acetyl-L-tryptophan 3,5-bis(trifluoromethyl)benzylester (L-732,138; 20 μg/injection)) attenuates spinal microglia and astrocyte marker and pro-inflammatory mediator immunoreactivity as well as hyperalgesia in withdrawn rats. Furthermore, covalent linkage of the opioid agonist with a Tachykinin NK₁ antagonist pharmacophore yielded a bivalent compound that did not augment spinal microglia or astrocyte marker or pro-inflammatory mediator immunoreactivity and did not cause paradoxical pain sensitization upon drug withdrawal. Thus, bivalent opioid/Tachykinin NK₁ receptor antagonists may provide a novel paradigm for long-term pain management.

  9. Long-lasting, distinct changes in central opioid receptor and urinary bladder functions in models of schizophrenia in rats.

    Kekesi, Orsolya; Tuboly, Gabor; Szucs, Maria; Birkas, Erika; Morvay, Zita; Benedek, Gyorgy; Horvath, Gyongyi

    2011-07-01

    Ketamine treatments and social isolation of rats reflect certain features of schizophrenia, among them altered pain sensitivity. To study the underlying mechanisms of these phenomena, rats were either housed individually or grouped for 33 days after weaning, and treated with either ketamine or saline for 14 days. After one month re-socialization, the urinary bladder capacity by ultrasound examination in the anesthetized animals, and changes of μ-opioid receptors by saturation binding experiments using a specific μ-opioid agonist [(3)H]DAMGO were determined. G-protein signaling was investigated in DAMGO-stimulated [(35)S]GTPγS functional assays. Ketamine treatment significantly decreased the bladder volume and isolation decreased the receptor density in cortical membranes. Among all groups, the only change in binding affinity was an increase induced by social isolation in the cortex. G-protein signaling was significantly decreased by either ketamine or social isolation in this tissue. Ketamine treatment, but not housing, significantly increased μ-opioid receptor densities in hippocampal membranes. Both ketamine and isolation increased the efficacy, while the potency of signaling was decreased by any treatment. Ketamine increased the receptor density and G-protein activation; while isolation decreased the efficacy of G-protein signaling in hippocampal membranes. The changes in the co-treated group were similar to those of the isolated animals in most tests. The distinct changes of opioid receptor functioning in different areas of the CNS may, at least partially, explain the augmented nociceptive threshold and morphine potency observed in these animals. Changes in the relative urinary bladder suggest a detrusor hyperreflexia, another sign of schizophrenia. Copyright © 2011 Elsevier B.V. All rights reserved.

  10. The role of the opioid system in alcohol dependence.

    Nutt, David J

    2014-01-01

    The role of the brain opioid system in alcohol dependence has been the subject of much research for over 25 years. This review explores the evidence: firstly describing the opioid receptors in terms of their individual subtypes, neuroanatomy, neurophysiology and ligands; secondly, summarising emerging data from specific neurochemical, behavioural and neuroimaging studies, explaining the characteristics of addiction with a focus on alcohol dependence and connecting the opioid system with alcohol dependence; and finally reviewing the known literature regarding opioid antagonists in clinical use for alcohol dependence. Further interrogation of how modulation of the opioid system, via use of MOP (mu), DOP (delta) and KOP (kappa) agents, restores the balance of a dysregulated system in alcohol dependence should increase our insight into this disease process and therefore guide better methods for understanding and treating alcohol dependence in the future.

  11. Evidence of morphine like substance and μ-opioid receptor expression in Toxacara canis (Nematoda: Ascaridae).

    Golabi, Mostafa; Naem, Soraya; Imani, Mehdi; Dalirezh, Nowruz

    2016-01-01

    Toxocara canis (Nematoda: Ascaridae) is an intestinal nematode parasite of dogs, which can also cause disease in humans. Transmission to humans usually occurs because of direct contact with T. canis eggs present in soil contaminated with the feces of infected dogs. This nematode has extraordinary abilities to survive for many years in different tissues of vertebrates, and develop to maturity in the intestinal tract of its definitive host. Survival of parasitic nematodes within a host requires immune evasion using complicated pathways. Morphine-like substance, as well as opioids, which are known as down regulating agents, can modulate both innate and acquired immune responses, and let the parasite survives in their hosts. In the present study, we aimed to find evidences of morphine-like substance and µ-opiate receptor expression in T. canis , using high performance liquid chromatography (HPLC) and reverse transcription polymerase chain reaction (RT-PCR). The results indicated that T. canis produced morphine-like substances at the level of 2.31± 0.26 ng g -1 wet weight, and expressed µ-opiate receptor as in expected size of 441 bp. According to our findings, it was concluded that T. canis , benefits using morphine-like substance to modulate host immunity.

  12. Ivy and neurogliaform interneurons are a major target of μ opioid receptor modulation

    Krook-Magnuson, Esther; Luu, Lillian; Lee, Sang-Hun; Varga, Csaba; Soltesz, Ivan

    2011-01-01

    Mu opioid receptors (μORs) are selectively expressed on interneurons in area CA1 of the hippocampus. Fast-spiking, parvalbumin expressing, basket cells express μORs, but circumstantial evidence suggests that another major, unidentified, GABAergic cell class must also be modulated by μORs. Here we report that the abundant, dendritically targeting, neurogliaform family of cells (Ivy and neurogliaform cells) is a previously unrecognized target of direct modulation by μORs. Ivy and neurogliaform cells are not only numerous, but also have unique properties, including promiscuous gap junctions formed with various interneuronal subtypes, volume transmission, and the ability to produce a postsynaptic GABAB response after a single presynaptic spike. Using a mouse line expressing green fluorescent protein under the neuropeptide Y promoter, we find that across all layers of CA1, activation of μORs hyperpolarizes Ivy and neurogliaform cells. Further, paired recordings between synaptically coupled Ivy and pyramidal cells show that Ivy cell terminals are dramatically inhibited by μOR-activation. Effects in Ivy and neurogliaform cells are seen at similar concentrations of agonist as those producing inhibition in fast-spiking PV basket cells. We also report that Ivy cells display the recently described phenomenon of persistent firing, a state of continued firing in the absence of continued input, and that induction of persistent firing is inhibited by μOR-activation. Together these findings identify a major, previously unrecognized, target of μOR-modulation. Given the prominence of this cell type in and beyond CA1, as well as its unique role in microcircuitry, opioid modulation of neurogliaform cells has wide implications. PMID:22016519

  13. Ivy and neurogliaform interneurons are a major target of μ-opioid receptor modulation.

    Krook-Magnuson, Esther; Luu, Lillian; Lee, Sang-Hun; Varga, Csaba; Soltesz, Ivan

    2011-10-19

    μ-Opioid receptors (μORs) are selectively expressed on interneurons in area CA1 of the hippocampus. Fast-spiking, parvalbumin-expressing, basket cells express μORs, but circumstantial evidence suggests that another major, unidentified, GABAergic cell class must also be modulated by μORs. Here we report that the abundant, dendritically targeting, neurogliaform family of cells (Ivy and neurogliaform cells) is a previously unrecognized target of direct modulation by μORs. Ivy and neurogliaform cells are not only numerous but also have unique properties, including promiscuous gap junctions formed with various interneuronal subtypes, volume transmission, and the ability to produce a postsynaptic GABA(B) response after a single presynaptic spike. Using a mouse line expressing green fluorescent protein under the neuropeptide Y promoter, we find that, across all layers of CA1, activation of μORs hyperpolarizes Ivy and neurogliaform cells. Furthermore, paired recordings between synaptically coupled Ivy and pyramidal cells show that Ivy cell terminals are dramatically inhibited by μOR activation. Effects in Ivy and neurogliaform cells are seen at similar concentrations of agonist as those producing inhibition in fast-spiking parvalbumin basket cells. We also report that Ivy cells display the recently described phenomenon of persistent firing, a state of continued firing in the absence of continued input, and that induction of persistent firing is inhibited by μOR activation. Together, these findings identify a major, previously unrecognized, target of μOR modulation. Given the prominence of this cell type in and beyond CA1, as well as its unique role in microcircuitry, opioid modulation of neurogliaform cells has wide implications.

  14. Ligand-induced internalization of neurotensin in transfected COS-7 cells: differential intracellular trafficking of ligand and receptor.

    Vandenbulcke, F; Nouel, D; Vincent, J P; Mazella, J; Beaudet, A

    2000-09-01

    The neuropeptide neurotensin (NT) is known to be internalized in a receptor-mediated fashion into its target cells. To gain insight into the mechanisms underlying this process, we monitored in parallel the migration of the NT1 neurotensin receptor subtype and a fluorescent analog of NT (fluo-NT) in COS-7 cells transfected with a tagged NT1 construct. Fluo-NT internalization was prevented by hypertonic sucrose, potassium depletion and cytosol acidification, demonstrating that it proceeded via clathrin-coated pits. Within 0-30 minutes, fluo-NT accumulated together with its receptor in Acridine Orange-positive, acidic organelles. These organelles concentrated transferrin and immunostained positively for rab 5A, therefore they were early endosomes. After 30-45 minutes, the ligand and its receptor no longer colocalized. Fluo-NT was first found in rab 7-positive late endosomes and later in a nonacidic juxtanuclear compartment identified as the Trans-Golgi Network (TGN) by virtue of its staining for syntaxin 6. This juxtanuclear compartment also stained positively for rab 7 and for the TGN/pericentriolar recycling endosome marker rab 11, suggesting that the ligand could have been recruited to the TGN from either late or recycling endosomes. By that time, internalized receptors were detected in Lamp-1-immunoreactive lysosomes. These results demonstrate that neurotensin/NT1 receptor complexes follow a recycling cycle that is unique among the G protein-coupled receptors studied to date, and provide the first evidence for the targeting of a nonendogenous protein from endosomes to the TGN.

  15. Role and psychological dependenci arrangement of opioid by type of reseptor opioid

    Arif Nurrochmad, Arif Nurrochmad

    2015-01-01

    Opioid receptor can be classified as p., 8, and K-opioid receptor that widely expressed in the CNS. The development of selective receptor agonist and cloning of each receptor have contributed greatly to our increasing knowledge of the neuropharmacological profile of each opioid receptor type. This review focuses on the functional interaction among these opioid receptor types that contribute to opioid dependence especially in psychological dependence. Several lines of evidence provide argument...

  16. Dermorphin-related peptides from the skin of Phyllomedusa bicolor and their amidated analogs activate two mu opioid receptor subtypes that modulate antinociception and catalepsy in the rat.

    Negri, L; Erspamer, G F; Severini, C; Potenza, R L; Melchiorri, P; Erspamer, V

    1992-01-01

    Three naturally occurring dermorphin-like peptides from the skin of the frog Phyllomedusa bicolor, the related carboxyl-terminal amides, and some substituted analogs were synthesized, their binding profiles to opioid receptors were determined, and their biological activities were studied in isolated organ preparations and intact animals. The opioid binding profile revealed a very high selectivity of these peptides for mu sites and suggested the existence of two receptor subtypes, of high and ...

  17. Modelling the interdependence between the stoichiometry of receptor oligomerization and ligand binding for a coexisting dimer/tetramer receptor system.

    Rovira, X; Vivó, M; Serra, J; Roche, D; Strange, P G; Giraldo, J

    2009-01-01

    Many G protein-coupled receptors have been shown to exist as oligomers, but the oligomerization state and the effects of this on receptor function are unclear. For some G protein-coupled receptors, in ligand binding assays, different radioligands provide different maximal binding capacities. Here we have developed mathematical models for co-expressed dimeric and tetrameric species of receptors. We have considered models where the dimers and tetramers are in equilibrium and where they do not interconvert and we have also considered the potential influence of the ligands on the degree of oligomerization. By analogy with agonist efficacy, we have considered ligands that promote, inhibit or have no effect on oligomerization. Cell surface receptor expression and the intrinsic capacity of receptors to oligomerize are quantitative parameters of the equations. The models can account for differences in the maximal binding capacities of radioligands in different preparations of receptors and provide a conceptual framework for simulation and data fitting in complex oligomeric receptor situations.

  18. δ-Opioid receptor-stimulated Akt signaling in neuroblastoma x glioma (NG108-15) hybrid cells involves receptor tyrosine kinase-mediated PI3K activation

    Heiss, Anika; Ammer, Hermann; Eisinger, Daniela A.

    2009-01-01

    δ-Opioid receptor (DOR) agonists possess cytoprotective properties, an effect associated with activation of the 'pro-survival' kinase Akt. Here we delineate the signal transduction pathway by which opioids induce Akt activation in neuroblastoma x glioma (NG108-15) hybrid cells. Exposure of the cells to both [D-Pen 2,5 ]enkephalin and etorphine resulted in a time- and dose-dependent increase in Akt activity, as measured by means of an activation-specific antibody recognizing phosphoserine-473. DOR-mediated Akt signaling is blocked by the opioid antagonist naloxone and involves inhibitory G i/o proteins, because pre-treatment with pertussis toxin, but not over-expression of the G q/11 scavengers EBP50 and GRK2-K220R, prevented this effect. Further studies with Wortmannin and LY294002 revealed that phophoinositol-3-kinase (PI3K) plays a central role in opioid-induced Akt activation. Opioids stimulate Akt activity through transactivation of receptor tyrosine kinases (RTK), because pre-treatment of the cells with inhibitors for neurotrophin receptor tyrosine kinases (AG879) and the insulin-like growth factor receptor IGF-1 (AG1024), but not over-expression of the Gβγ scavenger phosducin, abolished this effect. Activated Akt translocates to the nuclear membrane, where it promotes GSK3 phosphorylation and prevents caspase-3 cleavage, two key events mediating inhibition of cell apoptosis and enhancement of cell survival. Taken together, these results demonstrate that in NG108-15 hybrid cells DOR agonists possess cytoprotective properties mediated by activation of the RTK/PI3K/Akt signaling pathway.

  19. {delta}-Opioid receptor-stimulated Akt signaling in neuroblastoma x glioma (NG108-15) hybrid cells involves receptor tyrosine kinase-mediated PI3K activation

    Heiss, Anika; Ammer, Hermann [Institute of Pharmacology, Toxicology and Pharmacy Ludwig-Maximilians-University of Munich Koeniginstrasse 16 80539 Muenchen Federal Republic of Germany (Germany); Eisinger, Daniela A., E-mail: eisinger@pharmtox.vetmed.uni-muenchen.de [Institute of Pharmacology, Toxicology and Pharmacy Ludwig-Maximilians-University of Munich Koeniginstrasse 16 80539 Muenchen Federal Republic of Germany (Germany)

    2009-07-15

    {delta}-Opioid receptor (DOR) agonists possess cytoprotective properties, an effect associated with activation of the 'pro-survival' kinase Akt. Here we delineate the signal transduction pathway by which opioids induce Akt activation in neuroblastoma x glioma (NG108-15) hybrid cells. Exposure of the cells to both [D-Pen{sup 2,5}]enkephalin and etorphine resulted in a time- and dose-dependent increase in Akt activity, as measured by means of an activation-specific antibody recognizing phosphoserine-473. DOR-mediated Akt signaling is blocked by the opioid antagonist naloxone and involves inhibitory G{sub i/o} proteins, because pre-treatment with pertussis toxin, but not over-expression of the G{sub q/11} scavengers EBP50 and GRK2-K220R, prevented this effect. Further studies with Wortmannin and LY294002 revealed that phophoinositol-3-kinase (PI3K) plays a central role in opioid-induced Akt activation. Opioids stimulate Akt activity through transactivation of receptor tyrosine kinases (RTK), because pre-treatment of the cells with inhibitors for neurotrophin receptor tyrosine kinases (AG879) and the insulin-like growth factor receptor IGF-1 (AG1024), but not over-expression of the G{beta}{gamma} scavenger phosducin, abolished this effect. Activated Akt translocates to the nuclear membrane, where it promotes GSK3 phosphorylation and prevents caspase-3 cleavage, two key events mediating inhibition of cell apoptosis and enhancement of cell survival. Taken together, these results demonstrate that in NG108-15 hybrid cells DOR agonists possess cytoprotective properties mediated by activation of the RTK/PI3K/Akt signaling pathway.

  20. Ligand-independent Thrombopoietin Mutant Receptor Requires Cell Surface Localization for Endogenous Activity*

    Marty, Caroline; Chaligné, Ronan; Lacout, Catherine; Constantinescu, Stefan N.; Vainchenker, William; Villeval, Jean-Luc

    2009-01-01

    The activating W515L mutation in the thrombopoietin receptor (MPL) has been identified in primary myelofibrosis and essential thrombocythemia. MPL belongs to a subset of the cytokine receptor superfamily that requires the JAK2 kinase for signaling. We examined whether the ligand-independent MPLW515L mutant could signal intracellularly. Addition of the endoplasmic reticulum (ER) retention KDEL sequence to the receptor C terminus efficiently locked MPLW515L within its na...

  1. NNAlign: a platform to construct and evaluate artificial neural network models of receptor-ligand interactions

    Nielsen, Morten; Andreatta, Massimo

    2017-01-01

    Peptides are extensively used to characterize functional or (linear) structural aspects of receptor-ligand interactions in biological systems, e.g. SH2, SH3, PDZ peptide-recognition domains, the MHC membrane receptors and enzymes such as kinases and phosphatases. NNAlign is a method for the ident...... with insertions and deletions, encoding of receptor pseudo-sequences, and custom alphabets for the training sequences. The server is available at http://www.cbs.dtu.dk/services/NNAlign-2.0....

  2. Morphine reduces the threshold of helium preconditioning against myocardial infarction: the role of opioid receptors in rabbits

    Pagel, Paul S.; Krolikowski, John G.; Amour, Julien; Warltier, David C.; Weihrauch, Dorothee

    2015-01-01

    Objectives Brief, repetitive administration of helium before prolonged coronary artery occlusion and reperfusion protects myocardium against infarction. Opioid receptors mediate the cardioprotective effects of ischemic pre- and postconditioning, but whether these receptors also play a role in helium preconditioning is unknown. We tested the hypotheses that opioid receptors mediate helium preconditioning and that morphine (a μ1-opioid receptor agonist with δ1-opioid agonist properties) lowers the threshold of cardioprotection produced by helium in vivo. Design Randomized, prospective study. Setting University research laboratory. Participants Male New Zealand white rabbits. Interventions Rabbits (n=56) were instrumented for measurement of systemic hemodynamics and subjected to a 30 min left anterior descending coronary artery (LAD) occlusion and 3 h reperfusion. In separate experimental groups, rabbits (n=6 or 7 per group) received 0.9% saline (control), one or three cycles of 70% helium-30% oxygen administered for 5 min interspersed with 5 min of an air-oxygen mixture, morphine (0.1 mg/kg, i.v.), or the nonselective opioid antagonist naloxone (6 mg/kg, i.v.) before LAD occlusion. Other groups of rabbits received three cycles of helium or one cycle of helium plus morphine (0.1 mg/kg) in the absence or presence of naloxone (6 mg/kg) before ischemia and reperfusion. Statistical analysis of data was performed with analysis of variance for repeated measures followed by Bonferroni’s modification of Student’s t test. Measurements and Main Results Myocardial infarct size was determined using triphenyltetrazolium chloride staining and presented as a percentage of the left ventricular area at risk. Helium reduced myocardial infarct size in an exposure-related manner [36±6 (P>0.05) and 25±4% (P<0.05 versus control) for one and three cycles of helium, respectively; data are mean±SD] compared with control (44±7%). Morphine and naloxone alone did not affect infarct

  3. Changing the insulin receptor to possess insulin-like growth factor I ligand specificity

    Andersen, A.S.; Kjeldsen, T.; Wiberg, F.C.; Christensen, P.M.; Rasmussen, J.S.; Norris, K.; Moeller, K.B.; Moeller, N.P.H.

    1990-01-01

    To examine the role of the N-terminal part of the insulin-like growth factor I (IGF-I) receptor and insulin receptor in determining ligand specificity, the authors prepared an expression vector encoding a hybrid receptor where exon 1 (encoding the signal peptide and seven amino acids of the α-subunit), exon 2, and exon 3 of the insulin receptor were replaced with the corresponding IGF-I receptor cDNA (938 nucleotides). To allow direct quantitative comparison of the binding capabilities of this hybrid receptor with those of the human IGF-I receptor and the insulin receptor, all three receptors were expressed in baby hamster kidney (BHK) cells as soluble molecules and partially purified before characterization. The hybrid IGF-I/insulin receptor bound IGF-I with an affinity comparable to that of the wild-type IGF-I receptor. In contrast, the hybrid receptor no longer displayed high-affinity binding of insulin. These results directly demonstrate that it is possible to change the specificity of the insulin receptor to that of the IGF-I receptor and, furthermore, that the binding specificity for IGF-I is encoded within the nucleotide sequence from 135 to 938 of the IGF-I receptor cDNA. Since the hybrid receptor only bound insulin with low affinity, the insulin binding region is likely to be located within exons 2 and 3 of the insulin receptor

  4. Growth inhibition of thyroid follicular cell-derived cancers by the opioid growth factor (OGF) - opioid growth factor receptor (OGFr) axis

    McLaughlin, Patricia J; Zagon, Ian S; Park, Sunny S; Conway, Andrea; Donahue, Renee N; Goldenberg, David

    2009-01-01

    Carcinoma of the thyroid gland is an uncommon cancer, but the most frequent malignancy of the endocrine system. Most thyroid cancers are derived from the follicular cell. Follicular carcinoma (FTC) is considered more malignant than papillary thyroid carcinoma (PTC), and anaplastic thyroid cancer (ATC) is one of the most lethal human cancers. Opioid Growth Factor (OGF; chemical term - [Met 5 ]-enkephalin) and its receptor, OGFr, form an inhibitory axis regulating cell proliferation. Both the peptide and receptor have been detected in a wide variety of cancers, and OGF is currently used clinically as a biotherapy for some non-thyroid neoplasias. This study addressed the question of whether the OGF-OGFr axis is present and functional in human thyroid follicular cell - derived cancer. Utilizing human ATC (KAT-18), PTC (KTC-1), and FTC (WRO 82-1) cell lines, immunohistochemistry was employed to ascertain the presence and location of OGF and OGFr. The growth characteristics in the presence of OGF or the opioid antagonist naltrexone (NTX), and the specificity of opioid peptides for proliferation of ATC, were established in KAT-18 cells. Dependence on peptide and receptor were investigated using neutralization studies with antibodies and siRNA experiments, respectively. The mechanism of peptide action on DNA synthesis and cell survival was ascertained. The ubiquity of the OGF-OGFr axis in thyroid follicular cell-derived cancer was assessed in KTC-1 (PTC) and WRO 82-1 (FTC) tumor cells. OGF and OGFr were present in KAT-18 cells. Concentrations of 10 -6 M OGF inhibited cell replication up to 30%, whereas NTX increased cell growth up to 35% relative to cultures treated with sterile water. OGF treatment reduced cell number by as much as 38% in KAT-18 ATC in a dose-dependent and receptor-mediated manner. OGF antibodies neutralized the inhibitory effects of OGF, and siRNA knockdown of OGFr negated growth inhibition by OGF. Cell survival was not altered by OGF, but DNA synthesis

  5. Adrenergic Agonists Bind to Adrenergic-Receptor-Like Regions of the Mu Opioid Receptor, Enhancing Morphine and Methionine-Enkephalin Binding: A New Approach to “Biased Opioids”?

    Turke, Miah; Subhramanyam, Udaya K. Tiruttani; Churchill, Beth; Labahn, Joerg

    2018-01-01

    Extensive evidence demonstrates functional interactions between the adrenergic and opioid systems in a diversity of tissues and organs. While some effects are due to receptor and second messenger cross-talk, recent research has revealed an extracellular, allosteric opioid binding site on adrenergic receptors that enhances adrenergic activity and its duration. The present research addresses whether opioid receptors may have an equivalent extracellular, allosteric adrenergic binding site that has similar enhancing effects on opioid binding. Comparison of adrenergic and opioid receptor sequences revealed that these receptors share very significant regions of similarity, particularly in some of the extracellular and transmembrane regions associated with adrenergic binding in the adrenergic receptors. Five of these shared regions from the mu opioid receptor (muOPR) were synthesized as peptides and tested for binding to adrenergic, opioid and control compounds using ultraviolet spectroscopy. Adrenergic compounds bound to several of these muOPR peptides with low micromolar affinity while acetylcholine, histamine and various adrenergic antagonists did not. Similar studies were then conducted with purified, intact muOPR with similar results. Combinations of epinephrine with methionine enkephalin or morphine increased the binding of both by about half a log unit. These results suggest that muOPR may be allosterically enhanced by adrenergic agonists. PMID:29342106

  6. Adrenergic Agonists Bind to Adrenergic-Receptor-Like Regions of the Mu Opioid Receptor, Enhancing Morphine and Methionine-Enkephalin Binding: A New Approach to “Biased Opioids”?

    Robert Root-Bernstein

    2018-01-01

    Full Text Available Extensive evidence demonstrates functional interactions between the adrenergic and opioid systems in a diversity of tissues and organs. While some effects are due to receptor and second messenger cross-talk, recent research has revealed an extracellular, allosteric opioid binding site on adrenergic receptors that enhances adrenergic activity and its duration. The present research addresses whether opioid receptors may have an equivalent extracellular, allosteric adrenergic binding site that has similar enhancing effects on opioid binding. Comparison of adrenergic and opioid receptor sequences revealed that these receptors share very significant regions of similarity, particularly in some of the extracellular and transmembrane regions associated with adrenergic binding in the adrenergic receptors. Five of these shared regions from the mu opioid receptor (muOPR were synthesized as peptides and tested for binding to adrenergic, opioid and control compounds using ultraviolet spectroscopy. Adrenergic compounds bound to several of these muOPR peptides with low micromolar affinity while acetylcholine, histamine and various adrenergic antagonists did not. Similar studies were then conducted with purified, intact muOPR with similar results. Combinations of epinephrine with methionine enkephalin or morphine increased the binding of both by about half a log unit. These results suggest that muOPR may be allosterically enhanced by adrenergic agonists.

  7. The automated radiosynthesis and purification of the opioid receptor antagonist, [6-O-methyl-11C]diprenorphine on the GE TRACERlab FXFE radiochemistry module.

    Fairclough, Michael; Prenant, Christian; Brown, Gavin; McMahon, Adam; Lowe, Jonathan; Jones, Anthony

    2014-05-15

    [6-O-Methyl-(11)C]diprenorphine ([(11)C]diprenorphine) is a positron emission tomography ligand used to probe the endogenous opioid system in vivo. Diprenorphine acts as an antagonist at all of the opioid receptor subtypes, that is, μ (mu), κ (kappa) and δ (delta). The radiosynthesis of [(11)C]diprenorphine using [(11)C]methyl iodide produced via the 'wet' method on a home-built automated radiosynthesis set-up has been described previously. Here, we describe a modified synthetic method to [(11)C]diprenorphine performed using [(11)C]methyl iodide produced via the gas phase method on a GE TRACERlab FXFE radiochemistry module. Also described is the use of [(11)C]methyl triflate as the carbon-11 methylating agent for the [(11)C]diprenorphine syntheses. [(11)C]Diprenorphine was produced to good manufacturing practice standards for use in a clinical setting. In comparison to previously reported [(11)C]diprenorphine radiosyntheisis, the method described herein gives a higher specific activity product which is advantageous for receptor occupancy studies. The radiochemical purity of [(11)C]diprenorphine is similar to what has been reported previously, although the radiochemical yield produced in the method described herein is reduced, an issue that is inherent in the gas phase radiosynthesis of [(11)C]methyl iodide. The yields of [(11)C]diprenorphine are nonetheless sufficient for clinical research applications. Other advantages of the method described herein are an improvement to both reproducibility and reliability of the production as well as simplification of the purification and formulation steps. We suggest that our automated radiochemistry route to [(11)C]diprenorphine should be the method of choice for routine [(11)C]diprenorphine productions for positron emission tomography studies, and the production process could easily be transferred to other radiochemistry modules such as the TRACERlab FX C pro. Copyright © 2014 John Wiley & Sons, Ltd.

  8. Interaction of 3,8-diazabicyclo (3.2.1) octanes with mu and delta opioid receptors.

    Cignarella, G; Barlocco, D; Tranquillini, M E; Volterra, A; Brunello, N; Racagni, G

    1988-05-01

    A series of 3,8-diazabicyclo (3.2.1) octanes (DBO) (1) substituted at the nitrogen atoms by acyl and aralkenyl groups, were tested in in vitro binding assays towards mu and delta opioid receptors. The most representative terms (1a, 1d, 1g, 1j,) were also evaluated for the analgesic potency in vivo by the hot plate method. Among the compounds tested the most potent was the p.nitrocinnamyl DBO (1d) which displayed a mu/delta selectivity and an analgesic activity respectively 25 and 17 fold those of morphine. On the contrary, the m.hydroxycinnamyl DBO (1g) was markedly less active as agonist than the parent 1a, thus suggesting that structure 1 interacts with opioid receptors in a different fashion than morphine. Compound 1j isomer of 1a which is provided with high mu affinity, but lower analgesic potency, was found to possess a mixed agonist-antagonist activity.

  9. Discrete mapping of brain Mu and delta opioid receptors using selective peptides: Quantitative autoradiography, species differences and comparison with kappa receptors

    Sharif, N.A.; Hughes, J. (Addenbrookes Hospital Site, Cambridge (England))

    1989-05-01

    The opioid peptides, (3H)DAGO and (3H)DPDPE, bound to rat and guinea pig brain homogenates with a high, nanomolar affinity and to a high density of mu and delta receptors, respectively. (3H)DAGO binding to mu receptors was competitively inhibited by unlabelled opioids with the following rank order of potency: DAGO greater than morphine greater than DADLE greater than naloxone greater than etorphine much greater than U50488 much greater than DPDPE. In contrast, (3H)DPDPE binding to delta receptors was inhibited by compounds with the following rank order of potency: DPDPE greater than DADLE greater than etorphine greater than dynorphin(1-8) greater than naloxone much greater than U50488 much greater than DAGO. These profiles were consistent with specific labelling of the mu and delta opioid receptors, respectively. In vitro autoradiographic techniques coupled with computer-assisted image analyses revealed a discrete but differential anatomical localization of mu and delta receptors in the rat and guinea pig brain. In general, mu and delta receptor density in the rat exceeded that in the guinea pig brain and differed markedly from that of kappa receptors in these species. However, while mu receptors were distributed throughout the brain with hotspots in the fore-, mid- and hindbrain of the two rodents, the delta sites were relatively diffusely distributed, and were mainly concentrated in the forebrain with particularly high levels within the olfactory bulb (OB), n. accumbens and striatum. Notable regions of high density of mu receptors in the rat and guinea pig brain were the accessory olfactory bulb, striatal patches and streaks, amygdaloid nuclei, ventral hippocampal subiculum and dentate gyrus, numerous thalamic nuclei, geniculate bodies, central grey, superior and inferior colliculi, solitary and pontine nuclei and s. nigra.

  10. Local analgesic effect of tramadol is mediated by opioid receptors in late postoperative pain after plantar incision in rats.

    de Oliveira Junior, José Oswaldo; de Freitas, Milena Fernandes; Bullara de Andrade, Carolina; Chacur, Marucia; Ashmawi, Hazem Adel

    2016-01-01

    Tramadol is a drug used to treat moderate to severe pain. It is known to present a peripheral effect, but the local mechanisms underlying its actions remain unclear. The role of peripheral opioid receptors in postoperative pain is not well understood. In the present study, we examined the peripheral opioid receptors to determine the local effect of tramadol in a plantar incision pain model. Rats were subjected to plantar incision and divided into four groups on postoperative day (POD) 1: SF_SF, 0.9% NaCl injected into the right hindpaw; SF_TraI, 0.9% NaCl and tramadol injected into the right hindpaw; SF_TraC, 0.9% NaCl and tramadol injected into the contralateral hindpaw; and Nal_Tra, naloxone and tramadol injected into the ipsilateral hindpaw. To determine the animals' nociceptive threshold, mechanical hyperalgesia was measured before incision, on POD1 before treatment and at 15, 30, 45, and 60 minutes after the incision. The same procedure was repeated on the POD2. The expression levels of μ-opioid receptor (MOR) and δ-opioid receptor (DOR) were obtained through immunoblotting assays in the lumbar dorsal root ganglia (L3-L6) in naïve rats and 1, 2, 3, and 7 days after the incision. Our results showed that the plantar incision was able to cause an increase in mechanical hyperalgesia and that tramadol reversed this hyperalgesia on POD1 and POD2. Tramadol injections in the contralateral paw did not affect the animals' nociceptive threshold. Naloxone was able to antagonize the tramadol effect partially on POD1 and completely on POD2. The DOR expression increased on POD2, POD3, and POD7, whereas the MOR expression did not change. Together, our results show that tramadol promoted a local analgesic effect in the postoperative pain model that was antagonized by naloxone in POD2, alongside the increase of DOR expression.

  11. Identification of Receptor Ligands and Receptor Subtypes Using Antagonists in a Capillary Electrophoresis Single-Cell Biosensor Separation System

    Fishman, Harvey A.; Orwar, Owe; Scheller, Richard H.; Zare, Richard N.

    1995-08-01

    A capillary electrophoresis system with single-cell biosensors as a detector has been used to separate and identify ligands in complex biological samples. The power of this procedure was significantly increased by introducing antagonists that inhibited the cellular response from selected ligand-receptor interactions. The single-cell biosensor was based on the ligand-receptor binding and G-protein-mediated signal transduction pathways in PC12 and NG108-15 cell lines. Receptor activation was measured as increases in cytosolic free calcium ion concentration by using fluorescence microscopy with the intracellular calcium ion indicator fluo-3 acetoxymethyl ester. Specifically, a mixture of bradykinin (BK) and acetylcholine (ACh) was fractionated and the components were identified by inhibiting the cellular response with icatibant (HOE 140), a selective antagonist to the BK B_2 receptor subtype (B_2BK), and atropine, an antagonist to muscarinic ACh receptor subtypes. Structurally related forms of BK were also identified based on inhibiting B_2BK receptors. Applications of this technique include identification of endogenous BK in a lysate of human hepatocellular carcinoma cells (Hep G2) and screening for bioactivity of BK degradation products in human blood plasma. The data demonstrate that the use of antagonists with a single-cell biosensor separation system aids identification of separated components and receptor subtypes.

  12. Evaluation of the kappa-opioid receptor-selective tracer [{sup 11}C]GR103545 in awake rhesus macaques

    Schoultz, Bent W. [University of Oslo, Department of Chemistry, Oslo (Norway); Hjornevik, Trine; Willoch, Frode [University of Oslo, Centre for Molecular Biology and Neuroscience and Institute of Basic Medical Sciences, Oslo (Norway); Akershus University Hospital, Department of Nuclear Medicine, Loerenskog (Norway); Marton, Janos [ABX Advanced Biochemical Compounds GmbH, Radeberg (Germany); Noda, Akihiro; Murakami, Yoshihiro; Miyoshi, Sosuke; Nishimura, Shintaro [Medical and Pharmacological Research Center Foundation, Basic Research Department, Hakui City, Ishikawa (Japan); Aarstad, Erik [University College of London, Institute of Nuclear Medicine, London (United Kingdom); Drzezga, Alexander [Technische Universitaet Muenchen, Department of Nuclear Medicine, Klinikum rechts der Isar, Munich (Germany); Matsunari, Ichiro [Medical and Pharmacological Research Center Foundation, Clinical Research Department, Hakui City, Ishikawa (Japan); Henriksen, Gjermund [University of Oslo, Department of Chemistry, Oslo (Norway); Technische Universitaet Muenchen, Department of Nuclear Medicine, Klinikum rechts der Isar, Munich (Germany)

    2010-06-15

    The recent development in radiosynthesis of the {sup 11}C-carbamate function increases the potential of [{sup 11}C]GR103545, which for the last decade has been regarded as promising for imaging the kappa-opioid receptor ({kappa}-OR) with PET. In the present study, [{sup 11}C]GR103545 was evaluated in awake rhesus macaques. Separate investigations were performed to clarify the OR subtype selectivity of this compound. Regional brain uptake kinetics of [{sup 11}C]GR103545 was studied 0-120 min after injection. The binding affinity and opioid subtype selectivity of [{sup 11}C]GR103545 was determined in cells transfected with cloned human opioid receptors. In vitro binding assays demonstrated a high affinity of GR103545 for {kappa}-OR (K{sub i} = 0.02 {+-}0.01 nM) with excellent selectivity over {mu}-OR (6 x 10{sup 2}-fold) and {delta}-OR (2 x 10{sup 4}-fold). PET imaging revealed a volume of distribution (V{sub T}) pattern consistent with the known distribution of {kappa}-OR, with striatum = temporal cortex > cingulate cortex > frontal cortex > parietal cortex > thalamus > cerebellum. [{sup 11}C]GR103545 is selective for {kappa}-OR and holds promise for use to selectively depict and quantify this receptor in humans by means of PET. (orig.)

  13. Local analgesic effect of tramadol is not mediated by opioid receptors in early postoperative pain in rats

    Angela Maria Sousa

    2015-06-01

    Full Text Available BACKGROUND AND OBJECTIVES: Tramadol is known as a central acting analgesic drug, used for the treatment of moderate to severe pain. Local analgesic effect has been demonstrated, in part due to local anesthetic-like effect, but other mechanisms remain unclear. The role of peripheral opioid receptors in the local analgesic effect is not known. In this study, we examined role of peripheral opioid receptors in the local analgesic effect of tramadol in the plantar incision model. METHODS: Young male Wistar rats were divided into seven groups: control, intraplantar tramadol, intravenous tramadol, intravenous naloxone-intraplantar tramadol, intraplantar naloxone-intraplantar tramadol, intravenous naloxone-intravenous tramadol, and intravenous naloxone. After receiving the assigned drugs (tramadol 5 mg, naloxone 200 µg or 0.9% NaCl, rats were submitted to plantar incision, and withdrawal thresholds after mechanical stimuli with von Frey filaments were assessed at baseline, 10, 15, 30, 45 and 60 min after incision. RESULTS: Plantar incision led to marked mechanical hyperalgesia during the whole period of observation in the control group, no mechanical hyperalgesia were observed in intraplantar tramadol group, intraplantar naloxone-intraplantar tramadol group and intravenous naloxone-intraplantar tramadol. In the intravenous tramadol group a late increase in withdrawal thresholds (after 45 min was observed, the intravenous naloxone-intravenous tramadol group and intravenous naloxone remained hyperalgesic during the whole period. CONCLUSIONS: Tramadol presented an early local analgesic effect decreasing mechanical hyperalgesia induced by plantar incision. This analgesic effect was not mediated by peripheral opioid receptors.

  14. Evaluation of the kappa-opioid receptor-selective tracer [11C]GR103545 in awake rhesus macaques

    Schoultz, Bent W.; Hjornevik, Trine; Willoch, Frode; Marton, Janos; Noda, Akihiro; Murakami, Yoshihiro; Miyoshi, Sosuke; Nishimura, Shintaro; Aarstad, Erik; Drzezga, Alexander; Matsunari, Ichiro; Henriksen, Gjermund

    2010-01-01

    The recent development in radiosynthesis of the 11 C-carbamate function increases the potential of [ 11 C]GR103545, which for the last decade has been regarded as promising for imaging the kappa-opioid receptor (κ-OR) with PET. In the present study, [ 11 C]GR103545 was evaluated in awake rhesus macaques. Separate investigations were performed to clarify the OR subtype selectivity of this compound. Regional brain uptake kinetics of [ 11 C]GR103545 was studied 0-120 min after injection. The binding affinity and opioid subtype selectivity of [ 11 C]GR103545 was determined in cells transfected with cloned human opioid receptors. In vitro binding assays demonstrated a high affinity of GR103545 for κ-OR (K i = 0.02 ±0.01 nM) with excellent selectivity over μ-OR (6 x 10 2 -fold) and δ-OR (2 x 10 4 -fold). PET imaging revealed a volume of distribution (V T ) pattern consistent with the known distribution of κ-OR, with striatum = temporal cortex > cingulate cortex > frontal cortex > parietal cortex > thalamus > cerebellum. [ 11 C]GR103545 is selective for κ-OR and holds promise for use to selectively depict and quantify this receptor in humans by means of PET. (orig.)

  15. Nalfurafine hydrochloride, a selective κ opioid receptor agonist, has no reinforcing effect on intravenous self-administration in rhesus monkeys

    Kaoru Nakao

    2016-01-01

    Full Text Available Nalfurafine hydrochloride [(E-N-[17-(cyclopropylmethyl-4,5α-epoxy-3,14-dihydroxymorphinan-6β-yl]-3-(furan-3-yl-N-methylprop-2-enamide monohydrochloride; nalfurafine] is used in Japan as an antipruritic for the treatment of intractable pruritus in patients undergoing hemodialysis or with chronic liver disease. It is a potent and selective agonist at the κ opioid receptor, but also has weak and partial agonist activity at μ opioid receptors. Opioids, especially those acting at μ receptors, carry a risk of abuse. This is an important factor in the consideration of therapeutic risk vs. benefit in clinical use and the potential for misuse as a public health problem. It is therefore necessary to carefully evaluate the reinforcing effects of nalfurafine. To this end, we investigated intravenous self-administration of nalfurafine in rhesus monkeys. The number of self-administration of nalfurafine at doses of 0.0625, 0.125 and 0.25 μg/kg/infusion was not higher than that of saline in rhesus monkeys that frequently self-administered pentazocine (0.25 mg/kg/infusion. These results indicate that nalfurafine has no reinforcing effect in rhesus monkeys in the intravenous self-administration paradigm.

  16. Kappa opioid receptor antagonism and chronic antidepressant treatment have beneficial activities on social interactions and grooming deficits during heroin abstinence.

    Lalanne, L; Ayranci, G; Filliol, D; Gavériaux-Ruff, C; Befort, K; Kieffer, B L; Lutz, P-E

    2017-07-01

    Addiction is a chronic brain disorder that progressively invades all aspects of personal life. Accordingly, addiction to opiates severely impairs interpersonal relationships, and the resulting social isolation strongly contributes to the severity and chronicity of the disease. Uncovering new therapeutic strategies that address this aspect of addiction is therefore of great clinical relevance. We recently established a mouse model of heroin addiction in which, following chronic heroin exposure, 'abstinent' mice progressively develop a strong and long-lasting social avoidance phenotype. Here, we explored and compared the efficacy of two pharmacological interventions in this mouse model. Because clinical studies indicate some efficacy of antidepressants on emotional dysfunction associated with addiction, we first used a chronic 4-week treatment with the serotonergic antidepressant fluoxetine, as a reference. In addition, considering prodepressant effects recently associated with kappa opioid receptor signaling, we also investigated the kappa opioid receptor antagonist norbinaltorphimine (norBNI). Finally, we assessed whether fluoxetine and norBNI could reverse abstinence-induced social avoidance after it has established. Altogether, our results show that two interspaced norBNI administrations are sufficient both to prevent and to reverse social impairment in heroin abstinent animals. Therefore, kappa opioid receptor antagonism may represent a useful approach to alleviate social dysfunction in addicted individuals. © 2016 Society for the Study of Addiction.

  17. Discovery of a novel site of opioid action at the innate immune pattern-recognition receptor TLR4 and its role in addiction.

    Jacobsen, Jonathan Henry W; Watkins, Linda R; Hutchinson, Mark R

    2014-01-01

    Opioids have historically, and continue to be, an integral component of pain management. However, despite pharmacokinetic and dynamic optimization over the past 100 years, opioids continue to produce many undesirable side effects such as tolerance, reward, and dependence. As such, opioids are liable for addiction. Traditionally, opioid addiction was viewed as a solely neuronal process, and while substantial headway has been made into understanding the molecular and cellular mechanisms mediating this process, research has however, been relatively ambivalent to how the rest of the central nervous system (CNS) responds to opioids. Evidence over the past 20 years has clearly demonstrated the importance of the immunocompetent cells of the CNS (glia) in many aspects of opioid pharmacology. Particular focus has been placed on microglia and astrocytes, who in response to opioids, become activated and release inflammatory mediators. Importantly, the mechanism underlying immune activation is beginning to be elucidated. Evidence suggests an innate immune pattern-recognition receptor (toll-like receptor 4) as an integral component underlying opioid-induced glial activation. The subsequent proinflammatory response may be viewed akin to neurotransmission creating a process termed central immune signaling. Translationally, we are beginning to appreciate the importance of central immune signaling as it contributes to many behavioral actions of addiction including reward, withdrawal, and craving. As such, the aim of this chapter is to review and integrate the neuronal and central immune signaling perspective of addiction. © 2014 Elsevier Inc. All rights reserved.

  18. ALK receptor activation, ligands and therapeutic targeting in glioblastoma and in other cancers

    Wellstein, Anton

    2012-01-01

    The intracellular anaplastic lymphoma kinase (ALK) fragment shows striking homology with members of the insulin receptor family and was initially identified as an oncogenic fusion protein resulting from a translocation in lymphoma and more recently in a range of cancers. The full-length ALK transmembrane receptor of ~220 kDa was identified based on this initial work. This tyrosine kinase receptor and its ligands, the growth factors pleiotrophin (PTN) and midkine (MK) are highly expressed during development of the nervous system and other organs. Each of these genes has been implicated in malignant progression of different tumor types and shown to alter phenotypes as well as signal transduction in cultured normal and tumor cells. Beyond its role in cancer, the ALK receptor pathway is thought to contribute to nervous system development, function, and repair, as well as metabolic homeostasis and the maintenance of tissue regeneration. ALK receptor activity in cancer can be up-regulated by amplification, overexpression, ligand binding, mutations in the intracellular domain of the receptor and by activity of the receptor tyrosine phosphatase PTPRz. Here we discuss the evidence for ligand control of ALK activity as well as the potential prognostic and therapeutic implications from gene expression and functional studies. An analysis of 18 published gene expression data sets from different cancers shows that overexpression of ALK, its smaller homolog LTK (leukocyte tyrosine kinase) and the ligands PTN and MK in cancer tissues from patients correlate significantly with worse course and outcome of the disease. This observation together with preclinical functional studies suggests that this pathway could be a valid therapeutic target for which complementary targeting strategies with small molecule kinase inhibitors as well as antibodies to ligands or the receptors may be used.

  19. Interactions of ligands with active and inactive conformations of the dopamine D2 receptor.

    Malmberg, A; Mohell, N; Backlund Höök, B; Johansson, A M; Hacksell, U; Nordvall, G

    1998-04-10

    The affinities of 19 pharmacologically diverse dopamine D2 receptor ligands were determined for the active and inactive conformations of cloned human dopamine D2 receptors expressed in Ltk cells. The agonist [3H]quinpirole was used to selectively label the guanine nucleotide-binding protein-coupled, active receptor conformation. The antagonist [3H]raclopride, in the presence of the non-hydrolysable GTP-analogue Gpp(NH)p and sodium ions and in the absence of magnesium ions, was used to label the free inactive receptor conformation. The intrinsic activities of the ligands were determined in a forskolin-stimulated cyclic AMP assay using the same cells. An excellent correlation was shown between the affinity ratios (KR/KRG) of the ligands for the two receptor conformations and their intrinsic activity (r=0.96). The ligands included eight structurally related and enantiopure 2-aminotetralin derivatives; the enantiomers of 5-hydroxy-2-(dipropylamino)tetralin, 5-methoxy-2-(dipropylamino)tetralin, 5-fluoro-2-(dipropylamino)tetralin and 2-(dipropylamino)tetralin. The (S)-enantiomers behaved as full agonists in the cyclic AMP assay and displayed a large KR/KRG ratio. The (R)-enantiomers were classified as partial agonists and had lower ratios. The structure-affinity relationships of these compounds at the active and the inactive receptor conformations were analysed separately, and used in conjunction with a homology based receptor model of the dopamine D2 receptor. This led to proposed binding modes for agonists, antagonists and partial agonists in the 2-aminotetralin series. The concepts used in this study should be of value in the design of ligands with predetermined affinity and intrinsic activity.

  20. Generating "fragment-based virtual library" using pocket similarity search of ligand-receptor complexes.

    Khashan, Raed S

    2015-01-01

    As the number of available ligand-receptor complexes is increasing, researchers are becoming more dedicated to mine these complexes to aid in the drug design and development process. We present free software which is developed as a tool for performing similarity search across ligand-receptor complexes for identifying binding pockets which are similar to that of a target receptor. The search is based on 3D-geometric and chemical similarity of the atoms forming the binding pocket. For each match identified, the ligand's fragment(s) corresponding to that binding pocket are extracted, thus forming a virtual library of fragments (FragVLib) that is useful for structure-based drug design. The program provides a very useful tool to explore available databases.

  1. Mix-and-match: ligand-receptor pairs in stomatal development and beyond.

    Torii, Keiko U

    2012-12-01

    Stomata are small valves on the plant epidermis balancing gas exchange and water loss. Stomata are formed according to positional cues. In Arabidopsis, two EPIDERMAL PATTERNING FACTOR (EPF) peptides, EPF1 and EPF2, are secreted from stomatal precursors enforcing proper stomatal patterning. Here, I review recent studies revealing the ligand-receptor pairs and revising the previously predicted relations between receptors specifying stomatal patterning: ERECTA-family and TOO MANY MOUTHS (TMM). Furthermore, EPF-LIKE9 (EPFL9/Stomagen) promotes stomatal differentiation from internal tissues. Two EPFL peptides specify inflorescence architecture, a process beyond stomatal development, as ligands for ERECTA. Thus, broadly expressed receptor kinases may regulate multiple developmental processes through perceiving different peptide ligands, each with a specialized expression pattern. TMM in the epidermis may fine-tune multiple EPF/EPFL signals to prevent signal interference. Copyright © 2012 Elsevier Ltd. All rights reserved.

  2. Importance of the pharmacological profile of the bound ligand in enrichment on nuclear receptors: toward the use of experimentally validated decoy ligands.

    Lagarde, Nathalie; Zagury, Jean-François; Montes, Matthieu

    2014-10-27

    The evaluation of virtual ligand screening methods is of major importance to ensure their reliability. Taking into account the agonist/antagonist pharmacological profile should improve the quality of the benchmarking data sets since ligand binding can induce conformational changes in the nuclear receptor structure and such changes may vary according to the agonist/antagonist ligand profile. We indeed found that splitting the agonist and antagonist ligands into two separate data sets for a given nuclear receptor target significantly enhances the quality of the evaluation. The pharmacological profile of the ligand bound in the binding site of the target structure was also found to be an additional critical parameter. We also illustrate that active compound data sets for a given pharmacological activity can be used as a set of experimentally validated decoy ligands for another pharmacological activity to ensure a reliable and challenging evaluation of virtual screening methods.

  3. Structural Insights into Selective Ligand-Receptor Interactions Leading to Receptor Inactivation Utilizing Selective Melanocortin 3 Receptor Antagonists.

    Cai, Minying; Marelli, Udaya Kiran; Mertz, Blake; Beck, Johannes G; Opperer, Florian; Rechenmacher, Florian; Kessler, Horst; Hruby, Victor J

    2017-08-15

    Systematic N-methylated derivatives of the melanocortin receptor ligand, SHU9119, lead to multiple binding and functional selectivity toward melanocortin receptors. However, the relationship between N-methylation-induced conformational changes in the peptide backbone and side chains and melanocortin receptor selectivity is still unknown. We conducted comprehensive conformational studies in solution of two selective antagonists of the third isoform of the melanocortin receptor (hMC3R), namely, Ac-Nle-c[Asp-NMe-His 6 -d-Nal(2') 7 -NMe-Arg 8 -Trp 9 -Lys]-NH 2 (15) and Ac-Nle-c[Asp-His 6 -d-Nal(2') 7 -NMe-Arg 8 -NMe-Trp 9 -NMe-Lys]-NH 2 (17). It is known that the pharmacophore (His 6 -DNal 7 -Arg 8 -Trp 9 ) of the SHU-9119 peptides occupies a β II-turn-like region with the turn centered about DNal 7 -Arg 8 . The analogues with hMC3R selectivity showed distinct differences in the spatial arrangement of the Trp 9 side chains. In addition to our NMR studies, we also carried out molecular-level interaction studies of these two peptides at the homology model of hMC3R. Earlier chimeric human melanocortin 3 receptor studies revealed insights regarding the binding and functional sites of hMC3R selectivity. Upon docking of peptides 15 and 17 to the binding pocket of hMC3R, it was revealed that Arg 8 and Trp 9 side chains are involved in a majority of the interactions with the receptor. While Arg 8 forms polar contacts with D154 and D158 of hMC3R, Trp 9 utilizes π-π stacking interactions with F295 and F298, located on the transmembrane domain of hMC3R. It is hypothesized that as the frequency of Trp 9 -hMC3R interactions decrease, antagonistic activity increases. The absence of any interactions of the N-methyl groups with hMC3R suggests that their primary function is to modulate backbone conformations of the ligands.

  4. Assessment and Challenges of Ligand Docking into Comparative Models of G-Protein Coupled Receptors

    Nguyen, E.D.; Meiler, J.; Norn, C.

    2013-01-01

    screening and to design and optimize drug candidates. However, low sequence identity between receptors, conformational flexibility, and chemical diversity of ligands present an enormous challenge to molecular modeling approaches. It is our hypothesis that rapid Monte-Carlo sampling of protein backbone...... extracellular loop. Furthermore, these models are consistently correlated with low Rosetta energy score. To predict their binding modes, ligand conformers of the 14 ligands co-crystalized with the GPCRs were docked against the top ranked comparative models. In contrast to the comparative models themselves...

  5. Photoaffinity ligand for dopamine D2 receptors: azidoclebopride

    Niznik, H.B.; Guan, J.H.; Neumeyer, J.L.; Seeman, P.

    1985-01-01

    In order to label D2 dopamine receptors selectively and covalently by means of a photosensitive compound, azidoclebopride was synthesized directly from clebopride. The dissociation constant (KD) of clebopride for the D2 dopamine receptor (canine brain striatum) was 1.5 nM, while that for azidoclebopride was 21 nM. The affinities of both clebopride and azidoclebopride were markedly reduced in the absence of sodium chloride. In the presence of ultraviolet light, azidoclebopride inactivated D2 dopamine receptors irreversibly, as indicated by the inability of the receptors to bind [ 3 H]spiperone. Maximal photoinactivation of about 60% of the D2 dopamine receptors occurred at 1 microM azidoclebopride; 30% of the receptors were inactivated at 80 nM azidoclebopride (pseudo-IC50). Dopamine agonists selectively protected the D2 receptors from being inactivated by azidoclebopride, the order of potency being (-)-N-n-propylnorapomorphine greater than apomorphine greater than (+/-)-6,7-dihydroxy-2-aminotetralin greater than (+)-N-n-propylnorapomorphine greater than dopamine greater than noradrenaline greater than serotonin. Similarly, dopaminergic antagonists prevented the photoinactivation of D2 receptors by azidoclebopride with the following order of potency: spiperone greater than (+)-butaclamol greater than haloperidol greater than clebopride greater than (-)-sulpiride greater than (-)-butaclamol

  6. Aryl- and heteroaryl-substituted phenylalanines as AMPA receptor ligands

    Szymańska, Ewa; Chałupnik, Paulina; Johansen, Tommy Nørskov

    2017-01-01

    in radioligand binding assays at native rat ionotropic glutamate receptors. The most interesting compound in this series, (RS)-2-amino-3-(3'-hydroxy-5-(1H-pyrazol-4-yl)-[1,1'-biphenyl]-3-yl)propanoic acid 7e, showed the binding affinity of 4.6 µM for native AMPA receptors and almost 5-fold lower affinity...

  7. Fluoxetine reverses the behavioral despair induced by neurogenic stress in mice: role of N-methyl-d-aspartate and opioid receptors.

    Haj-Mirzaian, Arya; Kordjazy, Nastaran; Ostadhadi, Sattar; Amiri, Shayan; Haj-Mirzaian, Arvin; Dehpour, AhmadReza

    2016-06-01

    Opioid and N-methyl-d-aspartate (NMDA) receptors mediate different effects of fluoxetine. We investigated whether opioid and NMDA receptors are involved in the protective effect of fluoxetine against the behavioral despair induced by acute physical stress in male mice. We used the forced swimming test (FST), tail suspension test (TST), and open-field test (OFT) for behavioral evaluation. We used fluoxetine, naltrexone (opioid receptor antagonist), MK-801 (NMDA receptor antagonist), morphine (opioid receptor agonist), and NMDA (NMDA receptor agonist). Acute foot-shock stress (FSS) significantly induced behavioral despair (depressive-like) and anxiety-like behaviors in tests. Fluoxetine (5 mg/kg) reversed the depressant-like effect of FSS, but it did not alter the locomotion and anxiety-like behavior in animals. Acute administration of subeffective doses of naltrexone (0.3 mg/kg) or MK-801 (0.01 mg/kg) potentiated the antidepressant-like effect of fluoxetine, while subeffective doses of morphine (1 mg/kg) and NMDA (75 mg/kg) abolished this effect of fluoxetine. Also, co-administration of subeffective doses of naltrexone (0.05 mg/kg) and MK-801 (0.003 mg/kg) with fluoxetine (1 mg/kg) induced a significant decrease in the immobility time in FST and TST. Our results showed that opioid and NMDA receptors (alone or in combination) are involved in the antidepressant-like effect of fluoxetine against physical stress.

  8. Genomic organization, annotation, and ligand-receptor inferences of chicken chemokines and chemokine receptor genes based on comparative genomics

    Sze Sing-Hoi

    2005-03-01

    Full Text Available Abstract Background Chemokines and their receptors play important roles in host defense, organogenesis, hematopoiesis, and neuronal communication. Forty-two chemokines and 19 cognate receptors have been found in the human genome. Prior to this report, only 11 chicken chemokines and 7 receptors had been reported. The objectives of this study were to systematically identify chicken chemokines and their cognate receptor genes in the chicken genome and to annotate these genes and ligand-receptor binding by a comparative genomics approach. Results Twenty-three chemokine and 14 chemokine receptor genes were identified in the chicken genome. All of the chicken chemokines contained a conserved CC, CXC, CX3C, or XC motif, whereas all the chemokine receptors had seven conserved transmembrane helices, four extracellular domains with a conserved cysteine, and a conserved DRYLAIV sequence in the second intracellular domain. The number of coding exons in these genes and the syntenies are highly conserved between human, mouse, and chicken although the amino acid sequence homologies are generally low between mammalian and chicken chemokines. Chicken genes were named with the systematic nomenclature used in humans and mice based on phylogeny, synteny, and sequence homology. Conclusion The independent nomenclature of chicken chemokines and chemokine receptors suggests that the chicken may have ligand-receptor pairings similar to mammals. All identified chicken chemokines and their cognate receptors were identified in the chicken genome except CCR9, whose ligand was not identified in this study. The organization of these genes suggests that there were a substantial number of these genes present before divergence between aves and mammals and more gene duplications of CC, CXC, CCR, and CXCR subfamilies in mammals than in aves after the divergence.

  9. β-Endorphin via the Delta Opioid Receptor is a Major Factor in the Incubation of Cocaine Craving

    Dikshtein, Yahav; Barnea, Royi; Kronfeld, Noam; Lax, Elad; Roth-Deri, Ilana; Friedman, Alexander; Gispan, Iris; Elharrar, Einat; Levy, Sarit; Ben-Tzion, Moshe; Yadid, Gal

    2013-01-01

    Cue-induced cocaine craving intensifies, or ‘incubates', during the first few weeks of abstinence and persists over extended periods of time. One important factor implicated in cocaine addiction is the endogenous opioid β-endorphin. In the present study, we examined the possible involvement of β-endorphin in the incubation of cocaine craving. Rats were trained to self-administer cocaine (0.75 mg/kg, 10 days, 6 h/day), followed by either a 1-day or a 30-day period of forced abstinence. Subsequent testing for cue-induced cocaine-seeking behavior (without cocaine reinforcement) was performed. Rats exposed to the drug-associated cue on day 1 of forced abstinence demonstrated minimal cue-induced cocaine-seeking behavior concurrently with a significant increase in β-endorphin release in the nucleus accumbens (NAc). Conversely, exposure to the cue on day 30 increased cocaine seeking, while β-endorphin levels remained unchanged. Intra-NAc infusion of an anti-β-endorphin antibody (4 μg) on day 1 increased cue-induced cocaine seeking, whereas infusion of a synthetic β-endorphin peptide (100 ng) on day 30 significantly decreased cue response. Both intra-NAc infusions of the δ opioid receptor antagonist naltrindole (1 μg) on day 1 and naltrindole together with β-endorphin on day 30 increased cue-induced cocaine-seeking behavior. Intra-NAc infusion of the μ opioid receptor antagonist CTAP (30 ng and 3 μg) had no behavioral effect. Altogether, these results demonstrate a novel role for β-endorphin and the δ opioid receptor in the development of the incubation of cocaine craving. PMID:23800967

  10. Regional differences in mu and kappa opioid receptor G-protein activation in brain in male and female prairie voles.

    Martin, T J; Sexton, T; Kim, S A; Severino, A L; Peters, C M; Young, L J; Childers, S R

    2015-12-17

    Prairie voles are unusual mammals in that, like humans, they are capable of forming socially monogamous pair bonds, display biparental care, and engage in alloparental behaviors. Both mu and kappa opioid receptors are involved in behaviors that either establish and maintain, or result from pair bond formation in these animals. Mu and kappa opioid receptors both utilize inhibitory G-proteins in signal transduction mechanisms, however the efficacy by which these receptor subtypes stimulate G-protein signaling across the prairie vole neuraxis is not known. Utilizing [(35)S]GTPγS autoradiography, we characterized the efficacy of G-protein stimulation in coronal sections throughout male and female prairie vole brains by [D-Ala2,NMe-Phe4,Gly-ol5]-enkephalin (DAMGO) and U50,488H, selective mu and kappa opioid agonists, respectively. DAMGO stimulation was highest in the forebrain, similar to that found with other rodent species. U-50,488H produced greater stimulation in prairie voles than is typically seen in mice and rats, particularly in select forebrain areas. DAMGO produced higher stimulation in the core versus the shell of the nucleus accumbens (NAc) in females, while the distribution of U-50,488H stimulation was the opposite. There were no gender differences for U50,488H stimulation of G-protein activity across the regions examined, while DAMGO stimulation was greater in sections from females compared to those from males for NAc core, entopeduncular nucleus, and hippocampus. These data suggest that the kappa opioid system may be more sensitive to manipulation in prairie voles compared to mice and rats, and that female prairie voles may be more sensitive to mu agonists in select brain regions than males. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

  11. Analgesic Effects of Diluted Bee Venom Acupuncture Mediated by δ-Opioid and α2-Adrenergic Receptors in Osteoarthritic Rats.

    Huh, Jeong-Eun; Seo, Byung-Kwan; Lee, Jung-Woo; Kim, Chanyoung; Park, Yeon-Cheol; Lee, Jae-Dong; Baek, Yong-Hyeon

    2017-06-23

    Context • Pain from osteoarthritis is associated with peripheral nociception and central pain processing. Given the unmet need for innovative, effective, and well-tolerated therapies, many patients, after looking for more satisfactory alternatives, decide to use complementary and alternative modalities. The analgesic mechanism of subcutaneous injections of diluted bee venom into an acupoint is thought to be part of an anti-inflammatory effect and the central modulation of pain processing. Objectives • Using the rat model of collagenase-induced osteoarthritis (CIOA), the study intended to investigate the analgesic effects of bee venom acupuncture (BVA) as they are related to the acupuncture points and dosage used and to determine whether the analgesic mechanisms of BVA for pain were mediated by opioid or adrenergic receptors. Design • Male Sprague-Dawley rats were randomly assigned to one of 19 groups, with n = 10 for each group. Setting • The study was conducted at the East-West Bone and Joint Research Institute at Kyung Hee University (Seoul, South Korea). Intervention • All rats were intra-articularly injected with collagenase solution in the left knee, followed by a booster injection performed 4 d after the first injection. For the groups receiving BVA treatments, the treatment was administered into the ST-36 acupoint, except for 1 group that received the treatment into a nonacupoint. Three BVA intervention groups received no pretreatment with agonists or antagonists; 1 of them received a dose of 1 mg/kg of bee venom into acupoint ST-36, 1 received a dose of 2 mg/kg into acupoint ST-36, and 1 received a dose of 1 mg/kg into a nonacupoint location. For the intervention groups receiving pretreatments, the opioid-receptor or adrenergic-receptor agonists or antagonists were injected 20 min before the 1-mg/kg BVA treatments. Outcome Measures • Changes in the rats' pain thresholds were assessed by evaluation of pain-related behavior, using a tail flick

  12. Involvement of Opioid System, TRPM8, and ASIC Receptors in Antinociceptive Effect of Arrabidaea brachypoda (DC Bureau

    Vinícius Peixoto Rodrigues

    2017-11-01

    Full Text Available Arrabidaea brachypoda (DC Bureau is a medicinal plant found in Brazil. Known as “cipó-una”, it is popularly used as a natural therapeutic agent against pain and inflammation. This study evaluated the chemical composition and antinociceptive activity of the dichloromethane fraction from the roots of A. brachypoda (DEAB and its mechanism of action. The chemical composition was characterized by high-performance liquid chromatography, and this fraction is composed only of dimeric flavonoids. The antinociceptive effect was evaluated in formalin and hot plate tests after oral administration (10–100 mg/kg in male Swiss mice. We also investigated the involvement of TRPV1 (transient receptor potential vanilloid 1, TRPA1 (transient receptor potential ankyrin 1, TRPM8 (transient receptor potential melastatin 8, and ASIC (acid-sensing ion channel, as well as the opioidergic, glutamatergic, and supraspinal pathways. Moreover, the nociceptive response was reduced (30 mg/kg in the early and late phase of the formalin test. DEAB activity appears to involve the opioid system, TRPM8, and ASIC receptors, clearly showing that the DEAB alleviates acute pain in mice and suggesting the involvement of the TRPM8 and ASIC receptors and the opioid system in acute pain relief.

  13. Involvement of Opioid System, TRPM8, and ASIC Receptors in Antinociceptive Effect of Arrabidaea brachypoda (DC) Bureau.

    Rodrigues, Vinícius Peixoto; Rocha, Cláudia Quintino da; Périco, Larissa Lucena; Santos, Raquel de Cássia Dos; Ohara, Rie; Nishijima, Catarine Massucato; Ferreira Queiroz, Emerson; Wolfender, Jean-Luc; Rocha, Lúcia Regina Machado da; Santos, Adair Roberto Soares; Vilegas, Wagner; Hiruma-Lima, Clélia Akiko

    2017-11-02

    Arrabidaea brachypoda (DC) Bureau is a medicinal plant found in Brazil. Known as "cipó-una", it is popularly used as a natural therapeutic agent against pain and inflammation. This study evaluated the chemical composition and antinociceptive activity of the dichloromethane fraction from the roots of A. brachypoda (DEAB) and its mechanism of action. The chemical composition was characterized by high-performance liquid chromatography, and this fraction is composed only of dimeric flavonoids. The antinociceptive effect was evaluated in formalin and hot plate tests after oral administration (10-100 mg/kg) in male Swiss mice. We also investigated the involvement of TRPV1 (transient receptor potential vanilloid 1), TRPA1 (transient receptor potential ankyrin 1), TRPM8 (transient receptor potential melastatin 8), and ASIC (acid-sensing ion channel), as well as the opioidergic, glutamatergic, and supraspinal pathways. Moreover, the nociceptive response was reduced (30 mg/kg) in the early and late phase of the formalin test. DEAB activity appears to involve the opioid system, TRPM8, and ASIC receptors, clearly showing that the DEAB alleviates acute pain in mice and suggesting the involvement of the TRPM8 and ASIC receptors and the opioid system in acute pain relief.

  14. Characterization of the structure of the erythropoietin receptor by ligand blotting

    Atkins, H.L.; Broudy, V.C.; Papayannopoulou, T.

    1991-01-01

    Erythropoietin (Epo) regulates the growth and differentiation of erythroid cells by binding to a specific receptor. We characterized the native Epo receptor on erythroleukemia cell lines by ligand blotting. Solubilized cell membrane proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred onto nitrocellulose, and probed with 125I-Epo. Specificity was demonstrated by inhibition of 125I-Epo binding by unlabeled excess Epo but not other peptide growth factors and by the cellular distribution of the Epo binding protein. A single membrane protein of 61 Kd ± 4 Kd was sufficient to bind 125I Epo in both human (OCIM2, K562) and murine (GM979, Rauscher, DA-1) cell lines. This finding is consistent with the predicted size of the Epo receptor from the murine cDNA clone. However, chemical crosslinking of 125I-Epo to its receptor has identified two Epo binding proteins of 105 Kd and 85 Kd. This difference may occur because the receptor is size fractionated before Epo binding in the ligand blot, but after Epo binding in crosslinking studies. Ligand blotting demonstrates that the native Epo receptor is composed of a single 61-Kd Epo binding protein, and suggests the presence of additional proteins of 20 to 25 Kd that associate with the receptor after Epo binding

  15. Differential activation of G-proteins by μ-opioid receptor agonists

    Saidak, Zuzana; Blake-Palmer, Katherine; Hay, Debbie L; Northup, John K; Glass, Michelle

    2006-01-01

    We investigated the ability of the activated μ-opioid receptor (MOR) to differentiate between myristoylated Gαi1 and GαoA type Gα proteins, and the maximal activity of a range of synthetic and endogenous agonists to activate each Gα protein. Membranes from HEK293 cells stably expressing transfected MOR were chaotrope extracted to denature endogenous G-proteins and reconstituted with specific purified G-proteins. The Gα subunits were generated in bacteria and were demonstrated to be recognised equivalently to bovine brain purified Gα protein by CB1 cannabinoid receptors. The ability of agonists to catalyse the MOR-dependent GDP/[35S]GTPγS exchange was then compared for Gαi1 and GαoA. Activation of MOR by DAMGO produced a high-affinity saturable interaction for GαoA (Km=20±1 nM) but a low-affinity interaction with Gαi1 (Km=116±12 nM). DAMGO, met-enkephalin and leucine-enkephalin displayed maximal Gα activation among the agonists evaluated. Endomorphins 1 and 2, methadone and β-endorphin activated both Gα to more than 75% of the maximal response, whereas fentanyl partially activated both G-proteins. Buprenorphine and morphine demonstrated a statistically significant difference between the maximal activities between Gαi1 and GαoA. Interestingly, DAMGO, morphine, endomorphins 1 and 2, displayed significant differences in the potencies for the activation of the two Gα. Differences in maximal activity and potency, for Gαi1 versus GαoA, are both indicative of agonist selective activation of G-proteins in response to MOR activation. These findings may provide a starting point for the design of drugs that demonstrate greater selectivity between these two G-proteins and therefore produce a more limited range of effects. PMID:16415903

  16. Oxytocin and Opioid Receptor Gene Polymorphisms Associated with Greeting Behavior in Dogs

    Enikő Kubinyi

    2017-09-01

    Full Text Available Meeting humans is an everyday experience for most companion dogs, and their behavior in these situations and its genetic background is of major interest. Previous research in our laboratory reported that in German shepherd dogs the lack of G allele, and in Border collies the lack of A allele, of the oxytocin receptor gene (OXTR 19208A/G single nucleotide polymorphism (SNP was linked to increased friendliness, which suggests that although broad traits are affected by genetic variability, the specific links between alleles and behavioral variables might be breed-specific. In the current study, we found that Siberian huskies with the A allele approached a friendly unfamiliar woman less frequently in a greeting test, which indicates that certain polymorphisms are related to human directed behavior, but that the relationship patterns between polymorphisms and behavioral phenotypes differ between populations. This finding was further supported by our next investigation. According to primate studies, endogenous opioid peptide (e.g., endorphins receptor genes have also been implicated in social relationships. Therefore, we examined the rs21912990 of the OPRM1 gene. Firstly, we found that the allele frequencies of Siberian huskies and gray wolves were similar, but differed from that of Border collies and German shepherd dogs, which might reflect their genetic relationship. Secondly, we detected significant associations between the OPRM1 SNP and greeting behavior among German shepherd dogs and a trend in Border collies, but we could not detect an association in Siberian huskies. Although our results with OXTR and OPRM1 gene variants should be regarded as preliminary due to the relatively low sample size, they suggest that (1 OXTR and OPRM1 gene variants in dogs affect human-directed social behavior and (2 their effects differ between breeds.

  17. Oxytocin and Opioid Receptor Gene Polymorphisms Associated with Greeting Behavior in Dogs.

    Kubinyi, Enikő; Bence, Melinda; Koller, Dora; Wan, Michele; Pergel, Eniko; Ronai, Zsolt; Sasvari-Szekely, Maria; Miklósi, Ádám

    2017-01-01

    Meeting humans is an everyday experience for most companion dogs, and their behavior in these situations and its genetic background is of major interest. Previous research in our laboratory reported that in German shepherd dogs the lack of G allele, and in Border collies the lack of A allele, of the oxytocin receptor gene (OXTR) 19208A/G single nucleotide polymorphism (SNP) was linked to increased friendliness, which suggests that although broad traits are affected by genetic variability, the specific links between alleles and behavioral variables might be breed-specific. In the current study, we found that Siberian huskies with the A allele approached a friendly unfamiliar woman less frequently in a greeting test, which indicates that certain polymorphisms are related to human directed behavior, but that the relationship patterns between polymorphisms and behavioral phenotypes differ between populations. This finding was further supported by our next investigation. According to primate studies, endogenous opioid peptide (e.g., endorphins) receptor genes have also been implicated in social relationships. Therefore, we examined the rs21912990 of the OPRM1 gene. Firstly, we found that the allele frequencies of Siberian huskies and gray wolves were similar, but differed from that of Border collies and German shepherd dogs, which might reflect their genetic relationship. Secondly, we detected significant associations between the OPRM1 SNP and greeting behavior among German shepherd dogs and a trend in Border collies, but we could not detect an association in Siberian huskies. Although our results with OXTR and OPRM1 gene variants should be regarded as preliminary due to the relatively low sample size, they suggest that (1) OXTR and OPRM1 gene variants in dogs affect human-directed social behavior and (2) their effects differ between breeds.

  18. Regulation of ventilation and oxygen consumption by delta- and mu-opioid receptor agonists.

    Schaeffer, J I; Haddad, G G

    1985-09-01

    To study the effect of endorphins on metabolic rate and on the relationship between O2 consumption (VO2) and ventilation, we administered enkephalin analogues (relatively selective delta-receptor agonists) and a morphiceptin analogue (a highly selective mu-receptor agonist) intracisternally in nine unanesthetized chronically instrumented adult dogs. Both delta- and mu-agonists decreased VO2 by 40-60%. delta-Agonists induced a dose-dependent decrease in mean instantaneous minute ventilation (VT/TT) associated with periodic breathing. The decrease in VT/TT started and resolved prior to the decrease and returned to baseline of VO2, respectively. In contrast, the mu-agonists induced an increase in VT/TT associated with rapid shallow breathing. Arterial PCO2 increased and arterial PO2 decreased after both delta- and mu-agonists. Low doses of intracisternal naloxone (0.002-2.0 micrograms/kg) reversed the opioid effect on VT/TT but not on VO2; higher doses of naloxone (5-25 micrograms/kg) reversed both. Naloxone administered alone had no effect on VT/TT or VO2. These data suggest that 1) both delta- and mu-agonists induce alveolar hypoventilation despite a decrease in VO2, 2) this hypoventilation results from a decrease in VT/TT after delta-agonists but an increase in dead space ventilation after mu-agonists, and 3) endorphins do not modulate ventilation and metabolic rate tonically, but we speculate that they may do so in response to stressful stimulation.

  19. Nanoparticle-based receptors mimic protein-ligand recognition

    Riccardi, Laura; Gabrielli, Luca; Sun, Xiaohuan; Biasi, Federico De; Rastrelli, Federico; Mancin, Fabrizio; De Vivo, Marco

    2017-01-01

    Summary The self-assembly of a monolayer of ligands on the surface of noble-metal nanoparticles dictates the fundamental nanoparticle's behavior and its functionality. In this combined computational-experimental study, we analyze the structure, organization, and dynamics of functionalized coating thiols in monolayer-protected gold nanoparticles (AuNPs). We explain how functionalized coating thiols self-organize through a delicate and somehow counterintuitive balance of interactions within the...

  20. Heart Failure Therapeutics on the Basis of a Biased Ligand of the Angiotensin-2 Type 1 Receptor Rationale and Design of the BLAST-AHF Study (Biased Ligand of the Angiotensin Receptor Study in Acute Heart Failure)

    Felker, G. Michael; Butler, Javed; Collins, Sean P.; Cotter, Gad; Davison, Beth A.; Ezekowitz, Justin A.; Filippatos, Gerasimos; Levy, Phillip D.; Metra, Marco; Ponikowski, Piotr; Soergel, David G.; Teerlink, John R.; Violin, Jonathan D.; Voors, Adriaan A.; Pang, Peter S.

    The BLAST-AHF (Biased Ligand of the Angiotensin Receptor Study in Acute Heart Failure) study is designed to test the efficacy and safety of TRV027, a novel biased ligand of the angiotensin-2 type 1 receptor, in patients with acute heart failure (AHF). AHF remains a major public health problem, and

  1. Divergent Label-free Cell Phenotypic Pharmacology of Ligands at the Overexpressed β2-Adrenergic Receptors

    Ferrie, Ann M.; Sun, Haiyan; Zaytseva, Natalya; Fang, Ye

    2014-01-01

    We present subclone sensitive cell phenotypic pharmacology of ligands at the β2-adrenergic receptor (β2-AR) stably expressed in HEK-293 cells. The parental cell line was transfected with green fluorescent protein (GFP)-tagged β2-AR. Four stable subclones were established and used to profile a library of sixty-nine AR ligands. Dynamic mass redistribution (DMR) profiling resulted in a pharmacological activity map suggesting that HEK293 endogenously expresses functional Gi-coupled α2-AR and Gs-coupled β2-AR, and the label-free cell phenotypic activity of AR ligands are subclone dependent. Pathway deconvolution revealed that the DMR of epinephrine is originated mostly from the remodeling of actin microfilaments and adhesion complexes, to less extent from the microtubule networks and receptor trafficking, and certain agonists displayed different efficacy towards the cAMP-Epac pathway. We demonstrate that receptor signaling and ligand pharmacology is sensitive to the receptor expression level, and the organization of the receptor and its signaling circuitry.

  2. Dopamine D4 receptor counteracts morphine-induced changes in M opioid receptor signaling in the striosomes of the rat caudate putamen.

    Rivera, Alicia; Valderrama-Carvajal, Alejandra; Roales-Buján, Ruth; Suárez-Boomgaard, Diana; Medina-Luque, José; Shumilov, Kirill; De-la-Calle-Martin, Adelaida

    2015-01-01

    Morphine is one of the most potent analgesic drugs used to relieve moderate to severe pain. After long-term use of morphine, neuroadaptive changes in the brain promotes tolerance, which result in a reduced sensitivity to most of its effects with attenuation of analgesic efficacy, and dependence, revealed by drug craving and physical or psychological manifestations of drug withdrawal. The mu opioid receptor (MOR) is critical, not only in mediating morphine analgesia, but also in addictive beha...

  3. Role of neurotensin and opioid receptors in the cardiorespiratory effects of [Ile⁹]PK20, a novel antinociceptive chimeric peptide.

    Kaczyńska, Katarzyna; Szereda-Przestaszewska, Małgorzata; Kleczkowska, Patrycja; Lipkowski, Andrzej W

    2014-10-15

    Ile(9)PK20 is a novel hybrid of opioid-neurotensin peptides synthesized from the C-terminal hexapeptide of neurotensin and endomorphin-2 pharmacophore. This chimeric compound shows potent central and peripheral antinociceptive activity in experimental animals, however nothing is known about its influence on the respiratory and cardiovascular parameters. The present study was designed to determine the cardiorespiratory effects exerted by an intravenous injection (i.v.) of [Ile(9)]PK20. Share of the vagal afferentation and the contribution of NTS1 neurotensin and opioid receptors were tested. Intravenous injection of the hybrid at a dose of 100 μg/kg in the intact, anaesthetized rats provoked an increase in tidal volume preceded by a prompt short-lived decrease. Immediately after the end of injection brief acceleration of the respiratory rhythm appeared, and was ensued by the slowing down of breathing. Changes in respiration were concomitant with a bi-phasic response of the blood pressure: an immediate increase was followed by a sustained hypotension. Midcervical vagotomy eliminated the increase in tidal volume and respiratory rate responses. Antagonist of opioid receptors - naloxone hydrochloride eliminated only [Ile(9)]PK20-evoked decline in tidal volume response. Blockade of NTS1 receptors with an intravenous dose of SR 142,948, lessened the remaining cardiorespiratory effects. This study depicts that [Ile(9)]PK20 acting through neurotensin NTS1 receptors augments the tidal component of the breathing pattern and activates respiratory timing response through the vagal pathway. Blood pressure effects occur outside vagal afferentation and might result from activation of the central and peripheral vascular NTS1 receptors. In summary the respiratory effects of the hybrid appeared not to be profound, but they were accompanied with unfavourable prolonged hypotension. Copyright © 2014 Elsevier B.V. All rights reserved.

  4. GluVII:06--a highly conserved and selective anchor point for non-peptide ligands in chemokine receptors

    Rosenkilde, Mette M; Schwartz, Thue W

    2006-01-01

    ligands and that the two peripheral chemical moieties of the ligands from this central point in the receptor structure explore each of the two halves of the main ligand binding pocket. It is envisioned that knowledge of this binding mode can be exploited in structure-based discovery and design of novel...

  5. Ionotropic excitatory amino acid receptor ligands. Synthesis and pharmacology of a new amino acid AMPA antagonist

    Madsen, U; Sløk, F A; Stensbøl, T B

    2000-01-01

    We have previously described the potent and selective (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptor agonist, (RS)-2-amino-3-(3-carboxy-5-methyl-4-isoxazolyl)propionic acid (ACPA), and the AMPA receptor antagonist (RS)-2-amino-3-[3-(carboxymethoxy)-5-methyl-4...... excitatory amino acid (EAA) receptors using receptor binding and electrophysiological techniques, and for activity at metabotropic EAA receptors using second messenger assays. Compounds 1 and 4 were essentially inactive. (RS)-2-Amino-3-[3-(2-carboxyethyl)-5-methyl-4-isoxazolyl]propionic acid (ACMP, 2......-isoxazolyl]propionic acid (AMOA). Using these AMPA receptor ligands as leads, a series of compounds have been developed as tools for further elucidation of the structural requirements for activation and blockade of AMPA receptors. The synthesized compounds have been tested for activity at ionotropic...

  6. Radioreceptor opioid assay

    Miller, R.J.; Chang, K.-J.

    1981-01-01

    A radioreceptor assay is described for assaying opioid drugs in biological fluids. The method enables the assay of total opioid activity, being specific for opioids as a class but lacking specificity within the class. A radio-iodinated opioid and the liquid test sample are incubated with an opiate receptor material. The percentage inhibition of the binding of the radio-iodinated compound to the opiate receptor is calculated and the opioid activity of the test liquid determined from a standard curve. Examples of preparing radio-iodinated opioids and assaying opioid activity are given. A test kit for the assay is described. Compared to other methods, this assay is cheap, easy and rapid. (U.K.)

  7. Characterization of ligand binding to melanocortin 4 receptors using fluorescent peptides with improved kinetic properties.

    Link, Reet; Veiksina, Santa; Rinken, Ago; Kopanchuk, Sergei

    2017-03-15

    Melanocortin 4 (MC 4 ) receptors are important drug targets as they regulate energy homeostasis, eating behaviour and sexual functions. The ligand binding process to these G protein-coupled receptors is subject to considerable complexity. Different steps in the complex dynamic regulation can be characterized by ligand binding kinetics. Optimization of these kinetic parameters in terms of on-rate and residence time can increase the rapid onset of drug action and reduce off-target effects. Fluorescence anisotropy (FA) is one of the homogeneous fluorescence-based assays that enable continuous online monitoring of ligand binding kinetics. FA has been implemented for the kinetic study of melanocortin MC 4 receptors expressed on budded baculoviruses. However, the slow dissociation of the fluorescently labelled peptide NDP-α-MSH does not enable reaching equilibrium nor enable more in-depth study of the binding mechanisms. To overcome this problem, two novel red-shifted fluorescent ligands were designed. These cyclized heptapeptide derivatives (UTBC101 and UTBC102) exhibited nanomolar affinity toward melanocortin MC 4 receptors but had relatively different kinetic properties. The dissociation half-lives of UTBC101 (τ 1/2 =160min) and UTBC102 (τ 1/2 =7min) were shorter compared to that what was previously reported for Cy3B-NDP-α-MSH (τ 1/2 =224min). The significantly shorter dissociation half-life of UTBC102 enables equilibrium in screening assays, whereas the higher affinity of UTBC101 helps to resolve a wider range of competitor potencies. These two ligands are suitable for further kinetic screening of novel melanocortin MC 4 receptor specific ligands and could complement each other in these studies. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Association between Opioid Receptor mu 1 (OPRM1 Gene Polymorphisms and Tobacco and Alcohol Consumption in a Spanish Population

    Francesc Francès

    2015-04-01

    Full Text Available Evidence gained from animals and humans suggests that the encephalic opioid system might be involved in the development of drug addiction through its role in reward. Our aim is to assess the influence of genetic variations in the opioid receptor mu 1 on alcohol and tobacco consumption in a Spanish population. 763 unrelated individuals (465 women, 298 men aged 18-85 years were recruited between October 2011 and April 2012. Participants were requested to answer a 35-item questionnaire on tobacco and alcohol consumption, as well as to complete the AUDIT and Fagerström tests. Individuals were genotyped for three polymorphisms in the opioid receptor mu 1 (OPRM1 gene, using a TaqMan® protocol. In males, the rs10485057 polymorphism was associated with total pure ethanol intake and with the risk of being an alcohol consumer. Also, this polymorphism was significantly associated with higher Fagerström scores. Rs1799971 had a different influence on adaptive and maladaptive patterns of alcohol use. Despite the limited sample size, our study might enrich current knowledge on patterns of alcohol use, because it encompasses both extreme and adaptive phenotypes, providing thus a wider perspective on this subject.

  9. Preliminary Molecular Dynamic Simulations of the Estrogen Receptor Alpha Ligand Binding Domain from Antagonist to Apo

    Adrian E. Roitberg

    2008-06-01

    Full Text Available Estrogen receptors (ER are known as nuclear receptors. They exist in the cytoplasm of human cells and serves as a DNA binding transcription factor that regulates gene expression. However the estrogen receptor also has additional functions independent of DNA binding. The human estrogen receptor comes in two forms, alpha and beta. This work focuses on the alpha form of the estrogen receptor. The ERα is found in breast cancer cells, ovarian stroma cells, endometrium, and the hypothalamus. It has been suggested that exposure to DDE, a metabolite of DDT, and other pesticides causes conformational changes in the estrogen receptor. Before examining these factors, this work examines the protein unfolding from the antagonist form found in the 3ERT PDB crystal structure. The 3ERT PDB crystal structure has the estrogen receptor bound to the cancer drug 4-hydroxytamoxifen. The 4-hydroxytamoxifen ligand was extracted before the simulation, resulting in new conformational freedom due to absence of van der Waals contacts between the ligand and the receptor. The conformational changes that result expose the binding clef of the co peptide beside Helix 12 of the receptor forming an apo conformation. Two key conformations in the loops at either end of the H12 are produced resulting in the antagonist to apo conformation transformation. The results were produced over a 42ns Molecular Dynamics simulation using the AMBER FF99SB force field.

  10. Differential TAM receptor-ligand-phospholipid interactions delimit differential TAM bioactivities.

    Lew, Erin D; Oh, Jennifer; Burrola, Patrick G; Lax, Irit; Zagórska, Anna; Través, Paqui G; Schlessinger, Joseph; Lemke, Greg

    2014-09-29

    The TAM receptor tyrosine kinases Tyro3, Axl, and Mer regulate key features of cellular physiology, yet the differential activities of the TAM ligands Gas6 and Protein S are poorly understood. We have used biochemical and genetic analyses to delineate the rules for TAM receptor-ligand engagement and find that the TAMs segregate into two groups based on ligand specificity, regulation by phosphatidylserine, and function. Tyro3 and Mer are activated by both ligands but only Gas6 activates Axl. Optimal TAM signaling requires coincident TAM ligand engagement of both its receptor and the phospholipid phosphatidylserine (PtdSer): Gas6 lacking its PtdSer-binding 'Gla domain' is significantly weakened as a Tyro3/Mer agonist and is inert as an Axl agonist, even though it binds to Axl with wild-type affinity. In two settings of TAM-dependent homeostatic phagocytosis, Mer plays a predominant role while Axl is dispensable, and activation of Mer by Protein S is sufficient to drive phagocytosis.

  11. Nuclear receptor ligand-binding domains: reduction of helix H12 dynamics to favour crystallization

    Nahoum, Virginie; Lipski, Alexandra; Quillard, Fabien; Guichou, Jean-François [INSERM, U554, 34090 Montpellier (France); Université de Montpellier, CNRS, UMR5048, Centre de Biochimie Structurale (CBS), 34090 Montpellier (France); Boublik, Yvan [CNRS, UMR5237, Centre de Recherche de Biochimie Macromoléculaire (CRBM), 34293 Montpellier (France); Pérez, Efrèn [Universidade de Vigo, Departamento de Quimica Organica, Facultad de Química, 36310 Vigo (Spain); Germain, Pierre [Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), BP 10142, 67404 Illkirch CEDEX (France); Lera, Angel R. de [Universidade de Vigo, Departamento de Quimica Organica, Facultad de Química, 36310 Vigo (Spain); Bourguet, William, E-mail: bourguet@cbs.cnrs.fr [INSERM, U554, 34090 Montpellier (France); Université de Montpellier, CNRS, UMR5048, Centre de Biochimie Structurale (CBS), 34090 Montpellier (France)

    2008-07-01

    Attempts have been made to crystallize the ligand-binding domain of the human retinoid X receptor in complex with a variety of newly synthesized ligands. An inverse correlation was observed between the ‘crystallizability’ and the structural dynamics of the various receptor–ligand complexes. Crystallization trials of the human retinoid X receptor α ligand-binding domain (RXRα LBD) in complex with various ligands have been carried out. Using fluorescence anisotropy, it has been found that when compared with agonists these small-molecule effectors enhance the dynamics of the RXRα LBD C-terminal helix H12. In some cases, the mobility of this helix could be dramatically reduced by the addition of a 13-residue co-activator fragment (CoA). In keeping with these observations, crystals have been obtained of the corresponding ternary RXRα LBD–ligand–CoA complexes. In contrast, attempts to crystallize complexes with a highly mobile H12 remained unsuccessful. These experimental observations substantiate the previously recognized role of co-regulator fragments in facilitating the crystallization of nuclear receptor LBDs.

  12. LiCABEDS II. Modeling of ligand selectivity for G-protein-coupled cannabinoid receptors.

    Ma, Chao; Wang, Lirong; Yang, Peng; Myint, Kyaw Z; Xie, Xiang-Qun

    2013-01-28

    The cannabinoid receptor subtype 2 (CB2) is a promising therapeutic target for blood cancer, pain relief, osteoporosis, and immune system disease. The recent withdrawal of Rimonabant, which targets another closely related cannabinoid receptor (CB1), accentuates the importance of selectivity for the development of CB2 ligands in order to minimize their effects on the CB1 receptor. In our previous study, LiCABEDS (Ligand Classifier of Adaptively Boosting Ensemble Decision Stumps) was reported as a generic ligand classification algorithm for the prediction of categorical molecular properties. Here, we report extension of the application of LiCABEDS to the modeling of cannabinoid ligand selectivity with molecular fingerprints as descriptors. The performance of LiCABEDS was systematically compared with another popular classification algorithm, support vector machine (SVM), according to prediction precision and recall rate. In addition, the examination of LiCABEDS models revealed the difference in structure diversity of CB1 and CB2 selective ligands. The structure determination from data mining could be useful for the design of novel cannabinoid lead compounds. More importantly, the potential of LiCABEDS was demonstrated through successful identification of newly synthesized CB2 selective compounds.

  13. Interaction of cadmium with atrial natriuretic factor receptors: Ligand binding and cellular processing

    Giridhar, J.; Rathinavelu, A.; Isom, G.E.

    1990-01-01

    ANF is a peptide hormone secreted by the heart and produces potent diuresis and vascular smooth muscle relaxation. It is well known that Cd produces cardiovascular toxicity and is implicated in the pathogenesis of hypertension. Hence the effects of Cd on ANF receptor dynamics and ligand binding were studied in PC12 cells. Receptor internalization using 125 I-ANF as the ligand at 37 degree C displayed a decrease in endocytic rate constants (ERC) when either preincubated with Cd (500 μM for 30 min, ERC = 0.183/min) or coincubated with Cd (500 μM, ERC = 0.196) when compared to control value (ERC = 0.259/min). Ligand binding ( 125 I-ANF) was changed by Cd as reflected by a decrease in the number of binding sites/cell in both Cd preincubated (Kd = 3.81 x 10 -10 M, B max = 1 x 10 -10 M, binding sites/cell = 9333) and coincubated cells (Kd = 1.76 x 10 -10 M, B max = 3.92 x 10 -11 M, binding sites/cell = 5960) from control (Kd = 3.87 x 10 -10 M, B max = 9.58 x 10 -11 M, binding sites/cell = 12141). Photoaffinity labelling with 125 I-ANF as the ligand was used to measure receptor subtype binding. Coincubation of cells with Cd (500 μM) and ligand decreased both high and low mol. wt. receptor binding, whereas preincubation with Cd (500μM) for 60 min produced a slight decrease in binding of both receptor subtypes. These results indicate that the cardiovascular toxicity of Cd may be partially mediated by altered ANF receptor function

  14. Histamine H3 receptor ligands in the group of (homo)piperazine derivatives.

    Szczepanska, Katarzyna; Kuder, Kamil; Kiec-Kononowicz, Katarzyna

    2017-11-23

    Since its' discovery in 1983, followed by gene cloning in 1999, the histamine H3 receptor served as an outstanding target for drug discovery. The wide spectrum of possible therapeutic implications make H3R's one of the most researched areas in the vast GPCR ligands field - started from imidazole containing ligands, through various successful imidazole replacements, with recent introduction of Wakix® to pharmaceutical market. One of such replacements is piperazine moiety, a significant versatile scaffold in rational drug design for most of the GPCR ligands. Therefore, herein we review ligands built on piperazine, as well as its seven membered analogue azepine, that target H3R's and their potential therapeutical applications, in order to elucidate the current state of the art in this vast field. Due to a high level of structural divergence among compounds described herein, we decided to divide them into groups, where the key division element was the position of nitrogen basicity decreasing moieties in (homo)piperazine ring. Paying attention to a number of published structures and their overall high biological activity, one can realize that the (homo)piperazine scaffold bids a versatile template also for histamine H3 receptor ligands. With two possible substitution sites and therefore a number of possible structural combinations, piperazine derivatives stand as one of the largest group of high importance among H3R ligands. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  15. Structural basis for AMPA receptor activation and ligand selectivity

    Hogner, A; Kastrup, Jette Sandholm Jensen; Jin, R

    2002-01-01

    Glutamate is the principal excitatory neurotransmitter within the mammalian CNS, playing an important role in many different functions in the brain such as learning and memory. In this study, a combination of molecular biology, X-ray structure determinations, as well as electrophysiology...... with Br-HIBO and ACPA have allowed us to explain the molecular mechanism behind this selectivity and to identify key residues for ligand recognition. The agonists induce the same degree of domain closure as AMPA, except for Br-HIBO, which shows a slightly lower degree of domain closure. An excellent...

  16. 14-O-Methylmorphine: A Novel Selective Mu-Opioid Receptor Agonist with High Efficacy and Affinity.

    Zádor, Ferenc; Balogh, Mihály; Váradi, András; Zádori, Zoltán S; Király, Kornél; Szűcs, Edina; Varga, Bence; Lázár, Bernadette; Hosztafi, Sándor; Riba, Pál; Benyhe, Sándor; Fürst, Susanna; Al-Khrasani, Mahmoud

    2017-11-05

    14-O-methyl (14-O-Me) group in morphine-6-O-sulfate (M6SU) or oxymorphone has been reported to be essential for enhanced affinity, potency and antinociceptive effect of these opioids. Herein we report on the pharmacological properties (potency, affinity and efficacy) of the new compound, 14-O-methylmorphine (14-O-MeM) in in vitro. Additionally, we also investigated the antinociceptive effect of the novel compound, as well as its inhibitory action on gastrointestinal transit in in vivo. The potency and efficacy of test compound were measured by [ 35 S]GTPγS binding, isolated mouse vas deferens (MVD) and rat vas deferens (RVD) assays. The affinity of 14-O-MeM for opioid receptors was assessed by radioligand binding and MVD assays. The antinociceptive and gastrointestinal effects of the novel compound were evaluated in the rat tail-flick test and charcoal meal test, respectively. Morphine, DAMGO, Ile 5,6 deltorphin II, deltorphin II and U-69593 were used as reference compounds. 14-O-MeM showed higher efficacy (E max ) and potency (EC 50 ) than morphine in MVD, RVD or [ 35 S]GTPγS binding. In addition, 14-O-MeM compared to morphine showed higher affinity for μ-opioid receptor (MOR). In vivo, in rat tail-flick test 14-O-MeM proved to be stronger antinociceptive agent than morphine after peripheral or central administration. Additionally, both compounds inhibited the gastrointestinal peristalsis. However, when the antinociceptive and antitransit doses for each test compound are compared, 14-O-MeM proved to have slightly more favorable pharmacological profile. Our results affirm that 14-O-MeM, an opioid of high efficacy and affinity for MOR can be considered as a novel analgesic agent of potential clinical value. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Rapid agonist-induced loss of sup 125 I-. beta. -endorphin opioid receptor sites in NG108-15, but not SK-N-SH neuroblastoma cells

    Cone, R.I.; Lameh, J.; Sadee, W. (Univ. of California, San Francisco (United States))

    1991-01-01

    The authors have measured {mu} and {delta} opioid receptor sites on intact SK-N-SH and NG108-15 neuroblastoma cells, respectively, in culture. Use of {sup 125}I-{beta}-endorphin ({beta}E) as a tracer, together with {beta}E(6-31) to block high-affinity non-opioid binding in both cell lines, permitted the measurement of cell surface {mu} and {delta} opioid receptor sites. Labeling was at {delta} sites in NG108-15 cells and predominantly at {mu} sites in SK-N-SH cells. Pretreatment with the {mu} and {delta} agonist, DADLE, caused a rapid loss of cell surface {delta} receptor sites in NG108-15 cells, but failed to reduce significantly {mu} receptor density in SK-N-SH cells.

  18. Imaging GABAc Receptors with Ligand-Conjugated Quantum Dots

    Ian D. Tomlinson

    2007-01-01

    Full Text Available We report a methodology for labeling the GABAc receptor on the surface membrane of intact cells. This work builds upon our earlier work with serotonin-conjugated quantum dots and our studies with PEGylated quantum dots to reduce nonspecific binding. In the current approach, a PEGylated derivative of muscimol was synthesized and attached via an amide linkage to quantum dots coated in an amphiphilic polymer derivative of a modified polyacrylamide. These conjugates were used to image GABAC receptors heterologously expressed in Xenopus laevis oocytes.

  19. Major advances in the development of histamine H4 receptor ligands.

    Smits, Rogier A; Leurs, Rob; de Esch, Iwan J P

    2009-08-01

    The search for new and potent histamine H4 receptor ligands is leading to a steadily increasing number of scientific publications and patent applications. Several interesting and structurally diverse compounds have been found, but fierce IP competition for a preferred 2-aminopyrimidine scaffold is becoming apparent. Recent investigations into the role of the histamine H(4)R in (patho)physiology and the use of H4R ligands in in vivo disease models reveal enormous potential in the field of inflammation and allergy, among others. The development of ligands that display activity at two or more histamine receptor (HR) subtypes is another clinical opportunity that is currently being explored. Taken together, the histamine H4R field is gearing up for clinical studies and has the potential to deliver another generation of blockbuster drugs.

  20. Exogenous or endogenous Toll-like receptor ligands: which is the MVP in tumorigenesis?

    Yu, Li; Wang, Liantang; Chen, Shangwu

    2012-03-01

    Toll-like receptors (TLRs) are a class of pattern recognition receptors sensing microbial components and triggering an immune response against pathogens. In addition to their role in anti-infection immunity, increasing evidence indicates that engagement of TLRs can promote cancer cell survival and proliferation, induce tumor immune evasion, and enhance tumor metastasis and chemoresistance. Recent studies have demonstrated that endogenous molecules or damage-associated molecular patterns released from damaged/necrotic tissues are capable of activating TLRs and that the endogenous ligands-mediated TLR signaling is implicated in the tumor development and affects the therapeutic efficacy of tumors. Since both exogenous and endogenous TLR ligands can initiate TLR signaling, which is the most valuable player in tumor development becomes an interesting question. Here, we summarize the effect of TLR signaling on the development and progression of tumors, and discuss the role of exogenous and endogenous TLR ligands in the tumorigenesis.

  1. A multistep continuous-flow system for rapid on-demand synthesis of receptor ligands

    Petersen, Trine P; Ritzén, Andreas; Ulven, Trond

    2009-01-01

    A multistep continuous-flow system for synthesis of receptor ligands by assembly of three variable building blocks in a single unbroken flow is described. The sequence consists of three reactions and two scavenger steps, where a Cbz-protected diamine is reacted with an isocyanate, deprotected, an......, and reacted further with an alkylating agent....

  2. The molecular basis of ligand interaction at free fatty acid receptor 4 (FFA4/GPR120)

    Hudson, Brian D; Shimpukade, Bharat; Milligan, Graeme

    2014-01-01

    The long-chain fatty acid receptor FFA4 (previously GPR120) is receiving substantial interest as a novel target for the treatment of metabolic and inflammatory disease. This study examines for the first time the detailed mode of binding of both long-chain fatty acid and synthetic agonist ligands ...

  3. Docking of flexible ligands to flexible receptors in solution by molecular dynamics simulation

    Mangoni, R; Roccatano, D; Di Nola, A

    1999-01-01

    In this paper, a method of simulating the docking of small flexible ligands to flexible receptors in water is reported. The method is based on molecular dynamics simulations and is an extension of an algorithm previously reported by Di Nola et al, (Di Nola et al,, Proteins 1994;19:174-182), The

  4. Unnatural amino acids as probes of ligand-receptor interactions and their conformational consequences

    Pless, Stephan Alexander; Ahern, Christopher A

    2013-01-01

    -edge synthetic and chemical biological approaches. Here we summarize recent advances in the use of site-directed incorporation of unnatural amino acids and chemical probes to study ligand-receptor interactions, determine the location of binding sites, and examine the downstream conformational consequences...

  5. Aryl hydrocarbon receptor ligand effects in RBL2H3 cells

    Maaetoft-Udsen, Kristina; Shimoda, Lori M. N.; Frøkiær, Hanne

    2012-01-01

    The aryl hydrocarbon receptor (AHR) mediates toxic effects of dioxin and xenobiotic metabolism. AHR has an emerging role in the immune system, but its physiological ligands and functional role in immunocytes remain poorly understood. Mast cells are immunocytes that are central to inflammatory...

  6. Peroxisome Proliferator-Activated Receptor Ligands and Their Role in Chronic Myeloid Leukemia: Therapeutic Strategies.

    Yousefi, Bahman; Samadi, Nasser; Baradaran, Behzad; Shafiei-Irannejad, Vahid; Zarghami, Nosratollah

    2016-07-01

    Imatinib therapy remains the gold standard for treatment of chronic myeloid leukemia; however, the acquired resistance to this therapeutic agent in patients has urged the scientists to devise modalities for overcoming this chemoresistance. For this purpose, initially therapeutic agents with higher tyrosine kinase activity were introduced, which had the potential for inhibiting even mutant forms of Bcr-Abl. Furthermore, coupling imatinib with peroxisome proliferator-activated receptor ligands also showed beneficial effects in chronic myeloid leukemia cell proliferation. These combination protocols inhibited cell growth and induced apoptosis as well as differentiation in chronic myeloid leukemia cell lines. In addition, peroxisome proliferator-activated receptors ligands increased imatinib uptake by upregulating the expression of human organic cation transporter 1. Taken together, peroxisome proliferator-activated receptors ligands are currently being considered as novel promising therapeutic candidates for chronic myeloid leukemia treatment, because they can synergistically enhance the efficacy of imatinib. In this article, we reviewed the potential of peroxisome proliferator-activated receptors ligands for use in chronic myeloid leukemia treatment. The mechanism of action of these therapeutics modalities are also presented in detail. © 2016 John Wiley & Sons A/S.

  7. Nanoparticle-Based Receptors Mimic Protein-Ligand Recognition.

    Riccardi, Laura; Gabrielli, Luca; Sun, Xiaohuan; De Biasi, Federico; Rastrelli, Federico; Mancin, Fabrizio; De Vivo, Marco

    2017-07-13

    The self-assembly of a monolayer of ligands on the surface of noble-metal nanoparticles dictates the fundamental nanoparticle's behavior and its functionality. In this combined computational-experimental study, we analyze the structure, organization, and dynamics of functionalized coating thiols in monolayer-protected gold nanoparticles (AuNPs). We explain how functionalized coating thiols self-organize through a delicate and somehow counterintuitive balance of interactions within the monolayer itself and with the solvent. We further describe how the nature and plasticity of these interactions modulate nanoparticle-based chemosensing. Importantly, we found that self-organization of coating thiols can induce the formation of binding pockets in AuNPs. These transient cavities can accommodate small molecules, mimicking protein-ligand recognition, which could explain the selectivity and sensitivity observed for different organic analytes in NMR chemosensing experiments. Thus, our findings advocate for the rational design of tailored coating groups to form specific recognition binding sites on monolayer-protected AuNPs.

  8. Structure-activity relationships of constrained phenylethylamine ligands for the serotonin 5-ht2 receptors

    Isberg, Vignir; Paine, James; Leth-Petersen, Sebastian

    2013-01-01

    Serotonergic ligands have proven effective drugs in the treatment of migraine, pain, obesity, and a wide range of psychiatric and neurological disorders. There is a clinical need for more highly 5-HT2 receptor subtype-selective ligands and the most attention has been given to the phenethylamine...... about the bioactive conformation of the amine functionality. However, combined 1,2-constriction by cyclization has only been tested with one compound. Here, we present three new 1,2-cyclized phenylethylamines, 9-11, and describe their synthetic routes. Ligand docking in the 5-HT2B crystal structure...... but shift the placement of the core scaffold. The constraints in 9-11 resulted in docking poses with the 4-bromine in closer vicinity to 5.46, which is polar only in the human 5-HT2A subtype, for which 9-11 have the lowest affinity. The new ligands, conformational analysis and docking expand the structure...

  9. Elimination of a ligand gating site generates a supersensitive olfactory receptor.

    Sharma, Kanika; Ahuja, Gaurav; Hussain, Ashiq; Balfanz, Sabine; Baumann, Arnd; Korsching, Sigrun I

    2016-06-21

    Olfaction poses one of the most complex ligand-receptor matching problems in biology due to the unparalleled multitude of odor molecules facing a large number of cognate olfactory receptors. We have recently deorphanized an olfactory receptor, TAAR13c, as a specific receptor for the death-associated odor cadaverine. Here we have modeled the cadaverine/TAAR13c interaction, exchanged predicted binding residues by site-directed mutagenesis, and measured the activity of the mutant receptors. Unexpectedly we observed a binding site for cadaverine at the external surface of the receptor, in addition to an internal binding site, whose mutation resulted in complete loss of activity. In stark contrast, elimination of the external binding site generated supersensitive receptors. Modeling suggests this site to act as a gate, limiting access of the ligand to the internal binding site and thereby downregulating the affinity of the native receptor. This constitutes a novel mechanism to fine-tune physiological sensitivity to socially relevant odors.

  10. Kinetic modeling of receptor-ligand binding applied to positron emission tomographic studies with neuroleptic tracers

    Logan, J; Wolf, A P; Shiue, C Y; Fowler, J S

    1987-01-01

    Positron emission tomography (PET) with labeled neuroleptics has made possible the study of neurotransmitter-receptor systems in vivo. In this study we investigate the kinetics of the 3,4-dihydroxyphenylethylamine (dopamine) receptor-ligand binding using PET data from a series of experiments in the baboon with the /sup 18/F-labeled drugs spiperone, haloperidol, and benperidol. Models used to describe these systems are based on first-order kinetics which applies at high specific activity (low receptor occupancy). The parameters governing the uptake and loss of drug from the brain were found by fitting PET data from regions with little or no receptor concentration (cerebellum) and from experiments in which specific binding was blocked by pretreatment with the drug (+)-butaclamol. Receptor constants were determined by fitting data from receptor-containing structures. Correcting the arterial plasma activities (the model driving function) for the presence of drug metabolites was found to be important in the modeling of these systems.

  11. Structural basis of ligand recognition in 5-HT(3) receptors

    Kesters, D.; Thompson, A.J.; Brams, M.; van Elk, R.; Spurny, R.; Geitmann, M.; Villalgordo, J.M.; Guskov, A.; Danielson, U.H.; Lummis, S.C.R.; Smit, A.B.; Ulens, C.

    2013-01-01

    The 5-HT 3 receptor is a pentameric serotonin-gated ion channel, which mediates rapid excitatory neurotransmission and is the target of a therapeutically important class of anti-emetic drugs, such as granisetron. We report crystal structures of a binding protein engineered to recognize the agonist

  12. A python-based docking program utilizing a receptor bound ligand shape: PythDock.

    Chung, Jae Yoon; Cho, Seung Joo; Hah, Jung-Mi

    2011-09-01

    PythDock is a heuristic docking program that uses Python programming language with a simple scoring function and a population based search engine. The scoring function considers electrostatic and dispersion/repulsion terms. The search engine utilizes a particle swarm optimization algorithm. A grid potential map is generated using the shape information of a bound ligand within the active site. Therefore, the searching area is more relevant to the ligand binding. To evaluate the docking performance of PythDock, two well-known docking programs (AutoDock and DOCK) were also used with the same data. The accuracy of docked results were measured by the difference of the ligand structure between x-ray structure, and docked pose, i.e., average root mean squared deviation values of the bound ligand were compared for fourteen protein-ligand complexes. Since the number of ligands' rotational flexibility is an important factor affecting the accuracy of a docking, the data set was chosen to have various degrees of flexibility. Although PythDock has a scoring function simpler than those of other programs (AutoDock and DOCK), our results showed that PythDock predicted more accurate poses than both AutoDock4.2 and DOCK6.2. This indicates that PythDock could be a useful tool to study ligand-receptor interactions and could also be beneficial in structure based drug design.

  13. Analgesic efficacy of CR4056, a novel imidazoline-2 receptor ligand, in rat models of inflammatory and neuropathic pain

    Ferrari F

    2011-04-01

    Full Text Available Flora Ferrari1, Simonetta Fiorentino1, Laura Mennuni1, Paolo Garofalo1, Ornella Letari1, Stefano Mandelli2, Antonio Giordani3, Marco Lanza1, Gianfranco Caselli11Department of Pharmacology and Toxicology; 2Department of Medicinal Chemistry; 3R&D Chemistry Drug Development and OS, Rottapharm S.p.A., Monza (MB, ItalyAbstract: Two decades of investigations have failed to unequivocally clarify the functions and the molecular nature of imidazoline-2 receptors (I2R. However, there is robust pharmacological evidence for the functional modulation of monoamino oxidase (MAO and other important enzyme activities by I2 site ligands. Some compounds of this class proved to be active experimental tools in preventing both experimental pain and opioid tolerance and dependence. Unfortunately, even though these compounds bind with high potency to central I2 sites, they fail to represent a valid clinical opportunity due to their pharmacokinetic, selectivity or side-effects profile. This paper presents the preclinical profile of a novel I2 ligand (2-phenyl-6-(1H-imidazol-1ylquinazoline; [CR4056] that selectively inhibits the activity of human recombinant MAO-A in a concentration-dependent manner. A sub-chronic four day oral treatment of CR4056 increased norepinephrine (NE tissue levels both in the rat cerebral cortex (63.1% ± 4.2%; P<0.05 and lumbar spinal cord (51.3% ± 6.7%; P < 0.05. In the complete Freund's adjuvant (CFA rat model of inflammatory pain, CR4056 was found to be orally active (ED50 = 5.8 mg/kg, by mouth [p.o.]. In the acute capsaicin model, CR4056 completely blocked mechanical hyperalgesia in the injured hind paw (ED50 = 4.1 mg/kg, p.o.; ED100 = 17.9 mg/kg, p.o.. This effect was dose-dependently antagonized by the non-selective imidazoline I2/α2 antagonist idazoxan. In rat models of neuropathic pain, oral administration of CR4056 significantly attenuated mechanical hyperalgesia and allodynia. In summary, the present study suggests a novel

  14. Ligand-specific conformational changes in the alpha1 glycine receptor ligand-binding domain

    Pless, Stephan Alexander; Lynch, Joseph W

    2009-01-01

    , and by the antagonist, strychnine. Voltage-clamp fluorometry involves labeling introduced cysteines with environmentally sensitive fluorophores and inferring structural rearrangements from ligand-induced fluorescence changes. In the inner beta-sheet, we labeled residues in loop 2 and in binding domain loops D and E....... At each position, strychnine and glycine induced distinct maximal fluorescence responses. The pre-M1 domain responded similarly; at each of four labeled positions glycine produced a strong fluorescence signal, whereas strychnine did not. This suggests that glycine induces conformational changes...... in the inner beta-sheet and pre-M1 domain that may be important for activation, desensitization, or both. In contrast, most labeled residues in loops C and F yielded fluorescence changes identical in magnitude for glycine and strychnine. A notable exception was H201C in loop C. This labeled residue responded...

  15. Basal μ-opioid receptor availability in the amygdala predicts the inhibition of pain-related brain activity during heterotopic noxious counter-stimulation.

    Piché, Mathieu; Watanabe, Nobuhiro; Sakata, Muneyuki; Oda, Keiichi; Toyohara, Jun; Ishii, Kenji; Ishiwata, Kiichi; Hotta, Harumi

    2014-01-01

    The aim of this study was to investigate the association between the magnitude of anti-nociceptive effects induced by heterotopic noxious counter-stimulation (HNCS) and the basal μ-opioid receptor availability in the amygdala. In 8 healthy volunteers (4 females and 4 males), transcutaneous electrical stimulation was applied to the right sural nerve to produce the nociceptive flexion reflex (RIII-reflex), moderate pain, and scalp somatosensory evoked potentials (SEPs). Immersion of the left hand in cold water for 20min was used as HNCS. In a separate session, basal μ-opioid receptor availability was measured using positron emission tomography with the radiotracer [(11)C]carfentanil. HNCS produced a reduction of the P260 amplitude (pbasal μ-opioid receptor availability in the amygdala on the right (R(2)=0.55, p=0.03) with a similar trend on the left (R(2)=0.24, p=0.22). Besides, HNCS did not induce significant changes in pain and RIII-reflex amplitude (p>0.05). These results suggest that activation of μ-opioid receptors in the amygdala may contribute to the anti-nociceptive effects of HNCS. The lack of RIII-reflex modulation further suggests that μ-opioid receptor activation in the amygdala contributes to decrease pain-related brain activity through a cerebral mechanism independent of descending modulation. Copyright © 2014 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

  16. Effects of kappa opioid receptors on conditioned place aversion and social interaction in males and females

    Robles, Cindee F.; McMackin, Marissa Z.; Campi, Katharine L.; Doig, Ian E.; Takahashi, Elizabeth Y.; Pride, Michael; Trainor, Brian C.

    2014-01-01

    The effects of kappa opioid receptors (KOR) on motivated behavior are well established based on studies in male rodents, but relatively little is known about the effects of KOR in females. We examined the effects of KOR activation on conditioned place aversion and social interaction in the California mouse (Peromyscus californicus). Important differences were observed in long-term (place aversion) and short-term (social interaction) effects. Females but not males treated with a 2.5mg/kg dose of U50,488 formed a place aversion, whereas males but not females formed a place aversion at the 10 mg/kg dose. In contrast the short term effects of different doses of U50,488 on social interaction behavior were similar in males and females. Acute injection with 10 mg/kg of U50,488 (but not lower doses) reduced social interaction behavior in both males and females. The effects of U50,488 on phosphorylated extracellular signal regulated kinase (pERK) and p38 MAP kinase were cell type and region specific. Higher doses of U50,488 increased the number of pERK neurons in the ventrolateral bed nucleus of the stria terminals in males but not females, a nucleus implicated in male aggressive behavior. In contrast, both males and females treated with U50,488 had more activated p38 cells in the nucleus accumbens shell. Unexpectedly, cells expressing activated p38 co-expressed Iba-1, a widely used microglia marker. In summary we found strong sex differences in the effects of U50,488 on place aversion whereas the acute effects on U50,488 induced similar behavioral effects in males and females. PMID:24445073

  17. Effects of pharmacological manipulation of the kappa opioid receptors on the aversive effects of nicotine.

    Ward, Melissa; Norman, Haval; D'Souza, Manoranjan S

    2018-02-15

    Nicotine, an addictive component of tobacco smoke, produces both rewarding and aversive effects. Increasing the aversive effects of nicotine may help in promoting smoking cessation. However, neural targets mediating the aversive effects of nicotine have not been fully identified. In this study, we evaluated the role of kappa opioid receptors (KORs) in the aversive effects of nicotine (0.4 mg/kg, base; s.c.) using the nicotine-induced conditioned taste aversion (CTA) model in Wistar rats. The KORs were activated using the selective KOR agonist (±)U-50,488H (0, 0.03, 0.15 & 0.3mg/kg; s.c.) and inhibited using the KOR antagonist nor-binaltorphimine (nor-BNI; 0, 15 & 30mg/kg; s.c.) in separate groups of rats using a between-subjects design. Pretreatment with the KOR agonist (±)U-50,488H (0.3mg/kg) significantly increased aversion for the nicotine-associated solution. Additionally, (±)U-50,488H (0.3mg/kg) on its own induced aversion to the flavored solution associated with it even in the absence of nicotine, suggesting that the KOR agonist induced increase in nicotine-induced aversion was an additive effect. Interestingly, administration of the KOR antagonist nor-BNI (30mg/kg) prior to conditioning with nicotine/saline, but not after conditioning with nicotine/saline, attenuated nicotine-induced aversive effects compared to saline controls. Taken together, these data suggest a role for KORs in the aversive effects of nicotine. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Voluntary ethanol intake predicts κ-opioid receptor supersensitivity and regionally distinct dopaminergic adaptations in macaques.

    Siciliano, Cody A; Calipari, Erin S; Cuzon Carlson, Verginia C; Helms, Christa M; Lovinger, David M; Grant, Kathleen A; Jones, Sara R

    2015-04-15

    The dopaminergic projections from the ventral midbrain to the striatum have long been implicated in mediating motivated behaviors and addiction. Previously it was demonstrated that κ-opioid receptor (KOR) signaling in the striatum plays a critical role in the increased reinforcing efficacy of ethanol following ethanol vapor exposure in rodent models. Although rodents have been used extensively to determine the neurochemical consequences of chronic ethanol exposure, establishing high levels of voluntary drinking in these models has proven difficult. Conversely, nonhuman primates exhibit similar intake and pattern to humans in regard to drinking. Here we examine the effects of chronic voluntary ethanol self-administration on dopamine neurotransmission and the ability of KORs to regulate dopamine release in the dorsolateral caudate (DLC) and nucleus accumbens (NAc) core. Using voltammetry in brain slices from cynomolgus macaques after 6 months of ad libitum ethanol drinking, we found increased KOR sensitivity in both the DLC and NAc. The magnitude of ethanol intake predicted increases in KOR sensitivity in the NAc core, but not the DLC. Additionally, ethanol drinking increased dopamine release and uptake in the NAc, but decreased both of these measures in the DLC. These data suggest that chronic daily drinking may result in regionally distinct disruptions of striatal outputs. In concert with previous reports showing increased KOR regulation of drinking behaviors induced by ethanol exposure, the strong relationship between KOR activity and voluntary ethanol intake observed here gives further support to the hypothesis that KORs may provide a promising pharmacotherapeutic target in the treatment of alcoholism. Copyright © 2015 the authors 0270-6474/15/355959-10$15.00/0.

  19. Cortical delta-opioid receptors potentiate K+ homeostasis during anoxia and oxygen-glucose deprivation.

    Chao, Dongman; Donnelly, David F; Feng, Yin; Bazzy-Asaad, Alia; Xia, Ying

    2007-02-01

    Central neurons are extremely vulnerable to hypoxic/ischemic insult, which is a major cause of neurologic morbidity and mortality as a consequence of neuronal dysfunction and death. Our recent work has shown that delta-opioid receptor (DOR) is neuroprotective against hypoxic and excitotoxic stress, although the underlying mechanisms remain unclear. Because hypoxia/ischemia disrupts ionic homeostasis with an increase in extracellular K(+), which plays a role in neuronal death, we asked whether DOR activation preserves K(+) homeostasis during hypoxic/ischemic stress. To test this hypothesis, extracellular recordings with K(+)-sensitive microelectrodes were performed in mouse cortical slices under anoxia or oxygen-glucose deprivation (OGD). The main findings in this study are that (1) DOR activation with [D-Ala(2), D-Leu(5)]-enkephalinamide attenuated the anoxia- and OGD-induced increase in extracellular K(+) and decrease in DC potential in cortical slices; (2) DOR inhibition with naltrindole, a DOR antagonist, completely abolished the DOR-mediated prevention of increase in extracellular K(+) and decrease in DC potential; (3) inhibition of protein kinase A (PKA) with N-(2-[p-bromocinnamylamino]-ethyl)-5-isoquinolinesulfonamide dihydrochloride had no effect on the DOR protection; and (4) inhibition of protein kinase C (PKC) with chelerythrine chloride reduced the DOR protection, whereas the PKC activator (phorbol 12-myristate 13-acetate) mimicked the effect of DOR activation on K(+) homeostasis. These data suggest that activation of DOR protects the cortex against anoxia- or ODG-induced derangement of potassium homeostasis, and this protection occurs via a PKC-dependent and PKA-independent pathway. We conclude that an important aspect of DOR-mediated neuroprotection is its early action against derangement of K(+) homeostasis during anoxia or ischemia.

  20. Endogenous cannabinoid receptor ligand induces the migration of human natural killer cells.

    Kishimoto, Seishi; Muramatsu, Mayumi; Gokoh, Maiko; Oka, Saori; Waku, Keizo; Sugiura, Takayuki

    2005-02-01

    2-Arachidonoylglycerol is an endogenous ligand for the cannabinoid receptors (CB1 and CB2). Evidence is gradually accumulating which shows that 2-arachidonoylglycerol plays important physiological roles in several mammalian tissues and cells, yet the details remain ambiguous. In this study, we first examined the effects of 2-arachidonoylglycerol on the motility of human natural killer cells. We found that 2-arachidonoylglycerol induces the migration of KHYG-1 cells (a natural killer leukemia cell line) and human peripheral blood natural killer cells. The migration of natural killer cells induced by 2-arachidonoylglycerol was abolished by treating the cells with SR144528, a CB2 receptor antagonist, suggesting that the CB2 receptor is involved in the 2-arachidonoylglycerol-induced migration. In contrast to 2-arachidonoylglycerol, anandamide, another endogenous cannabinoid receptor ligand, did not induce the migration. Delta9-tetrahydrocannabinol, a major psychoactive constituent of marijuana, also failed to induce the migration; instead, the addition of delta9-tetrahydrocannabinol together with 2-arachidonoylglycerol abolished the migration induced by 2-arachidonoylglycerol. It is conceivable that the endogenous ligand for the cannabinoid receptor, that is, 2-arachidonoylglycerol, affects natural killer cell functions such as migration, thereby contributing to the host-defense mechanism against infectious viruses and tumor cells.

  1. Ligand-Induced Dynamics of Neurotrophin Receptors Investigated by Single-Molecule Imaging Approaches

    Marchetti, Laura; Luin, Stefano; Bonsignore, Fulvio; de Nadai, Teresa; Beltram, Fabio; Cattaneo, Antonino

    2015-01-01

    Neurotrophins are secreted proteins that regulate neuronal development and survival, as well as maintenance and plasticity of the adult nervous system. The biological activity of neurotrophins stems from their binding to two membrane receptor types, the tropomyosin receptor kinase and the p75 neurotrophin receptors (NRs). The intracellular signalling cascades thereby activated have been extensively investigated. Nevertheless, a comprehensive description of the ligand-induced nanoscale details of NRs dynamics and interactions spanning from the initial lateral movements triggered at the plasma membrane to the internalization and transport processes is still missing. Recent advances in high spatio-temporal resolution imaging techniques have yielded new insight on the dynamics of NRs upon ligand binding. Here we discuss requirements, potential and practical implementation of these novel approaches for the study of neurotrophin trafficking and signalling, in the framework of current knowledge available also for other ligand-receptor systems. We shall especially highlight the correlation between the receptor dynamics activated by different neurotrophins and the respective signalling outcome, as recently revealed by single-molecule tracking of NRs in living neuronal cells. PMID:25603178

  2. Are peripheral opioid antagonists the solution to opioid side effects?

    Bates, John J

    2012-02-03

    Opioid medication is the mainstay of therapy for severe acute and chronic pain. Unfortunately, the side effects of these medications can affect patient comfort and safety, thus limiting their proven therapeutic potential. Whereas the main analgesic effects of opioids are centrally mediated, many of the common side effects are mediated via peripheral receptors. Novel peripheral opioid antagonists have been recently introduced that can block the peripheral actions of opioids without affecting centrally mediated analgesia. We review the clinical and experimental evidence of their efficacy in ameliorating opioid side effects and consider what further information might be useful in defining their role. IMPLICATIONS: The major analgesic effects of opioid medication are mediated within the brain and spinal cord. Many of the side effects of opioids are caused by activation of receptors outside these areas. Recently developed peripherally restricted opioid antagonists have the ability to block many opioid side effects without affecting analgesia.

  3. A2A adenosine receptor ligand binding and signalling is allosterically modulated by adenosine deaminase.

    Gracia, Eduard; Pérez-Capote, Kamil; Moreno, Estefanía; Barkešová, Jana; Mallol, Josefa; Lluís, Carme; Franco, Rafael; Cortés, Antoni; Casadó, Vicent; Canela, Enric I

    2011-05-01

    A2ARs (adenosine A2A receptors) are highly enriched in the striatum, which is the main motor control CNS (central nervous system) area. BRET (bioluminescence resonance energy transfer) assays showed that A2AR homomers may act as cell-surface ADA (adenosine deaminase; EC 3.5.4.4)-binding proteins. ADA binding affected the quaternary structure of A2ARs present on the cell surface. ADA binding to adenosine A2ARs increased both agonist and antagonist affinity on ligand binding to striatal membranes where these proteins are co-expressed. ADA also increased receptor-mediated ERK1/2 (extracellular-signal-regulated kinase 1/2) phosphorylation. Collectively, the results of the present study show that ADA, apart from regulating the concentration of extracellular adenosine, may behave as an allosteric modulator that markedly enhances ligand affinity and receptor function. This powerful regulation may have implications for the physiology and pharmacology of neuronal A2ARs.

  4. Foreign or Domestic CARs: Receptor Ligands as Antigen-Binding Domains

    Donald R. Shaffer

    2014-01-01

    Full Text Available Chimeric antigen receptors (CARs are increasingly being used in clinical trials to treat a variety of malignant conditions and recent results with CD19-specific CARs showing complete tumor regressions has sparked the interest of researchers and the public alike. Traditional CARs have been generated using single-chain variable fragments (scFv, often derived from murine monoclonal antibodies, for antigen specificity. As the clinical experience with CAR T cells grows, so does the potential for unwanted immune responses against the foreign transgene. Strategies that may reduce the immunogenicity of CAR T cells are humanization of the scFv and the use of naturally occurring receptor ligands as antigen-binding domains. Herein, we review the experience with alternatively designed CARs that contain receptor ligands rather than scFv. While most of the experiences have been in the pre-clinical setting, clinical data is also emerging.

  5. Application of 3D-QSAR in the rational design of receptor ligands and enzyme inhibitors.

    Mor, Marco; Rivara, Silvia; Lodola, Alessio; Lorenzi, Simone; Bordi, Fabrizio; Plazzi, Pier Vincenzo; Spadoni, Gilberto; Bedini, Annalida; Duranti, Andrea; Tontini, Andrea; Tarzia, Giorgio

    2005-11-01

    Quantitative structure-activity relationships (QSARs) are frequently employed in medicinal chemistry projects, both to rationalize structure-activity relationships (SAR) for known series of compounds and to help in the design of innovative structures endowed with desired pharmacological actions. As a difference from the so-called structure-based drug design tools, they do not require the knowledge of the biological target structure, but are based on the comparison of drug structural features, thus being defined ligand-based drug design tools. In the 3D-QSAR approach, structural descriptors are calculated from molecular models of the ligands, as interaction fields within a three-dimensional (3D) lattice of points surrounding the ligand structure. These descriptors are collected in a large X matrix, which is submitted to multivariate analysis to look for correlations with biological activity. Like for other QSARs, the reliability and usefulness of the correlation models depends on the validity of the assumptions and on the quality of the data. A careful selection of compounds and pharmacological data can improve the application of 3D-QSAR analysis in drug design. Some examples of the application of CoMFA and CoMSIA approaches to the SAR study and design of receptor or enzyme ligands is described, pointing the attention to the fields of melatonin receptor ligands and FAAH inhibitors.

  6. Activation of peroxisome proliferator-activated receptors (PPARs) by their ligands and protein kinase A activators

    Lazennec, Gwendal; Canaple, Laurence; Saugy, Damien; Wahli, Walter

    2000-01-01

    The nuclear peroxisome proliferator-activated receptors (PPARs) α, β and γ activate the transcription of multiple genes involved in lipid metabolism. Several natural and synthetic ligands have been identified for each PPAR isotype but little is known about the phosphorylation state of these receptors. We show here that activators of protein kinase A (PKA) can enhance mouse PPAR activity in the absence and the presence of exogenous ligands in transient transfection experiments. The activation function 1 (AF-1) of PPARs was dispensable for transcriptional enhancement, whereas the activation function 2 (AF-2) was required for this effect. We also show that several domains of PPAR can be phosphorylated by PKA in vitro. Moreover, gel experiments suggest that PKA stabilizes binding of the liganded PPAR to DNA. PKA inhibitors decreased not only the kinase dependent induction of PPARs but also their ligand-dependent induction, suggesting that the ligands may also mobilize the PKA pathway to lead to maximal transcriptional induction by PPARs. Moreover, comparing PPARα KO with PPARα wild-type mice, we show that the expression of the ACO gene can be regulated by PKA-activated PPARα in liver. These data demonstrate that the PKA pathway is an important modulator of PPAR activity and we propose a model associating this pathway in the control of fatty acid β-oxidation under conditions of fasting, stress and exercise. PMID:11117527

  7. Acetylation of pregnane X receptor protein determines selective function independent of ligand activation

    Biswas, Arunima; Pasquel, Danielle; Tyagi, Rakesh Kumar; Mani, Sridhar

    2011-01-01

    Research highlights: → Pregnane X receptor (PXR), a major regulatory protein, is modified by acetylation. → PXR undergoes dynamic deacetylation upon ligand-mediated activation. → SIRT1 partially mediates PXR deacetylation. → PXR deacetylation per se induces lipogenesis mimicking ligand-mediated activation. -- Abstract: Pregnane X receptor (PXR), like other members of its class of nuclear receptors, undergoes post-translational modification [PTM] (e.g., phosphorylation). However, it is unknown if acetylation (a major and common form of protein PTM) is observed on PXR and, if it is, whether it is of functional consequence. PXR has recently emerged as an important regulatory protein with multiple ligand-dependent functions. In the present work we show that PXR is indeed acetylated in vivo. SIRT1 (Sirtuin 1), a NAD-dependent class III histone deacetylase and a member of the sirtuin family of proteins, partially mediates deacetylation of PXR. Most importantly, the acetylation status of PXR regulates its selective function independent of ligand activation.

  8. A multi-protein receptor-ligand complex underlies combinatorial dendrite guidance choices in C. elegans

    Zou, Wei; Shen, Ao; Dong, Xintong; Tugizova, Madina; Xiang, Yang K; Shen, Kang

    2016-01-01

    Ligand receptor interactions instruct axon guidance during development. How dendrites are guided to specific targets is less understood. The C. elegans PVD sensory neuron innervates muscle-skin interface with its elaborate dendritic branches. Here, we found that LECT-2, the ortholog of leukocyte cell-derived chemotaxin-2 (LECT2), is secreted from the muscles and required for muscle innervation by PVD. Mosaic analyses showed that LECT-2 acted locally to guide the growth of terminal branches. Ectopic expression of LECT-2 from seam cells is sufficient to redirect the PVD dendrites onto seam cells. LECT-2 functions in a multi-protein receptor-ligand complex that also contains two transmembrane ligands on the skin, SAX-7/L1CAM and MNR-1, and the neuronal transmembrane receptor DMA-1. LECT-2 greatly enhances the binding between SAX-7, MNR-1 and DMA-1. The activation of DMA-1 strictly requires all three ligands, which establishes a combinatorial code to precisely target and pattern dendritic arbors. DOI: http://dx.doi.org/10.7554/eLife.18345.001 PMID:27705746

  9. Introduction to the College on Problems of Drug Dependence special issue: contemporary advances in opioid neuropharmacology.

    Walsh, Sharon L; Unterwald, Ellen M; Izenwasser, Sari

    2010-05-01

    Opioid receptors are critical therapeutic targets for medications development relevant to the treatment of drug dependence and pain. With recent advances in molecular neurobiology, it has become evident that the functional activity of opioid receptors, as ligand-regulated protein complexes, is modulated by multifarious intracellular and extracellular events, that there is genetic variation in coding for receptors, and that the activity of endogenous opioid systems may underlie actions common to other addictive disorders. This supplemental issue of Drug and Alcohol Dependence, arising from an invited symposium at the 71st Annual Meeting of the College on Problems of Drug Dependence, provides a series of contemporary reviews focused on recent advances in opioid neuropharmacology. Each speaker provides herein an invited comprehensive review of the state of knowledge on a specific topic in opioid neuropharmacology. Evans and colleagues describe the multi-faceted control of the opioid G-protein coupled receptor as a dynamic "sensor" complex and identify novel targets for drug development. von Zastrow focuses on opioid receptor-mediated events regulated by endocytosis and membrane trafficking through the endocytic pathway and differential responses to opioid agonists. Blendy and colleague provide a review of human association studies on the functional relevance of the mu opioid receptor variant, A118G, and presents data from the A112G knock-in model, an analogous mouse variant to A118G. Finally, Maldonado and colleagues provide a broader systems review from genetic, pharmacologic and behavioral studies implicating the endogenous opioid systems as a substrate for the mediation of substance use disorders spanning pharmacological classes.

  10. Changes in mu-opioid receptor expression and function in the mesolimbic system after long-term access to a palatable diet.

    Pitman, Kimberley A; Borgland, Stephanie L

    2015-10-01

    The incidence of obesity in both adults and children is rising. In order to develop effective treatments for obesity, it is important to understand how diet can induce changes in the brain that could promote excessive intake of high-calorie foods and alter the efficacy of therapeutic targets. The mu-opioid receptor is involved in regulating the motivation for and hedonic reaction to food. Here, we review the literature examining changes in the expression and function of mu-opioid receptors in the mesolimbic system of rodents after extended access to a high-fat diet. We also review how maternal diet can induce long-term changes in the expression or function of mu-opioid receptors in the mesolimbic system of offspring. Understanding the behavioural and therapeutic implications of these changes requires further study. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. Brain catalase activity inhibition as well as opioid receptor antagonism increases ethanol-induced HPA axis activation.

    Pastor, Raúl; Sanchis-Segura, Carles; Aragon, Carlos M G

    2004-12-01

    Growing evidence indicates that brain catalase activity is involved in the psychopharmacological actions of ethanol. Recent data suggest that participation of this enzymatic system in some ethanol effects could be mediated by the endogenous opioid system. The present study assessed whether brain catalase has a role in ethanol-induced activation of the HPA axis, a neuroendocrine system modulated by the endogenous opioid neurotransmission. Swiss male mice received an intraperitoneal injection of the catalase inhibitor 3-amino-1,2,4-triazole (AT; 0-1 g/kg), and 0 to 20 hr after this administration, animals received an ethanol (0-4 g/kg; intraperitoneally) challenge. Thirty, 60, or 120 min after ethanol administration, plasma corticosterone levels were determined immunoenzymatically. In addition, we tested the effects of 45 mg/kg of cyanamide (another catalase inhibitor) and 0 to 2 mg/kg of naltrexone (nonselective opioid receptor antagonist) on ethanol-induced enhancement in plasma corticosterone values. The present study revealed that AT boosts ethanol-induced increase in plasma corticosterone levels in a dose- and time-dependent manner. However, it did not affect corticosterone values when measured after administration of saline, cocaine (4 mg/kg, intraperitoneally), or morphine (30 mg/kg, intraperitoneally). The catalase inhibitor cyanamide (45 mg/kg, intraperitoneally) also increased ethanol-related plasma corticosterone levels. These effects of AT and cyanamide on ethanol-induced corticosterone values were observed under treatment conditions that decreased significantly brain catalase activity. Indeed, a significant correlation between effects of catalase manipulations on both variables was found. Finally, we found that the administration of naltrexone enhanced the levels of plasma corticosterone after the administration of saline or ethanol. This study shows that the inhibition of brain catalase increases ethanol-induced plasma corticosterone levels. Results are

  12. Long-term antagonism of κ opioid receptors prevents escalation of and increased motivation for heroin intake.

    Schlosburg, Joel E; Whitfield, Timothy W; Park, Paula E; Crawford, Elena F; George, Olivier; Vendruscolo, Leandro F; Koob, George F

    2013-12-04

    The abuse of opioid drugs, both illicit and prescription, is a persistent problem in the United States, accounting for >1.2 million users who require treatment each year. Current treatments rely on suppressing immediate withdrawal symptoms and replacing illicit drug use with long-acting opiate drugs. However, the mechanisms that lead to preventing opiate dependence are still poorly understood. We hypothesized that κ opioid receptor (KOR) activation during chronic opioid intake contributes to negative affective states associated with withdrawal and the motivation to take increasing amounts of heroin. Using a 12 h long-access model of heroin self-administration, rats showed escalation of heroin intake over several weeks. This was prevented by a single high dose (30 mg/kg) of the long-acting KOR antagonist norbinaltorphimine (nor-BNI), paralleled by reduced motivation to respond for heroin on a progressive-ratio schedule of reinforcement, a measure of compulsive-like responding. Systemic nor-BNI also significantly decreased heroin withdrawal-associated anxiety-like behavior. Immunohistochemical analysis showed prodynorphin content increased in the nucleus accumbens core in all heroin-exposed rats, but selectively increased in the nucleus accumbens shell in long-access rats. Local infusion of nor-BNI (4 μg/side) into accumbens core altered the initial intake of heroin but not the rate of escalation, while local injection into accumbens shell selectively suppressed increases in heroin intake over time without altering initial intake. These data suggest that dynorphin activity in the nucleus accumbens mediates the increasing motivation for heroin taking and compulsive-like responding for heroin, suggesting that KOR antagonists may be promising targets for the treatment of opioid addiction.

  13. Intrauterine growth restriction modifies the hedonic response to sweet taste in newborn pups - Role of the accumbal μ-opioid receptors.

    Laureano, D P; Dalle Molle, R; Alves, M B; Luft, C; Desai, M; Ross, M G; Silveira, P P

    2016-05-13

    Intrauterine growth restriction (IUGR) is associated with increased preference for palatable foods. The hedonic response to sweet taste, modulated by the nucleus accumbens μ-opioid-receptors, may be involved. We investigated hedonic responses and receptor levels in IUGR and Control animals. From pregnancy day 10, Sprague-Dawley dams received either an ad libitum (Control), or a 50% food restricted (FR) diet. At birth, pups were cross-fostered, and nursed by Adlib fed dams. The hedonic response was evaluated at 1 day after birth and at 90 days of life, by giving sucrose solution or water and analyzing the hedonic facial responses (within 60s). Control pups exposed either to water or sucrose resolved their hedonic responses after 16 and 18s, respectively, while FR hedonic responses to sucrose persisted over 20s. FR pups had deceased phospho-μ-opioid-receptor (p=0.009) and reduced phosphor:total mu opioid receptor ratio compared to controls pups (p=0.003). In adults, there was an interaction between group and solution at the end of the evaluation (p=0.044): Control decreased the response after sucrose solution, FR did not change over time. There were no differences in phosphorylation of μ-opioid-receptor in adults. These results demonstrate IUGR newborn rats exhibit alterations in hedonic response accompanied by a decrease in μ-opioid-receptor phosphorylation, though these alterations do not persist at 3 months of age. Opioid system alterations in early life may contribute to the development of preference for highly palatable foods and contribute to rapid weight gain and obesity in IUGR offspring. Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

  14. Delta opioid receptor on equine sperm cells: subcellular localization and involvement in sperm motility analyzed by computer assisted sperm analyzer (CASA

    Lacalandra Giovanni M

    2010-06-01

    Full Text Available Abstract Background Opioid receptors and endogenous opioid peptides act not only in the control of nociceptive pathways, indeed several reports demonstrate the effects of opiates on sperm cell motility and morphology suggesting the importance of these receptors in the modulation of reproduction in mammals. In this study we investigated the expression of delta opioid receptors on equine spermatozoa by western blot/indirect immunofluorescence and its relationship with sperm cell physiology. Methods We analyzed viability, motility, capacitation, acrosome reaction and mitochondrial activity in the presence of naltrindole and DPDPE by means of a computer assisted sperm analyzer and a fluorescent confocal microscope. The evaluation of viability, capacitation and acrosome reaction was carried out by the double CTC/Hoechst staining, whereas mitochondrial activity was assessed by means of MitoTracker Orange dye. Results We showed that in equine sperm cells, delta opioid receptor is expressed as a doublet of 65 and 50 kDa molecular mass and is localized in the mid piece of tail; we also demonstrated that naltrindole, a delta opioid receptor antagonist, could be utilized in modulating several physiological parameters of the equine spermatozoon in a dose-dependent way. We also found that low concentrations of the antagonist increase sperm motility whereas high concentrations show the opposite effect. Moreover low concentrations hamper capacitation, acrosome reaction and viability even if the percentage of cells with active mitochondria seems to be increased; the opposite effect is exerted at high concentrations. We have also observed that the delta opioid receptor agonist DPDPE is scarcely involved in affecting the same parameters at the employed concentrations. Conclusions The results described in this paper add new important details in the comprehension of the mammalian sperm physiology and suggest new insights for improving reproduction and for

  15. Effects of stress and. beta. -funal trexamine pretreatment on morphine analgesia and opioid binding in rats

    Adams, J.U.; Andrews, J.S.; Hiller, J.M.; Simon, E.J.; Holtzman, S.G.

    1987-12-28

    This study was essentially an in vivo protection experiment designed to test further the hypothesis that stress induces release of endogenous opiods which then act at opioid receptors. Rats that were either subjected to restraint stress for 1 yr or unstressed were injected ICV with either saline or 2.5 ..mu..g of ..beta..-funaltrexamine (..beta..-FNA), an irreversible opioid antagonist that alkylates the mu-opioid receptor. Twenty-four hours later, subjects were tested unstressed for morphine analgesia or were sacrificed and opioid binding in brain was determined. (/sup 3/H)D-Ala/sup 2/NMePhe/sup 4/-Gly/sup 5/(ol)enkephalin (DAGO) served as a specific ligand for mu-opioid receptors, and (/sup 3/H)-bremazocine as a general ligand for all opioid receptors. Rats injected with saline while stressed were significantly less sensitive to the analgesic action of morphine 24 hr later than were their unstressed counterparts. ..beta..-FNA pretreatment attenuated morphine analgesia in an insurmountable manner. Animals pretreated with ..beta..-FNA while stressed were significantly more sensitive to the analgesic effect of morphine than were animals that received ..beta..-FNA while unstressed. ..beta..-FNA caused small and similar decreases in (/sup 3/H)-DAGO binding in brain of both stressed and unstressed animals. 35 references, 2 figures, 2 tables.

  16. Molecular mechanism of ligand recognition by NR3 subtype glutamate receptors

    Yao, Yongneng; Harrison, Chris B.; Freddolino, Peter L.; Schulten, Klaus; Mayer, Mark L. (UIUC); (NIH)

    2008-10-27

    NR3 subtype glutamate receptors have a unique developmental expression profile, but are the least well-characterized members of the NMDA receptor gene family, which have key roles in synaptic plasticity and brain development. Using ligand binding assays, crystallographic analysis, and all atom MD simulations, we investigate mechanisms underlying the binding by NR3A and NR3B of glycine and D-serine, which are candidate neurotransmitters for NMDA receptors containing NR3 subunits. The ligand binding domains of both NR3 subunits adopt a similar extent of domain closure as found in the corresponding NR1 complexes, but have a unique loop 1 structure distinct from that in all other glutamate receptor ion channels. Within their ligand binding pockets, NR3A and NR3B have strikingly different hydrogen bonding networks and solvent structures from those found in NR1, and fail to undergo a conformational rearrangement observed in NR1 upon binding the partial agonist ACPC. MD simulations revealed numerous interdomain contacts, which stabilize the agonist-bound closed-cleft conformation, and a novel twisting motion for the loop 1 helix that is unique in NR3 subunits.

  17. [3H]CGP 61594, the first photoaffinity ligand for the glycine site of NMDA receptors

    Benke, D.; Honer, M.; Mohler, H.; Heckendorn, R.; Pozza, M.F.; Allgeier, H.; Angst, C.

    1999-01-01

    Activation of NMDA receptors requires the presence of glycine as a coagonist which binds to a site that is allosterically linked to the glutamate binding site. To identify the protein constituents of the glycine binding site in situ the photoaffinity label [ 3 H]CGP 61594 was synthesized. In reversible binding assays using crude rat brain membranes, [ 3 H]CGP 61594 labeled with high affinity (K D =23 nM) the glycine site of the NMDA receptor. This was evident from the Scatchard analysis, the displacing potencies of various glycine site ligands and the allosteric modulation of [ 3 H]CGP 61594 binding by ligands of the glutamate and polyamine sites. Electrophysiological experiments in a neocortical slice preparation identified CGP 61594 as a glycine antagonist. Upon UV-irradiation, a protein band of 115 kDa was specifically photolabeled by [ 3 H]CGP 61594 in brain membrane preparations. The photolabeled protein was identified as the NR1 subunit of the NMDA receptor by NR1 subunit-specific immunoaffinity chromatography. Thus, [ 3 H]CGP 61594 is the first photoaffinity label for the glycine site of NMDA receptors. It will serve as a tool for the identification of structural elements that are involved in the formation of the glycine binding domain of NMDA receptors in situ and will thereby complement the mutational analysis of recombinant receptors. (Copyright (c) 1999 Elsevier Science B.V., Amsterdam. All rights reserved.)

  18. Metabotropic glutamatergic receptors and their ligands in drug addiction.

    Pomierny-Chamioło, Lucyna; Rup, Kinga; Pomierny, Bartosz; Niedzielska, Ewa; Kalivas, Peter W; Filip, Małgorzata

    2014-06-01

    Glutamatergic excitatory transmission is implicated in physiological and pathological conditions like learning, memory, neuronal plasticity and emotions, while glutamatergic abnormalities are reported in numerous neurological and psychiatric disorders, including neurodegenerative diseases, epilepsy, stroke, traumatic brain injury, depression, anxiety, schizophrenia and pain. Also, several lines of evidence have accumulated indicating a pivotal role for glutamatergic neurotransmission in mediating addictive behaviors. Among the proteins regulating glutamatergic transmission, the metabotropic glutamate receptors (mGluR) are being developed as pharmacological targets for treating many neuropsychiatric disorders, including drug addiction. In this review we describe the molecular structure of mGluRs and their distribution, physiology and pharmacology in the central nervous system, as well as their use as targets in preclinical studies of drug addiction. Copyright © 2013 Elsevier Inc. All rights reserved.

  19. Effects of serotonin (5-HT)1B receptor ligands on amphetamine-seeking behavior in rats.

    Miszkiel, Joanna; Przegaliński, Edmund

    2013-01-01

    Numerous studies have indicated that serotonin (5-HT)1B receptor ligands affect the behavioral effects of psychostimulants (cocaine, amphetamine), including the reinforcing activities of these drugs. To substantiate a role for those receptors in incentive motivation for amphetamine, we used the extinction/reinstatement model to examine the effects of the 5-HT1B receptor ligands on the reinstatement of extinguished amphetamine-seeking behavior. Rats trained to self-administer amphetamine (0.06 mg/kg/infusion) subsequently underwent the extinction procedure. These rats were then tested for the amphetamine-primed or amphetamine-associated cue-induced reinstatement of extinguished amphetamine-seeking behavior. The 5-HT1B receptor antagonist SB 216641 (5-7.5 mg/kg) attenuated the amphetamine (1.5 mg/kg)- and the amphetamine-associated cue combined with the threshold dose of amphetamine (0.5 mg/kg)-induced reinstatement of amphetamine-seeking behavior. The 5-HT1B receptor agonist CP 94253 (1.25-5 mg/kg) also inhibited the amphetamine-seeking behavior induced by amphetamine (1.5 mg/kg) but not by the cue combined with the threshold dose of amphetamine. The inhibitory effect of CP94253 on amphetamine-seeking behavior remained unaffected by the 5-HT1B receptor antagonist. Our results indicate that tonic activation of 5-HT1B receptors is involved in amphetamine- and cue-induced reinstatement of amphetamine-seeking behavior and that the inhibitory effects of 5-HT1B receptor antagonists on these phenomena are directly related to the motivational aspects of amphetamine abuse. The inhibitory effect of CP 94253 on amphetamine-seeking behavior seems to be unrelated to 5-HT1B receptor activation and may result from a general reduction of motivation.

  20. Preparation and evaluation of an astatine-211-labeled sigma receptor ligand for alpha radionuclide therapy

    Ogawa, Kazuma; Mizuno, Yoshiaki; Washiyama, Kohshin; Shiba, Kazuhiro; Takahashi, Naruto; Kozaka, Takashi; Watanabe, Shigeki; Shinohara, Atsushi; Odani, Akira

    2015-01-01

    Introduction: Sigma receptors are overexpressed in a variety of human tumors, making them potential targets for radionuclide receptor therapy. We have previously synthesized and evaluated 131 I-labeled (+)-2-[4-(4-iodophenyl)piperidino]cyclohexanol [(+)-[ 131 I]pIV], which has a high affinity for sigma receptors. Therefore, (+)-[ 131 I]pIV significantly inhibited tumor cell proliferation in tumor-bearing mice. In the present study, we report the synthesis and the in vitro and in vivo characterization of (+)-[ 211 At]pAtV, an 211 At-labeled sigma receptor ligand, that has potential use in alpha-radionuclide receptor therapy. Methods: The radiolabeled sigma receptor ligand (+)-[ 211 At]pAtV was prepared using a standard halogenation reaction generating a 91% radiochemical yield with 98% purity after HPLC purification. The partition coefficient of (+)-[ 211 At]pAtV was measured. Cellular uptake experiments and in vivo biodistribution experiments were performed using a mixed solution of (+)-[ 211 At]pAtV and (+)-[ 125 I]pIV; the human prostate cancer cell line DU-145, which expresses high levels of the sigma receptors, and DU-145 tumor-bearing mice. Results: The lipophilicity of (+)-[ 211 At]pAtV was similar to that of (+)-[ 125 I]pIV. DU-145 cellular uptake and the biodistribution patterns in DU-145 tumor-bearing mice at 1 h post-injection were also similar between (+)-[ 211 At]pAtV and (+)-[ 125 I]pIV. Namely, (+)-[ 211 At]pAtV demonstrated high uptake and retention in tumor via binding to sigma receptors. Conclusion: These results indicate that (+)-[ 211 At]pAtV could function as an new agent for alpha-radionuclide receptor therapy.

  1. Preparation and evaluation of an astatine-211-labeled sigma receptor ligand for alpha radionuclide therapy.

    Ogawa, Kazuma; Mizuno, Yoshiaki; Washiyama, Kohshin; Shiba, Kazuhiro; Takahashi, Naruto; Kozaka, Takashi; Watanabe, Shigeki; Shinohara, Atsushi; Odani, Akira

    2015-11-01

    Sigma receptors are overexpressed in a variety of human tumors, making them potential targets for radionuclide receptor therapy. We have previously synthesized and evaluated (131)I-labeled (+)-2-[4-(4-iodophenyl)piperidino]cyclohexanol [(+)-[(131)I]pIV], which has a high affinity for sigma receptors. Therefore, (+)-[(131)I]pIV significantly inhibited tumor cell proliferation in tumor-bearing mice. In the present study, we report the synthesis and the in vitro and in vivo characterization of (+)-[(211)At]pAtV, an (211)At-labeled sigma receptor ligand, that has potential use in alpha-radionuclide receptor therapy. The radiolabeled sigma receptor ligand (+)-[(211)At]pAtV was prepared using a standard halogenation reaction generating a 91% radiochemical yield with 98% purity after HPLC purification. The partition coefficient of (+)-[(211)At]pAtV was measured. Cellular uptake experiments and in vivo biodistribution experiments were performed using a mixed solution of (+)-[(211)At]pAtV and (+)-[(125)I]pIV; the human prostate cancer cell line DU-145, which expresses high levels of the sigma receptors, and DU-145 tumor-bearing mice. The lipophilicity of (+)-[(211)At]pAtV was similar to that of (+)-[(125)I]pIV. DU-145 cellular uptake and the biodistribution patterns in DU-145 tumor-bearing mice at 1h post-injection were also similar between (+)-[(211)At]pAtV and (+)-[(125)I]pIV. Namely, (+)-[(211)At]pAtV demonstrated high uptake and retention in tumor via binding to sigma receptors. These results indicate that (+)-[(211)At]pAtV could function as an new agent for alpha-radionuclide receptor therapy. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. Novel chalcone-based fluorescent human histamine H3 receptor ligands as pharmacological tools

    Holger eStark

    2012-03-01

    Full Text Available Novel fluorescent chalcone-based ligands at human histamine H3 receptors (hH3R have been designed, synthesized and characterized. Compounds described are non-imidazole analogues of ciproxifan with a tetralone motif. Tetralones as chemical precursors and related fluorescent chalcones exhibit affinities at hH3R in the same concentration range like that of the reference antagonist ciproxifan (hH3R pKi value of 7.2. Fluorescence characterization of our novel ligands shows emission maxima about 570 nm for yellow fluorescent chalcones and ≥600 nm for the red fluorescent derivatives. Interferences to cellular autofluorescence could be excluded. All synthesized chalcone compounds could be taken to visualize hH3R proteins in stably transfected HEK-293 cells using confocal laser scanning fluorescence microscopy. These novel fluorescent ligands possess high potential to be used as pharmacological tools for hH3R visualization in different tissues.

  3. Peptides identify multiple hotspots within the ligand binding domain of the TNF receptor 2

    Lennick Michael

    2003-01-01

    Full Text Available Abstract Background Hotspots are defined as the minimal functional domains involved in protein:protein interactions and sufficient to induce a biological response. Results Here we describe the use of complex and high diversity phage display libraries to isolate peptides (called Hotspot Ligands or HSPLs which sub-divide the ligand binding domain of the tumor necrosis factor receptor 2 (TNFR2; p75 into multiple hotspots. We have shown that these libraries could generate HSPLs which not only subdivide hotspots on protein and non-protein targets but act as agonists or antagonists. Using this approach, we generated peptides which were specific for human TNFR2, could be competed by the natural ligands, TNFα and TNFβ and induced an unexpected biological response in a TNFR2-specific manner. Conclusions To our knowledge, this is the first report describing the dissection of the TNFR2 into biologically active hotspots with the concomitant identification of a novel and unexpected biological activity.

  4. Ligand-specific regulation of the extracellular surface of a G-protein-coupled receptor

    Bokoch, Michael P; Zou, Yaozhong; Rasmussen, Søren Gøgsig Faarup

    2010-01-01

    extending from the orthosteric ligand-binding site in the transmembrane core to the cytoplasmic G-protein-coupling domains. In contrast, the extracellular surface (ECS) of GPCRs is remarkably diverse and is therefore an ideal target for the discovery of subtype-selective drugs. However, little is known...... conformational coupling between the ECS and the orthosteric binding site, showing that drugs targeting this diverse surface could function as allosteric modulators with high subtype selectivity. Moreover, these studies provide a new insight into the dynamic behaviour of GPCRs not addressable by static, inactive...... about the functional role of the ECS in receptor activation, or about conformational coupling of this surface to the native ligand-binding pocket. Here we use NMR spectroscopy to investigate ligand-specific conformational changes around a central structural feature in the ECS of the beta(2) adrenergic...

  5. Titration ELISA as a Method to Determine the Dissociation Constant of Receptor Ligand Interaction.

    Eble, Johannes A

    2018-02-15

    The dissociation constant describes the interaction between two partners in the binding equilibrium and is a measure of their affinity. It is a crucial parameter to compare different ligands, e.g., competitive inhibitors, protein isoforms and mutants, for their binding strength to a binding partner. Dissociation constants are determined by plotting concentrations of bound versus free ligand as binding curves. In contrast, titration curves, in which a signal that is proportional to the concentration of bound ligand is plotted against the total concentration of added ligand, are much easier to record. The signal can be detected spectroscopically and by enzyme-linked immunosorbent assay (ELISA). This is exemplified in a protocol for a titration ELISA that measures the binding of the snake venom-derived rhodocetin to its immobilized target domain of α2β1 integrin. Titration ELISAs are versatile and widely used. Any pair of interacting proteins can be used as immobilized receptor and soluble ligand, provided that both proteins are pure, and their concentrations are known. The difficulty so far has been to determine the dissociation constant from a titration curve. In this study, a mathematical function underlying titration curves is introduced. Without any error-prone graphical estimation of a saturation yield, this algorithm allows processing of the raw data (signal intensities at different concentrations of added ligand) directly by mathematical evaluation via non-linear regression. Thus, several titration curves can be recorded simultaneously and transformed into a set of characteristic parameters, among them the dissociation constant and the concentration of binding-active receptor, and they can be evaluated statistically. When combined with this algorithm, titration ELISAs gain the advantage of directly presenting the dissociation constant. Therefore, they may be used more efficiently in the future.

  6. Ligand-selective activation of heterologously-expressed mammalian olfactory receptor.

    Ukhanov, K; Bobkov, Y; Corey, E A; Ache, B W

    2014-10-01

    Mammalian olfactory receptors (ORs) appear to have the capacity to couple to multiple G protein-coupled signaling pathways in a ligand-dependent selective manner. To better understand the mechanisms and molecular range of such ligand selectivity, we expressed the mouse eugenol OR (mOR-EG) in HEK293T cells together with Gα15 to monitor activation of the phospholipase-C (PLC) signaling pathway and/or Gαolf to monitor activation of the adenylate cyclase (AC) signaling pathway, resulting in intracellular Ca(2+) release and/or Ca(2+) influx through a cyclic nucleotide-gated channel, respectively. PLC-dependent responses differed dynamically from AC-dependent responses, allowing them to be distinguished when Gα15 and Gαolf were co-expressed. The dynamic difference in readout was independent of the receptor, the heterologous expression system, and the ligand concentration. Of 17 reported mOR-EG ligands tested, including eugenol, its analogs, and structurally dissimilar compounds (mousse cristal, nootkatone, orivone), some equally activated both signaling pathways, some differentially activated both signaling pathways, and some had no noticeable effect even at 1-5mM. Our findings argue that mOR-EG, when heterologously expressed, can couple to two different signaling pathways in a ligand selective manner. The challenge now is to determine the potential of mOR-EG, and perhaps other ORs, to activate multiple signaling pathways in a ligand selective manner in native ORNs. Copyright © 2014 Elsevier Ltd. All rights reserved.

  7. Anti-nociceptive effect of patchouli alcohol: Involving attenuation of cyclooxygenase 2 and modulation of mu-opioid receptor.

    Yu, Xuan; Wang, Xin-Pei; Yan, Xiao-Jin; Jiang, Jing-Fei; Lei, Fan; Xing, Dong-Ming; Guo, Yue-Ying; Du, Li-Jun

    2017-08-09

    To explore the anti-nociceptive effect of patchouli alcohol (PA), the essential oil isolated from Pogostemon cablin (Blanco) Bent, and determine the mechanism in molecular levels. The acetic acid-induced writhing test and formalin-induced plantar injection test in mice were employed to confifirm the effect in vivo. Intracellular calcium ion was imaged to verify PA on mu-opioid receptor (MOR). Cyclooxygenase 2 (COX2) and MOR of mouse brain were expressed for determination of PA's target. Cellular experiments were carried out to find out COX2 and MOR expression induced by PA. PA significantly reduced latency period of visceral pain and writhing induced by acetic acid saline solution (Peffect of PA. A decrease in the intracellular calcium level (Peffect. PA showed the characters of enhancing the MOR expression and reducing the intracellular calcium ion similar to opioid effect. Both COX2 and MOR are involved in the mechanism of PA's anti-nociceptive effect, and the up-regulation of the receptor expression and the inhibition of intracellular calcium are a new perspective to PA's effect on MOR.

  8. Development of N-substituted quinolinimides, as potential PET tracers for the visualisation of δ-opioid receptors

    Bourdier, Th.

    2005-12-01

    In order to develop radiotracers for in vivo studies of δ-opioid receptors by Positron Emission Tomography (PET) or Single Photon Emission computed Tomography (SPECT), we undertook the synthesis of halogenated analogues (chlorinated and brominated) of compound 12. These analogues were prepared by a convergent synthesis and from these novel structures a halogen exchange reaction has been performed to complete this series. These molecules were tested to determine their in vitro affinity and selectivity toward δ opioid receptors. The compounds 12 and 15 were labelled with carbon-11. The radiosynthesis of compound 12, in weak radioactivity chemistry, was performed first by the Stille reaction and second by a new methodology based on the transfer reaction of [ 11 C]-methyl group. This new methodology used a mono-organotin compound prepared by addition of [ 11 C]-iodomethane onto Lappert's stannylene. The compound [ 11 C]-12 was obtained with 60 and 10% radiochemical yield respectively. In order to produce higher radioactivity quantities, the Stille reaction was automated. The compounds [ 11 C]-12 and [ 11 C]-15 were obtained in 40 minutes with a specific radioactivity ranging from 322 to 747 mCi/μmol. (author)

  9. Local analgesic effect of tramadol is not mediated by opioid receptors in early postoperative pain in rats

    Angela Maria Sousa

    2015-05-01

    Full Text Available Background and objectives: Tramadol is known as a central acting analgesic drug, used for the treatment of moderate to severe pain. Local analgesic effect has been demonstrated, in part due to local anesthetic-like effect, but other mechanisms remain unclear. The role of peripheral opioid receptors in the local analgesic effect is not known. In this study, we examined role of peripheral opioid receptors in the local analgesic effect of tramadol in the plantar incision model. Methods: Young male Wistar rats were divided into seven groups: control, intraplantar tramadol, intravenous tramadol, intravenous naloxone-intraplantar tramadol, intraplantar naloxone-intraplantar tramadol, intravenous naloxone-intravenous tramadol, and intravenous naloxone. After receiving the assigned drugs (tramadol 5 mg, naloxone 200 μg or 0.9% NaCl, rats were submitted to plantar incision, and withdrawal thresholds after mechanical stimuli with von Frey filaments were assessed at baseline, 10, 15, 30, 45 and 60 min after incision. Results: Plantar incision led to marked mechanical hyperalgesia during the whole period of observation in the control group, no mechanical hyperalgesia were observed in intraplantar tramadol group, intraplantar naloxone-intraplantar tramadol group and intravenous naloxone-intraplantar tramadol. In the intravenous tramadol group a late increase in withdrawal thresholds (after 45 min was observed, the intravenous naloxone-intravenous tramadol group and intravenous naloxone remained hyperalgesic during the whole period. Conclusions: Tramadol presented an early local analgesic effect decreasing mechanical hyperalgesia induced by plantar incision. This analgesic effect was not mediated by peripheral opioid receptors. Resumo: Justificativa e objetivos: Tramadol é conhecido como um fármaco analgésico de ação central, usado para o tratamento de dor moderada a grave. O efeito analgésico local foi demonstrado, em parte devido ao efeito

  10. Spinal antinociceptive effects of [D-Ala2]deltorphin II, a novel and highly selective delta-opioid receptor agonist.

    Improta, G; Broccardo, M

    1992-01-01

    Pharmacological assays in isolated tissues and binding tests have recently shown that two peptides, with the sequence Tyr-D-Ala-Phe-Asp-(or Glu)- Val-Val-Gly-NH2, isolated from skin extracts of Phyllomedusa bicolor and named [D-Ala2]deltorphin I and II, respectively, possess a higher affinity and selectivity for delta-opioid receptors than any other known natural compound. Since much evidence supports the role of spinal delta-opioid sites in producing antinociceptive effects, we investigated whether analgesia might be detected by direct spinal cord administration of [D-Ala2]deltorphin II (DADELT II) in the rat. The thermal antinociceptive effects of intrathecal DADELT II and dermorphin, a potent mu-selective agonist, were compared at different postinjection times by means of the tail-flick test. The DADELT II produced a dose-related inhibition of the tail-flick response, which lasted 10-60 min depending on the dose and appeared to be of shorter duration than the analgesia produced in rats after intrathecal injection of dermorphin (20-120 min). The analgesic effect of infused or injected DADELT II was completely abolished by naltrindole, the highly selective delta antagonist. These results confirm the involvement of delta receptors in spinal analgesic activity in the rat.

  11. Antidiabetic actions of a phosphatidylcholine ligand for nuclear receptor LRH-1

    Lee, Jae Man; Lee, Yoon Kwang; Mamrosh, Jennifer L.; Busby, Scott A.; Griffin, Patrick R.; Pathak, Manish C.; Ortlund, Eric A.; Moore, David D.

    2011-01-01

    Nuclear hormone receptors regulate diverse metabolic pathways and the orphan nuclear receptor LRH-1 (NR5A2) regulates bile acid biosynthesis1,2. Structural studies have identified phospholipids as potential LRH-1 ligands3–5, but their functional relevance is unclear. Here we show that an unusual phosphatidylcholine species with two saturated 12 carbon fatty acid acyl side chains (dilauroyl phosphatidylcholine, DLPC) is an LRH-1 agonist ligand in vitro. DLPC treatment induces bile acid biosynthetic enzymes in mouse liver, increases bile acid levels, and lowers hepatic triglycerides and serum glucose. DLPC treatment also decreases hepatic steatosis and improves glucose homeostasis in two mouse models of insulin resistance. Both the antidiabetic and lipotropic effects are lost in liver specific Lrh-1 knockouts. These findings identify an LRH-1 dependent phosphatidylcholine signaling pathway that regulates bile acid metabolism and glucose homeostasis. PMID:21614002

  12. submitter Emerging importance of chemokine receptor CXCR3 and its ligands in cardiovascular diseases

    Altara, R; Brandao, R D; Zeidan, A; Booz, G W; Zouein, F A

    2016-01-01

    The CXC chemokines, CXCL4, -9, -10, -11, CXCL4L1, and the CC chemokine CCL21, activate CXC chemokine receptor 3 (CXCR3), a cell-surface G protein-coupled receptor expressed mainly by Th1 cells, cytotoxic T (Tc) cells and NK cells that have a key role in immunity and inflammation. However, CXCR3 is also expressed by vascular smooth muscle and endothelial cells, and appears to be important in controlling physiological vascular function. In the last decade, evidence from pre-clinical and clinical studies has revealed the participation of CXCR3 and its ligands in multiple cardiovascular diseases (CVDs) of different aetiologies including atherosclerosis, hypertension, cardiac hypertrophy and heart failure, as well as in heart transplant rejection and transplant coronary artery disease (CAD). CXCR3 ligands have also proven to be valid biomarkers for the development of heart failure and left ventricular dysfunction, suggesting an underlining pathophysiological relation between levels of these chemokines and the deve...

  13. Design of ligands for the nicotinic acetylcholine receptors: the quest for selectivity.

    Bunnelle, William H; Dart, Michael J; Schrimpf, Michael R

    2004-01-01

    In the last decade, nicotinic acetylcholine receptors (nAChRs) have emerged as important targets for drug discovery. The therapeutic potential of nicotinic agonists depends substantially on the ability to selectively activate certain receptor subtypes that mediate beneficial effects. The design of such compounds has proceeded in spite of a general shortage of data pertaining to subtype selectivity. Medicinal chemistry efforts have been guided principally by binding affinities to the alpha4beta2 and/or alpha7 subtypes, even though these are not predictive of agonist activity at either subtype. Nevertheless, a diverse family of nAChR ligands has been developed, and several analogs with promising therapeutic potential have now advanced to human clinical trials. This paper provides an overview of the structure-affinity relationships that continue to drive development of new nAChR ligands.

  14. Labeling of receptor ligands with bromine radionuclides. Progress report, March 1, 1981-February 28, 1982

    Welch, M.J.

    1981-10-01

    In recent years there has been an interest in the use of various radioisotopes of bromine as labels for radiopharmaceuticals. Although radioisotopes of iodine have been used extensively as radiopharmaceutical labels, there are several advantages associated with the use of radiobromine as a label, due primarily to increased stability of bonds to the radiohalide and smaller steric perturbation resulting from substitution of the radiohalide. Methods of attaching radiobromine to receptor ligands with the potential of mapping estrogen receptors in mammary tumors and uteri were studied. Two ligands were studied extensively in vitro and in animal models; preliminary studies were also carried out in humans. To date, the only radioisotope of bromine used was bromine-77. In addition, a series of model compounds were labeled with bromine-77 using a recently described method for rapid bromination; the scope and limitations of this new rapid radiobromination technique were evaluated

  15. Dopamine D4 Receptor Counteracts Morphine-Induced Changes in µ Opioid Receptor Signaling in the Striosomes of the Rat Caudate Putamen

    Diana Suárez-Boomgaard

    2014-01-01

    Full Text Available The mu opioid receptor (MOR is critical in mediating morphine analgesia. However, prolonged exposure to morphine induces adaptive changes in this receptor leading to the development of tolerance and addiction. In the present work we have studied whether the continuous administration of morphine induces changes in MOR protein levels, its pharmacological profile, and MOR-mediated G-protein activation in the striosomal compartment of the rat CPu, by using immunohistochemistry and receptor and DAMGO-stimulated [35S]GTPγS autoradiography. MOR immunoreactivity, agonist binding density and its coupling to G proteins are up-regulated in the striosomes by continuous morphine treatment in the absence of changes in enkephalin and dynorphin mRNA levels. In addition, co-treatment of morphine with the dopamine D4 receptor (D4R agonist PD168,077 fully counteracts these adaptive changes in MOR, in spite of the fact that continuous PD168,077 treatment increases the [3H]DAMGO Bmax values to the same degree as seen after continuous morphine treatment. Thus, in spite of the fact that both receptors can be coupled to Gi/0 protein, the present results give support for the existence of antagonistic functional D4R-MOR receptor-receptor interactions in the adaptive changes occurring in MOR of striosomes on continuous administration of morphine.

  16. The effects of opioid drugs on dopamine mediated locomotor activity in rats

    Leathern, L.L.

    1986-12-01

    Opioid drugs influence various behavioural parameters including locomotor activity in experimental animals. The interaction between the opioid and dopaminergic systems is one possible explanation for the effect of opioid drugs on locomotor activity. In this study behavioural and biochemical assays were done to investigate the interaction between the opioid and dopaminergic systems. Behavioural studies were done by measurement of locomotor activity (LA) of rats after acute or chronic pretreatment with opioid and/or dopaminergic drugs. Biochemical studies were in the form of radioligand binding assays, the effect on the number (Bmax) and affinity (K D ) of receptors was measured after chronic pretreatment with opioid and/or dopaminergic drugs. The opioid drugs used are morphine, nalbuphine and naloxone. Dopaminergic drugs used included: agonists-apomorphine and piribedil; antagonists-pimozide, haloperidol, chlorpromazine. In the acute situation increased LA was obtained with morphine and the DA agonists. A correlation between the behavioural and biochemical assays was found. Chronic pretreatment with morphine enhanced apomorphine induced LA, this supersensitivity was also measured as an increased receptor density (Bmax) of D2 receptors in the striatum. Chronic morphine pretreatment caused a decrease in morphine induced LA, while this subsensitivity was not apparent in the ligand binding assays - where no change in receptor number was observed. Chronic naloxone pretreatment enhanced morphine induced LA, as well as increased the Bmax of opioid receptors in the whole brain. It is concluded that an interaction between the opioid and dopaminergic systems does exist, and may account for the mechanism of action of the opioids

  17. Anions mediate ligand binding in Adineta vaga glutamate receptor ion channels.

    Lomash, Suvendu; Chittori, Sagar; Brown, Patrick; Mayer, Mark L

    2013-03-05

    AvGluR1, a glutamate receptor ion channel from the primitive eukaryote Adineta vaga, is activated by alanine, cysteine, methionine, and phenylalanine, which produce lectin-sensitive desensitizing responses like those to glutamate, aspartate, and serine. AvGluR1 LBD crystal structures reveal an unusual scheme for binding dissimilar ligands that may be utilized by distantly related odorant/chemosensory receptors. Arginine residues in domain 2 coordinate the γ-carboxyl group of glutamate, whereas in the alanine, methionine, and serine complexes a chloride ion acts as a surrogate ligand, replacing the γ-carboxyl group. Removal of Cl(-) lowers affinity for these ligands but not for glutamate or aspartate nor for phenylalanine, which occludes the anion binding site and binds with low affinity. AvGluR1 LBD crystal structures and sedimentation analysis also provide insights into the evolutionary link between prokaryotic and eukaryotic iGluRs and reveal features unique to both classes, emphasizing the need for additional structure-based studies on iGluR-ligand interactions. Copyright © 2013 Elsevier Ltd. All rights reserved.

  18. Thyroid hormone and retinoic acid nuclear receptors: specific ligand-activated transcription factors

    Brtko, J.

    1998-01-01

    Transcriptional regulation by both the thyroid hormone and the vitamin A-derived 'retinoid hormones' is a critical component in controlling many aspects of higher vertebrate development and metabolism. Their functions are mediated by nuclear receptors, which comprise a large super-family of ligand-inducible transcription factors. Both the thyroid hormone and the retinoids are involved in a complex arrangement of physiological and development responses in many tissues of higher vertebrates. The functions of 3,5,3'-triiodothyronine (T 3 ), the thyromimetically active metabolite of thyroxine as well as all-trans retinoic acid, the biologically active vitamin A metabolite are mediated by nuclear receptor proteins that are members of the steroid/thyroid/retinoid hormone receptor family. The functions of all members of the receptor super family are discussed. (authors)

  19. Synthesis and radiofluorination of putative NMDA receptor ligands

    Kronenberg, U

    2011-01-15

    In the course of this work on the synthesis of radioligands for the NMDA receptor the authentic standards and labeling precursors of four compounds with an amidine structure was performed. Synthesis of the precursors followed reaction conditions given in the literature and was successful. The imidoesters used for the synthesis were obtained from their nitriles in a Pinner synthesis, while 2-hydroxybenzylamine was synthesized in a reduction of 2-hydroxybenzonitrile using borane as a reducing agent. After a coupling reaction of the amine and the imidoester in DMF using triethylamine as base the precursors were obtained in good yields and purified by crystallization from methanol. The cyclic standard compound was synthesized directly from 2-(bromomethyl)- benzonitrile and 2-hydroxybenzylamine in a ring closing reaction. Similar to the other precursors, crystallization from methanol produced a pure compound. The authentic standards were synthesized starting from salicylaldehyde. In a four step synthesis the desired ortho-fluoroethoxybenzylamine was obtained in good yield. Coupling of the amine with the respective imidoester or in the case of the cyclic compound 2-(bromomethyl)-benzonitrile gave the desired product which was then purified by column chromatography or by crystallization from ethanol and water. For the labeling procedure 1-bromo-2-[{sub 18}F]fluoroethane was synthesized following a previously published pathway starting from 1,2-dibromoethane. An alternative route of radiosynthesis for this prosthetic group was tested using ethyleneglycole- 1,2-ditosylate. The labeling reaction was performed on one of the precursors testing both DMF and DMSO as solvents and using NaOH as base. Yields of N-(2-fluoroethoxybenzyl)- cinnamamidine were about 78 % at 80 C after 30 minutes in DMSO. The desired product can now be synthesized in sufficient yields for in vitro and in vivo evaluation studies. Labeling on the cyclic precursor was attempted utilizing DMSO as solvent

  20. Synthesis and radiofluorination of putative NMDA receptor ligands

    Kronenberg, U.

    2011-01-01

    In the course of this work on the synthesis of radioligands for the NMDA receptor the authentic standards and labeling precursors of four compounds with an amidine structure was performed. Synthesis of the precursors followed reaction conditions given in the literature and was successful. The imidoesters used for the synthesis were obtained from their nitriles in a Pinner synthesis, while 2-hydroxybenzylamine was synthesized in a reduction of 2-hydroxybenzonitrile using borane as a reducing agent. After a coupling reaction of the amine and the imidoester in DMF using triethylamine as base the precursors were obtained in good yields and purified by crystallization from methanol. The cyclic standard compound was synthesized directly from 2-(bromomethyl)- benzonitrile and 2-hydroxybenzylamine in a ring closing reaction. Similar to the other precursors, crystallization from methanol produced a pure compound. The authentic standards were synthesized starting from salicylaldehyde. In a four step synthesis the desired ortho-fluoroethoxybenzylamine was obtained in good yield. Coupling of the amine with the respective imidoester or in the case of the cyclic compound 2-(bromomethyl)-benzonitrile gave the desired product which was then purified by column chromatography or by crystallization from ethanol and water. For the labeling procedure 1-bromo-2-[ 18 F]fluoroethane was synthesized following a previously published pathway starting from 1,2-dibromoethane. An alternative route of radiosynthesis for this prosthetic group was tested using ethyleneglycole- 1,2-ditosylate. The labeling reaction was performed on one of the precursors testing both DMF and DMSO as solvents and using NaOH as base. Yields of N-(2-fluoroethoxybenzyl)- cinnamamidine were about 78 % at 80 C after 30 minutes in DMSO. The desired product can now be synthesized in sufficient yields for in vitro and in vivo evaluation studies. Labeling on the cyclic precursor was attempted utilizing DMSO as solvent, but no

  1. Rapid, portable detection of endocrine disrupting chemicals through ligand-nuclear hormone receptor interactions.

    Hunt, J Porter; Schinn, Song-Min; Jones, Matthew D; Bundy, Bradley C

    2017-12-04

    Endocrine disrupting chemicals (EDC) are structurally diverse compounds that can interact with nuclear hormone receptors, posing significant risk to human and ecological health. Unfortunately, many conventional biosensors have been too structure-specific, labor-intensive or laboratory-oriented to detect broad ranges of EDC effectively. Recently, several technological advances are providing more rapid, portable, and affordable detection of endocrine-disrupting activity through ligand-nuclear hormone receptor interactions. Here, we overview these recent advances applied to EDC biosensors - including cell lyophilization, cell immobilization, cell-free systems, smartphone-based signal detection, and improved competitive binding assays.

  2. Ligand Modulation of the Epstein-Barr Virus-induced Seven-transmembrane Receptor EBI2

    Benned-Jensen, Tau; Smethurst, Christopher; Holst, Peter Johannes

    2011-01-01

    The Epstein-Barr virus-induced receptor 2 (EBI2) is a constitutively active seven-transmembrane receptor, which was recently shown to orchestrate the positioning of B cells in the follicle. To date, no ligands, endogenously or synthetic, have been identified that modulate EBI2 activity. Here we...... with similar potency. Overexpression of EBI2 profoundly potentiated antibody-stimulated ex vivo proliferation of murine B cells compared with WT cells, whereas this was equivalently reduced for EBI2-deficient B cells. Inhibition of EBI2 constitutive activity suppressed the proliferation in all cases...

  3. Quantitative autoradiography of ligands for dopamine receptors and transporters in brain of Göttingen minipig

    Minuzzi, Luciano; Olsen, Aage Kristian; Bender, Dirk

    2006-01-01

    The pig has been used as animal model for positron emission tomography (PET) studies of dopamine (DA) receptors and pharmacological perturbations of DA neurotransmission. However, the binding properties of DA receptors and transporters in pig brain have not been characterized in vitro. Therefore...... in young and old pigs, and were close to those reported for rat and human brain. Furthermore, gradients in the concentrations of D1 and D2/3 sites in striatum measured in vitro agreed with earlier findings in PET studies. However, the dopamine transporter (DAT) ligand [3H]GBR12935 did not bind in pig brain...

  4. Effects of coumestrol on lipid and glucose metabolism as a farnesoid X receptor ligand

    Takahashi, Miki; Kanayama, Tomohiko; Yashiro, Takuya; Kondo, Hidehiko; Murase, Takatoshi; Hase, Tadashi; Tokimitsu, Ichiro; Nishikawa, Jun-ichi; Sato, Ryuichiro

    2008-01-01

    In the course of an effort to identify novel agonists of the farnesoid X receptor (FXR), coumestrol was determined to be one such ligand. Reporter and in vitro coactivator interaction assays revealed that coumestrol bound and activated FXR. Treatment of Hep G2 cells with coumestrol stimulated the expression of FXR target genes, thereby regulating the expression of target genes of the liver X receptor and hepatocyte nuclear factor-4α. Through these actions, coumestrol is expected to exert beneficial effects on lipid and glucose metabolism

  5. Dopamine D3 receptor ligands for drug addiction treatment: update on recent findings.

    Le Foll, Bernard; Collo, Ginetta; Rabiner, Eugenii A; Boileau, Isabelle; Merlo Pich, Emilio; Sokoloff, Pierre

    2014-01-01

    The dopamine D3 receptor is located in the limbic area and apparently mediates selective effects on motivation to take drugs and drug-seeking behaviors, so that there has been considerable interest on the possible use of D3 receptor ligands to treat drug addiction. However, only recently selective tools allowing studying this receptor have been developed. This chapter presents an overview of findings that were presented at a symposium on the conference Dopamine 2013 in Sardinia in May 2013. Novel neurobiological findings indicate that drugs of abuse can lead to significant structural plasticity in rodent brain and that this is dependent on the availability of functional dopamine D3 autoreceptor, whose activation increased phosphorylation in the ERK pathway and in the Akt/mTORC1 pathway indicating the parallel engagement of a series of intracellular signaling pathways all involved in cell growth and survival. Preclinical findings using animal models of drug-seeking behaviors confirm that D3 antagonists have a promising profile to treat drug addiction across drugs of abuse type. Imaging the D3 is now feasible in human subjects. Notably, the development of (+)-4-propyl-9-hydroxynaphthoxazine ligand used in positron emission tomography (PET) studies in humans allows to measure D3 and D2 receptors based on the area of the brain under study. This PET ligand has been used to confirm up-regulation of D3 sites in psychostimulant users and to reveal that tobacco smoking produces elevation of dopamine at the level of D3 sites. There are now novel antagonists being developed, but also old drugs such as buspirone, that are available to test the D3 hypothesis in humans. The first results of clinical investigations are now being provided. Overall, those recent findings support further exploration of D3 ligands to treat drug addiction. © 2014 Elsevier B.V. All rights reserved.

  6. Microchemical synthesis of the serotonin receptor ligand, 125I-LSD

    Hartig, P.R.; Krohn, A.M.; Hirschman, S.A.

    1985-01-01

    The synthesis and properties of 2-[ 125 I]-lysergic acid diethylamide, the first 125 I-labeled serotonin receptor ligand, are described. A novel microsynthesis apparatus was developed for this synthesis. The apparatus employs a micromanipulator and glass micro tools to handle microliter to nanoliter volumes on a microscope stage. This apparatus should be generally useful for the synthesis of radioligands and other compounds when limited amounts of material must be handled in small volumes

  7. Different amounts of ejaculatory activity, a natural rewarding behavior, induce differential mu and delta opioid receptor internalization in the rat's ventral tegmental area.

    Garduño-Gutiérrez, René; León-Olea, Martha; Rodríguez-Manzo, Gabriela

    2013-12-06

    Opioid receptors internalize upon specific agonist stimulation. The in vivo significance of receptor internalization is not well established, partly due to the limited in vivo models used to study this phenomenon. Ejaculation promotes endogenous opioid release which activates opioid receptors at the brain, including the mesolimbic system and medial preoptic area. The objective of the present work was to analyze if there was a correlation between the degree of in vivo mu (MOR) and delta opioid receptor (DOR) internalization in the ventral tegmental area and the execution of different amounts of ejaculatory behavior of male rats. To this aim, we analyzed the brains of rats that ejaculated once or six successive times and of sexually exhausted rats with an established sexual inhibition, using immunofluorescence and confocal microscopy. Results showed that MOR and DOR internalization increased as a consequence of ejaculation. There was a relationship between the amount of sexual activity executed and the degree of internalization for MOR, but not for DOR. MOR internalization was larger in rats that ejaculated repeatedly than in animals ejaculating only once. Significant DOR internalization was found only in animals ejaculating once. Changes in MOR, DOR and beta arrestin2 detection, associated to sexual activity, were also found. It is suggested that copulation to satiety might be useful as a model system to study the biological significance of receptor internalization. © 2013 Published by Elsevier B.V.

  8. Monitoring ligand-dependent assembly of receptor ternary complexes in live cells by BRETFect.

    Cotnoir-White, David; El Ezzy, Mohamed; Boulay, Pierre-Luc; Rozendaal, Marieke; Bouvier, Michel; Gagnon, Etienne; Mader, Sylvie

    2018-03-13

    There is currently an unmet need for versatile techniques to monitor the assembly and dynamics of ternary complexes in live cells. Here we describe bioluminescence resonance energy transfer with fluorescence enhancement by combined transfer (BRETFect), a high-throughput technique that enables robust spectrometric detection of ternary protein complexes based on increased energy transfer from a luciferase to a fluorescent acceptor in the presence of a fluorescent intermediate. Its unique donor-intermediate-acceptor relay system is designed so that the acceptor can receive energy either directly from the donor or indirectly via the intermediate in a combined transfer, taking advantage of the entire luciferase emission spectrum. BRETFect was used to study the ligand-dependent cofactor interaction properties of the estrogen receptors ERα and ERβ, which form homo- or heterodimers whose distinctive regulatory properties are difficult to dissect using traditional methods. BRETFect uncovered the relative capacities of hetero- vs. homodimers to recruit receptor-specific cofactors and regulatory proteins, and to interact with common cofactors in the presence of receptor-specific ligands. BRETFect was also used to follow the assembly of ternary complexes between the V2R vasopressin receptor and two different intracellular effectors, illustrating its use for dissection of ternary protein-protein interactions engaged by G protein-coupled receptors. Our results indicate that BRETFect represents a powerful and versatile technique to monitor the dynamics of ternary interactions within multimeric complexes in live cells.

  9. The Prelude on Novel Receptor and Ligand Targets Involved in the Treatment of Diabetes Mellitus

    Venu Gopal Jonnalagadda

    2014-05-01

    Full Text Available Metabolic disorders are a group of disorders, due to the disruption of the normal metabolic process at a cellular level. Diabetes Mellitus and Tyrosinaemia are the majorly reported metabolic disorders. Among them, Diabetes Mellitus is a one of the leading metabolic syndrome, affecting 5 to 7 % of the population worldwide and mainly characterised by elevated levels of glucose and is associated with two types of physiological event disturbances such as impaired insulin secretion and insulin resistance. Up to now, various treatment strategies are like insulin, alphaglucosidase inhibitors, biguanides, incretins were being followed. Concurrently, various novel therapeutic strategies are required to advance the therapy of Diabetes mellitus. For the last few decades, there has been an extensive research in understanding the metabolic pathways involved in Diabetes Mellitus at the cellular level and having the profound knowledge on cell-growth, cell-cycle, and apoptosis at a molecular level provides new targets for the treatment of Diabetes Mellitus. Receptor signalling has been involved in these mechanisms, to translate the information coming from outside. To understand the various receptors involved in these pathways, we must have a sound knowledge on receptors and ligands involved in it. This review mainly summarises the receptors and ligands which are involved the Diabetes Mellitus. Finally, researchers have to develop the alternative chemical moieties that retain their affinity to receptors and efficacy. Diabetes Mellitus being a metabolic disorder due to the glucose surfeit, demands the need for regular exercise along with dietary changes.

  10. Characterizing ligand-gated ion channel receptors with genetically encoded Ca2++ sensors.

    John G Yamauchi

    2011-01-01

    Full Text Available We present a cell based system and experimental approach to characterize agonist and antagonist selectivity for ligand-gated ion channels (LGIC by developing sensor cells stably expressing a Ca(2+ permeable LGIC and a genetically encoded Förster (or fluorescence resonance energy transfer (FRET-based calcium sensor. In particular, we describe separate lines with human α7 and human α4β2 nicotinic acetylcholine receptors, mouse 5-HT(3A serotonin receptors and a chimera of human α7/mouse 5-HT(3A receptors. Complete concentration-response curves for agonists and Schild plots of antagonists were generated from these sensors and the results validate known pharmacology of the receptors tested. Concentration-response relations can be generated from either the initial rate or maximal amplitudes of FRET-signal. Although assaying at a medium throughput level, this pharmacological fluorescence detection technique employs a clonal line for stability and has versatility for screening laboratory generated congeners as agonists or antagonists on multiple subtypes of ligand-gated ion channels. The clonal sensor lines are also compatible with in vivo usage to measure indirectly receptor activation by endogenous neurotransmitters.

  11. Blocking opioid receptors alters short-term feed intake and oro-sensorial preferences in weaned calves.

    Montoro, C; Ipharraguerre, I R; Bach, A

    2012-05-01

    Opioid peptides may participate in the control of feed intake through mechanisms involving pleasure reward linked to consumption of palatable feed. The objective of this study was to determine whether blocking opioid receptors might void oro-sensorial preferences of calves, and affect circulating glucose, insulin, and anorexigenic hormones in fasted and fed calves. Two experiments involved 32 Holstein calves [body weight (BW)=86.5±1.73 kg, age=72±0.6 d]. In experiment 1, all calves received an ad libitum choice of the same feed either unflavored or flavored with a sweetener (Luctarom SFS-R, Lucta, Montornès del Vallès, Spain). Feed consumption was recorded every 2 h from 0800 to 1400 h for 3 consecutive days to verify the establishment of an oro-sensorial preference for sweet feed (SF). The SF was preferred over the control feed (CF) at all recorded times. In experiment 2, calves were subjected to a 2 × 2 factorial design to study the interaction between opioid activity and metabolic state. Half of the calves were fasted for 14 h (FAS), whereas the other half remained well fed (FED). Within each of these groups, at feeding time (0800 h), half of the calves received an i.v. injection of naloxone (NAL, an opioid receptor antagonist; 1 mg/kg of BW) and the other half was injected with saline solution (SAL; 0.9% NaCl). Therefore, treatments were FED-NAL, FED-SAL, FAS-NAL, and FAS-SAL. Blood samples were taken at -10, 20, 180, and 240 min relative to NAL or SAL injections. As expected, cumulative consumption of starter feed was greater in FAS than in FED calves. Total feed consumption 2 h after feeding was lower in NAL than in SAL calves. Calves in the FAS group did not discern between CF and SF during the first 4 h after feed offer. Preference for SF was greater in SAL than in NAL calves. Calves in the FED-SAL treatment preferred SF at 2 and 6 h after feed offer and tended to prefer SF at 4 h after feeding. However, FED-NAL calves did not discern between SF and CF

  12. General Linker Diversification Approach to Bivalent Ligand Assembly: Generation of an Array of Ligands for the Cation-Independent Mannose 6-Phosphate Receptor.

    Fei, Xiang; Zavorka, Megan E; Malik, Guillaume; Connelly, Christopher M; MacDonald, Richard G; Berkowitz, David B

    2017-08-18

    A generalized strategy is presented for the rapid assembly of a set of bivalent ligands with a variety of linking functionalities from a common monomer. Herein, an array of phosphatase-inert mannose-6-phosphonate-presenting ligands for the cation-independent-mannose 6-phosphate receptor (CI-MPR) is constructed. Receptor binding affinity varies with linking functionality-the simple amide and 1,5-triazole(tetrazole) being preferred over the 1,4-triazole. This approach is expected to find application across chemical biology, particularly in glycoscience, wherein multivalency often governs molecular recognition.

  13. Peripheral-type benzodiazepine receptor: a protein of mitochondrial outer membranes utilizing porphyrins as endogenous ligands

    Snyder, S.H.; Verma, A.; Trifiletti, R.R.

    1987-01-01

    The peripheral-type benzodiazepine receptor is a site identified by its nanomolar affinity for [ 3 H]diazepam, similar to the affinity of diazepam for the central-type benzodiazepine receptor in the brain. The peripheral type benzodiazepine receptor occurs in many peripheral tissues but has discrete localizations as indicated by autoradiographic studies showing uniquely high densities of the receptors in the adrenal cortex and in Leydig cells of the testes. Subcellular localization studies reveal a selective association of the receptors with the outer membrane of mitochondria. Photoaffinity labeling of the mitochondrial receptor with [ 3 H]flunitrazepam reveals two discrete labeled protein bands of 30 and 35 kDa, respectively. The 35-kDa band appears to be identical with the voltage-dependent anion channel protein porin. Fractionation of numerous peripheral tissues reveals a single principal endogenous ligand for the receptor, consisting of porphyrins, which display nanomolar affinity. Interactions of porphyrins with the mitochondrial receptor may clarify its physiological role and account for many pharmacological actions of benzodiazepines

  14. Attenuation of eph receptor kinase activation in cancer cells by coexpressed ephrin ligands.

    Giulia Falivelli

    Full Text Available The Eph receptor tyrosine kinases mediate juxtacrine signals by interacting "in trans" with ligands anchored to the surface of neighboring cells via a GPI-anchor (ephrin-As or a transmembrane segment (ephrin-Bs, which leads to receptor clustering and increased kinase activity. Additionally, soluble forms of the ephrin-A ligands released from the cell surface by matrix metalloproteases can also activate EphA receptor signaling. Besides these trans interactions, recent studies have revealed that Eph receptors and ephrins coexpressed in neurons can also engage in lateral "cis" associations that attenuate receptor activation by ephrins in trans with critical functional consequences. Despite the importance of the Eph/ephrin system in tumorigenesis, Eph receptor-ephrin cis interactions have not been previously investigated in cancer cells. Here we show that in cancer cells, coexpressed ephrin-A3 can inhibit the ability of EphA2 and EphA3 to bind ephrins in trans and become activated, while ephrin-B2 can inhibit not only EphB4 but also EphA3. The cis inhibition of EphA3 by ephrin-B2 implies that in some cases ephrins that cannot activate a particular Eph receptor in trans can nevertheless inhibit its signaling ability through cis association. We also found that an EphA3 mutation identified in lung cancer enhances cis interaction with ephrin-A3. These results suggest a novel mechanism that may contribute to cancer pathogenesis by attenuating the tumor suppressing effects of Eph receptor signaling pathways activated by ephrins in trans.

  15. Allosteric ligands and their binding sites define γ-aminobutyric acid (GABA) type A receptor subtypes.

    Olsen, Richard W

    2015-01-01

    GABAA receptors (GABA(A)Rs) mediate rapid inhibitory transmission in the brain. GABA(A)Rs are ligand-gated chloride ion channel proteins and exist in about a dozen or more heteropentameric subtypes exhibiting variable age and brain regional localization and thus participation in differing brain functions and diseases. GABA(A)Rs are also subject to modulation by several chemotypes of allosteric ligands that help define structure and function, including subtype definition. The channel blocker picrotoxin identified a noncompetitive channel blocker site in GABA(A)Rs. This ligand site is located in the transmembrane channel pore, whereas the GABA agonist site is in the extracellular domain at subunit interfaces, a site useful for low energy coupled conformational changes of the functional channel domain. Two classes of pharmacologically important allosteric modulatory ligand binding sites reside in the extracellular domain at modified agonist sites at other subunit interfaces: the benzodiazepine site and the high-affinity, relevant to intoxication, ethanol site. The benzodiazepine site is specific for certain GABA(A)R subtypes, mainly synaptic, while the ethanol site is found at a modified benzodiazepine site on different, extrasynaptic, subtypes. In the transmembrane domain are allosteric modulatory ligand sites for diverse chemotypes of general anesthetics: the volatile and intravenous agents, barbiturates, etomidate, propofol, long-chain alcohols, and neurosteroids. The last are endogenous positive allosteric modulators. X-ray crystal structures of prokaryotic and invertebrate pentameric ligand-gated ion channels, and the mammalian GABA(A)R protein, allow homology modeling of GABA(A)R subtypes with the various ligand sites located to suggest the structure and function of these proteins and their pharmacological modulation. © 2015 Elsevier Inc. All rights reserved.

  16. Multitarget-directed tricyclic pyridazinones as G protein-coupled receptor ligands and cholinesterase inhibitors.

    Pau, Amedeo; Catto, Marco; Pinna, Giovanni; Frau, Simona; Murineddu, Gabriele; Asproni, Battistina; Curzu, Maria M; Pisani, Leonardo; Leonetti, Francesco; Loza, Maria Isabel; Brea, José; Pinna, Gérard A; Carotti, Angelo

    2015-06-01

    By following a multitarget ligand design approach, a library of 47 compounds was prepared, and they were tested as binders of selected G protein-coupled receptors (GPCRs) and inhibitors of acetyl and/or butyryl cholinesterase. The newly designed ligands feature pyridazinone-based tricyclic scaffolds connected through alkyl chains of variable length to proper amine moieties (e.g., substituted piperazines or piperidines) for GPCR and cholinesterase (ChE) molecular recognition. The compounds were tested at three different GPCRs, namely serotoninergic 5-HT1A, adrenergic α1A, and dopaminergic D2 receptors. Our main goal was the discovery of compounds that exhibit, in addition to ChE inhibition, antagonist activity at 5-HT1A because of its involvement in neuronal deficits typical of Alzheimer's and other neurodegenerative diseases. Ligands with nanomolar affinity for the tested GPCRs were discovered, but most of them behaved as dual antagonists of α1A and 5-HT1A receptors. Nevertheless, several compounds displaying this GPCR affinity profile also showed moderate to good inhibition of AChE and BChE, thus deserving further investigations to exploit the therapeutic potential of such unusual biological profiles. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Tyrosine Kinase Ligand-Receptor Pair Prediction by Using Support Vector Machine

    Masayuki Yarimizu

    2015-01-01

    Full Text Available Receptor tyrosine kinases are essential proteins involved in cellular differentiation and proliferation in vivo and are heavily involved in allergic diseases, diabetes, and onset/proliferation of cancerous cells. Identifying the interacting partner of this protein, a growth factor ligand, will provide a deeper understanding of cellular proliferation/differentiation and other cell processes. In this study, we developed a method for predicting tyrosine kinase ligand-receptor pairs from their amino acid sequences. We collected tyrosine kinase ligand-receptor pairs from the Database of Interacting Proteins (DIP and UniProtKB, filtered them by removing sequence redundancy, and used them as a dataset for machine learning and assessment of predictive performance. Our prediction method is based on support vector machines (SVMs, and we evaluated several input features suitable for tyrosine kinase for machine learning and compared and analyzed the results. Using sequence pattern information and domain information extracted from sequences as input features, we obtained 0.996 of the area under the receiver operating characteristic curve. This accuracy is higher than that obtained from general protein-protein interaction pair predictions.

  18. Non-Ligand-Induced Dimerization is Sufficient to Initiate the Signalling and Endocytosis of EGF Receptor

    George Kourouniotis

    2016-07-01

    Full Text Available The binding of epidermal growth factor (EGF to EGF receptor (EGFR stimulates cell mitogenesis and survival through various signalling cascades. EGF also stimulates rapid EGFR endocytosis and its eventual degradation in lysosomes. The immediate events induced by ligand binding include receptor dimerization, activation of intrinsic tyrosine kinase and autophosphorylation. However, in spite of intensified efforts, the results regarding the roles of these events in EGFR signalling and internalization is still very controversial. In this study, we constructed a chimeric EGFR by replacing its extracellular domain with leucine zipper (LZ and tagged a green fluorescent protein (GFP at its C-terminus. We showed that the chimeric LZ-EGFR-GFP was constitutively dimerized. The LZ-EGFR-GFP dimer autophosphorylated each of its five well-defined C-terminal tyrosine residues as the ligand-induced EGFR dimer does. Phosphorylated LZ-EGFR-GFP was localized to both the plasma membrane and endosomes, suggesting it is capable of endocytosis. We also showed that LZ-EGFR-GFP activated major signalling proteins including Src homology collagen-like (Shc, extracellular signal-regulated kinase (ERK and Akt. Moreover, LZ-EGFR-GFP was able to stimulate cell proliferation. These results indicate that non-ligand induced dimerization is sufficient to activate EGFR and initiate cell signalling and EGFR endocytosis. We conclude that receptor dimerization is a critical event in EGF-induced cell signalling and EGFR endocytosis.

  19. Ligand-specific regulation of the extracellular surface of a G-protein-coupled receptor

    Bokoch, Michael P.; Zou, Yaozhong; Rasmussen, Søren G.F.; Liu, Corey W.; Nygaard, Rie; Rosenbaum, Daniel M.; Fung, Juan José; Choi, Hee-Jung; Thian, Foon Sun; Kobilka, Tong Sun; Puglisi, Joseph D.; Weis, William I.; Pardo, Leonardo; Prosser, R. Scott; Mueller, Luciano; Kobilka, Brian K. (Stanford-MED); (Toronto); (BMS); (UAB, Spain)

    2010-01-14

    G-protein-coupled receptors (GPCRs) are seven-transmembrane proteins that mediate most cellular responses to hormones and neurotransmitters. They are the largest group of therapeutic targets for a broad spectrum of diseases. Recent crystal structures of GPCRs have revealed structural conservation extending from the orthosteric ligand-binding site in the transmembrane core to the cytoplasmic G-protein-coupling domains. In contrast, the extracellular surface (ECS) of GPCRs is remarkably diverse and is therefore an ideal target for the discovery of subtype-selective drugs. However, little is known about the functional role of the ECS in receptor activation, or about conformational coupling of this surface to the native ligand-binding pocket. Here we use NMR spectroscopy to investigate ligand-specific conformational changes around a central structural feature in the ECS of the {beta}{sub 2} adrenergic receptor: a salt bridge linking extracellular loops 2 and 3. Small-molecule drugs that bind within the transmembrane core and exhibit different efficacies towards G-protein activation (agonist, neutral antagonist and inverse agonist) also stabilize distinct conformations of the ECS. We thereby demonstrate conformational coupling between the ECS and the orthosteric binding site, showing that drugs targeting this diverse surface could function as allosteric modulators with high subtype selectivity. Moreover, these studies provide a new insight into the dynamic behaviour of GPCRs not addressable by static, inactive-state crystal structures.

  20. Effect of Tramadol (μ-opioid receptor agonist on orthodontic tooth movements in a rat model

    E. Javadi

    2012-01-01

    Full Text Available Objective: Tramadol is a synthetic analgesic of opioids which has more flexible mechanisms of action than typical opioids. Since it has been reported in previous study that typical opioids like morphine can affect the bone homeostasis, it is worthwhile to examine the effects of tramadol on tooth movement. In this study we investigated effects of tramadol on orthodontic tooth movement in rats.Materials and Methods: 30 male wistar rats were selected and received orthodontic appliance. 3 groups were designed based on the substance that they received daily injections of during a 2-week orthodontic treatment. 1. Control group with no injection.2.Control group with normal saline injection.3. the tramadol group. After the two-week treatment period the amount of tooth movement were measured in all the groups. Also the histological analysis was performed assessing the root resorption, osteoclasts numbers and bone resorption.Results: The amount of tooth movement was not significantl in the tramadol group comparing to the other groups (P>0.05.The results of 3 histological parameters (amount of root resorption, osteoclastic numbers and bone resorption were statistically insignificant (P>0.05.Conclusion: Tramadol as an atypical opioid does not interfere with the process of bone remodeling and tooth movement in rat. Tramadol does not affect osteoclastic activity and bone resorption and it does not cause to change the resulted root resorption either.

  1. Sex differences in subcellular distribution of delta opioid receptors in the rat hippocampus in response to acute and chronic stress

    Sanoara Mazid

    2016-12-01

    Full Text Available Drug addiction requires associative learning processes that critically involve hippocampal circuits, including the opioid system. We recently found that acute and chronic stress, important regulators of addictive processes, affect hippocampal opioid levels and mu opioid receptor trafficking in a sexually dimorphic manner. Here, we examined whether acute and chronic stress similarly alters the levels and trafficking of hippocampal delta opioid receptors (DORs. Immediately after acute immobilization stress (AIS or one-day after chronic immobilization stress (CIS, the brains of adult female and male rats were perfusion-fixed with aldehydes. The CA3b region and the dentate hilus of the dorsal hippocampus were quantitatively analyzed by light microscopy using DOR immunoperoxidase or dual label electron microscopy for DOR using silver intensified immunogold particles (SIG and GABA using immunoperoxidase. At baseline, females compared to males had more DORs near the plasmalemma of pyramidal cell dendrites and about 3 times more DOR-labeled CA3 dendritic spines contacted by mossy fibers. In AIS females, near-plasmalemmal DOR-SIGs decreased in GABAergic hilar dendrites. However, in AIS males, near-plasmalemmal DOR-SIGs increased in CA3 pyramidal cell and hilar GABAergic dendrites and the percentage of CA3 dendritic spines contacted by mossy fibers increased to about half that seen in unstressed females. Conversely, after CIS, near-plasmalemmal DOR-SIGs increased in hilar GABA-labeled dendrites of females whereas in males plasmalemmal DOR-SIGs decreased in CA3 pyramidal cell dendrites and near-plasmalemmal DOR-SIGs decreased hilar GABA-labeled dendrites. As CIS in females, but not males, redistributed DOR-SIGs near the plasmalemmal of hilar GABAergic dendrites, a subsequent experiment examined the acute affect of oxycodone on the redistribution of DOR-SIGs in a separate cohort of CIS females. Plasmalemmal DOR-SIGs were significantly elevated on hilar

  2. Ligand recognition and domain structure of Vps10p, a vacuolar protein sorting receptor in Saccharomyces cerevisiae

    Jørgensen, M U; Emr, S D; Winther, Jakob R.

    1999-01-01

    Vp10p is a receptor that sorts several different vacuolar proteins by cycling between a late Golgi compartment and the endosome. The cytoplasmic tail of Vps10p is necessary for the recycling, whereas the lumenal domain is predicted to interact with the soluble ligands. We have studied ligand bind...

  3. Effects of cypermethrin on the ligand-independent interaction between androgen receptor and steroid receptor coactivator-1

    Pan, Chen; Liu, Ya-Peng; Li, Yan-Fang; Hu, Jin-Xia; Zhang, Jin-Peng; Wang, Hong-Mei; Li, Jing; Xu, Li-Chun

    2012-01-01

    The pyrethroid insecticide, cypermethrin has been considered as an environmental anti-androgen by interfering with the androgen receptor (AR) transactivation. In order to clarify the effects of cypermethrin on the ligand-independent interaction between the AR and SRC-1, the mammalian two-hybrid assay has been developed in the study. The AR N-terminal domain 1–660 amino acid residues were subcloned into the plasmid pVP16 to construct the vector pVP16-ARNTD. The SRC-1 C-terminal domain 989–1240 amino acid residues were subcloned into the plasmid pM to construct the vector pM-SRC-1. The fusion vectors pVP16-ARNTD, pM-SRC-1 and the pG5CAT Reporter Vector were cotransfected into the CV-1 cells. The AR AF1 interacted with SRC-1 in the absence of exogenous ligand 5α-dihydrotestosterone (DHT). Furthermore, DHT did not enhance the interaction between AR AF-1 and SRC-1 at the concentrations from 10 −10 M to 10 −8 M. Cypermethrin inhibited the interaction between the AR AF1 and SRC-1, and the significant reduction was detected at the concentration of 10 −5 M. It is suggested that the interaction between the AR AF1 and SRC-1 is ligand-independent. Cypermethrin inhibits AR activity by disrupting the ligand-independent AR–SRC-1 interaction.

  4. Chemically engineering ligand selectivity at the free fatty acid receptor 2 based on pharmacological variation between species orthologs

    Hudson, Brian D; Christiansen, Elisabeth; Tikhonova, Irina G

    2012-01-01

    When it is difficult to develop selective ligands within a family of related G-protein-coupled receptors (GPCRs), chemically engineered receptors activated solely by synthetic ligands (RASSLs) are useful alternatives for probing receptor function. In the present work, we explored whether a RASSL...... on this receptor and demonstrates that exploitation of pharmacological variation between species orthologs is a powerful method to generate novel chemically engineered GPCRs.-Hudson, B. D., Christiansen, E., Tikhonova, I. G., Grundmann, M., Kostenis, E., Adams, D. R., Ulven, T., Milligan, G. Chemically engineering...

  5. Synthesis of the possible receptor Ligand [125I]-spiperone for D2-dopamine receptor and in-vivo biodistribution

    Amin, A.M.; Shoukry, M.; Abd EL-Bary, A.

    2009-01-01

    The spiperone is a selective D2-dopamine receptor antagonist radioiodination of spiperone is of interest for dopamine (DA) receptor studies both in vivo and in vitro. The labeling of spiperone with iodine-125 was extremely done in a neutral ph 7, using chloramine-T as oxidizing agent via heating the reaction mixture at 70 C (degree) for 10 - 15 minutes producing radiochemical yield of 97 %. In vivo biodistribution studies showed that the initial brain uptake correlated fairly well with the brain uptake index and that the kinetics of the radioactivity specifically bound to the striatum were strongly influenced by the dopamine receptor binding affinity of the compound. The brain uptake of 125 I-Spiperone was high and equal to 3.5, 3.25,2.75 and 1.7 % per gram tissue at 5, 30, 60 and 120 minutes post injection, respectively. 125 I-Spiperone binds with high affinity to dopamine receptors in vivo. Specific binding is about 65% of the total binding as is displaced stereo-specifically by clozapine. 125 I-spiperone may prove to be a useful ligand in studies examining D2-dopamine receptors. Furthermore iodinated spiperone may be useful in radioreceptor assays of neuroleptic drug levels and, in a 123 I-labeled form, for imaging of dopamine receptors, in vivo, using single photon tomography.

  6. Novel aza-analogous ergoline derived scaffolds as potent serotonin 5-HT6 and dopamine D2 receptor ligands

    Krogsgaard-Larsen, Niels; Jensen, Anders A.; Schrøder, T.J.

    2014-01-01

    By introducing distal substituents on a tetracyclic scaffold resembling the ergoline structure, two series of analogues were achieved exhibiting subnanomolar receptor binding affinities for the dopamine D2 and serotonin 5-HT6 receptor subtype, respectively. While the 5-HT6 ligands were antagonists......, the D2 ligands displayed intrinsic activities ranging from full agonism to partial agonism with low intrinsic activity. These structures could potentially be interesting for treatment of neurological diseases such as schizophrenia, Parkinson’s disease, and cognitive deficits....

  7. A Killer Immunoglobulin - Like Receptor Gene - Content Haplotype and A Cognate Human Leukocyte Antigen Ligand are Associated with Autism

    Torres, Anthony; Westover, Jonna; Benson, Michael; Johnson, Randall; Dykes, Annelise

    2016-01-01

    The killing activity of natural killer cells is largely regulated by the binding of class I human leukocyte antigen cognate ligands to killer cell immunoglobulin - like receptor proteins. The killer cell immunoglobulin - like receptor gene - complex contains genes that activate and others that inhibit the killing state of natural killer cells depending on the binding of specific human leukocyte antigen cognate ligands. It has been suggested in previous publications that activating human leuko...

  8. Evaluation of 3-Ethyl-3-(phenylpiperazinylbutyl)oxindoles as PET Ligands for the Serotonin 5-HT7 Receptor

    Herth, Matthias M; Andersen, Valdemar L; Hansen, Hanne D

    2015-01-01

    We have investigated several oxindole derivatives in the pursuit of a 5-HT7 receptor PET ligand. Herein the synthesis, chiral separation, and pharmacological profiling of two possible PET candidates toward a wide selection of CNS-targets are detailed. Subsequent (11)C-labeling and in vivo evaluat...... evaluation in Danish landrace pigs showed that both ligands displayed high brain uptake. However, neither of the radioligands could be displaced by the 5-HT7 receptor selective inverse agonist SB-269970....

  9. Effects of different ligands on epidermal growth factor receptor (EGFR) nuclear translocation

    Faria, Jerusa A.Q.A.; Andrade, Carolina de; Goes, Alfredo M. [Department of Biochemistry and Immunology, Universidade Federal de Minas Gerais, Av. Antonio Carlos, 6627, Belo Horizonte, MG, 31270-901 (Brazil); Rodrigues, Michele A. [Department of Biochemistry and Immunology, Universidade Federal de Minas Gerais, Av. Antonio Carlos, 6627, Belo Horizonte, MG, 31270-901 (Brazil); Department of General Pathology, Universidade Federal de Minas Gerais, Av. Antonio Carlos, 6627, Belo Horizonte, MG, 31270-901 (Brazil); Gomes, Dawidson A., E-mail: dawidson@ufmg.br [Department of Biochemistry and Immunology, Universidade Federal de Minas Gerais, Av. Antonio Carlos, 6627, Belo Horizonte, MG, 31270-901 (Brazil)

    2016-09-09

    The epidermal growth factor receptor (EGFR) is activated through binding to specific ligands and generates signals for proliferation, differentiation, migration, and cell survival. Recent data show the role of nuclear EGFR in tumors. Although many EGFR ligands are upregulated in cancers, little is known about their effects on EGFR nuclear translocation. We have compared the effects of six EGFR ligands (EGF, HB-EGF, TGF-α, β-Cellulin, amphiregulin, and epiregulin) on nuclear translocation of EGFR, receptor phosphorylation, migration, and proliferation. Cell fractionation and confocal immunofluorescence detected EGFR in the nucleus after EGF, HB-EGF, TGF-α and β-Cellulin stimulation in a dose-dependent manner. In contrast, amphiregulin and epiregulin did not generate nuclear translocation of EGFR. EGF, HB-EGF, TGF-α and β-Cellulin showed correlations between a higher rate of wound closure and increased phosphorylation of residues in the carboxy-terminus of EGFR, compared to amphiregulin and epiregulin. The data indicate that EGFR is translocated to the nucleus after stimulation with EGF, HB-EGF, TGF-α and β-Cellulin, and that these ligands are related to increased phosphorylation of EGFR tyrosine residues, inducing migration of SkHep-1 cells. - Highlights: • EGF, HB-EGF, TGF-α, β-Cellulin are involved in the EGFR nuclear translocation. • Amphiregulin and epiregulin did not promote nuclear translocation of EGFR. • EGF, HB-EGF, TGF-α and β-Cellulin have a role in SkHep-1 cells migration. • EGFR ligands associated with better prognosis don't stimulate EGFR translocation.

  10. Effects of different ligands on epidermal growth factor receptor (EGFR) nuclear translocation

    Faria, Jerusa A.Q.A.; Andrade, Carolina de; Goes, Alfredo M.; Rodrigues, Michele A.; Gomes, Dawidson A.

    2016-01-01

    The epidermal growth factor receptor (EGFR) is activated through binding to specific ligands and generates signals for proliferation, differentiation, migration, and cell survival. Recent data show the role of nuclear EGFR in tumors. Although many EGFR ligands are upregulated in cancers, little is known about their effects on EGFR nuclear translocation. We have compared the effects of six EGFR ligands (EGF, HB-EGF, TGF-α, β-Cellulin, amphiregulin, and epiregulin) on nuclear translocation of EGFR, receptor phosphorylation, migration, and proliferation. Cell fractionation and confocal immunofluorescence detected EGFR in the nucleus after EGF, HB-EGF, TGF-α and β-Cellulin stimulation in a dose-dependent manner. In contrast, amphiregulin and epiregulin did not generate nuclear translocation of EGFR. EGF, HB-EGF, TGF-α and β-Cellulin showed correlations between a higher rate of wound closure and increased phosphorylation of residues in the carboxy-terminus of EGFR, compared to amphiregulin and epiregulin. The data indicate that EGFR is translocated to the nucleus after stimulation with EGF, HB-EGF, TGF-α and β-Cellulin, and that these ligands are related to increased phosphorylation of EGFR tyrosine residues, inducing migration of SkHep-1 cells. - Highlights: • EGF, HB-EGF, TGF-α, β-Cellulin are involved in the EGFR nuclear translocation. • Amphiregulin and epiregulin did not promote nuclear translocation of EGFR. • EGF, HB-EGF, TGF-α and β-Cellulin have a role in SkHep-1 cells migration. • EGFR ligands associated with better prognosis don't stimulate EGFR translocation.

  11. Importance of the extracellular loops in G protein-coupled receptors for ligand recognition and receptor activation.

    Peeters, M C; van Westen, G J P; Li, Q; IJzerman, A P

    2011-01-01

    G protein-coupled receptors (GPCRs) are the major drug target of medicines on the market today. Therefore, much research is and has been devoted to the elucidation of the function and three-dimensional structure of this large family of membrane proteins, which includes multiple conserved transmembrane domains connected by intra- and extracellular loops. In the last few years, the less conserved extracellular loops have garnered increasing interest, particularly after the publication of several GPCR crystal structures that clearly show the extracellular loops to be involved in ligand binding. This review will summarize the recent progress made in the clarification of the ligand binding and activation mechanism of class-A GPCRs and the role of extracellular loops in this process. Copyright © 2010 Elsevier Ltd. All rights reserved.

  12. Crystal structure of the ligand-bound glucagon-like peptide-1 receptor extracellular domain.

    Runge, Steffen; Thøgersen, Henning; Madsen, Kjeld; Lau, Jesper; Rudolph, Rainer

    2008-04-25

    The glucagon-like peptide-1 receptor (GLP-1R) belongs to Family B1 of the seven-transmembrane G protein-coupled receptors, and its natural agonist ligand is the peptide hormone glucagon-like peptide-1 (GLP-1). GLP-1 is involved in glucose homeostasis, and activation of GLP-1R in the plasma membrane of pancreatic beta-cells potentiates glucose-dependent insulin secretion. The N-terminal extracellular domain (nGLP-1R) is an important ligand binding domain that binds GLP-1 and the homologous peptide Exendin-4 with differential affinity. Exendin-4 has a C-terminal extension of nine amino acid residues known as the "Trp cage", which is absent in GLP-1. The Trp cage was believed to interact with nGLP-1R and thereby explain the superior affinity of Exendin-4. However, the molecular details that govern ligand binding and specificity of nGLP-1R remain undefined. Here we report the crystal structure of human nGLP-1R in complex with the antagonist Exendin-4(9-39) solved by the multiwavelength anomalous dispersion method to 2.2A resolution. The structure reveals that Exendin-4(9-39) is an amphipathic alpha-helix forming both hydrophobic and hydrophilic interactions with nGLP-1R. The Trp cage of Exendin-4 is not involved in binding to nGLP-1R. The hydrophobic binding site of nGLP-1R is defined by discontinuous segments including primarily a well defined alpha-helix in the N terminus of nGLP-1R and a loop between two antiparallel beta-strands. The structure provides for the first time detailed molecular insight into ligand binding of the human GLP-1 receptor, an established target for treatment of type 2 diabetes.

  13. Plasma membrane cholesterol level and agonist-induced internalization of delta-opioid receptors; colocalization study with intracellular membrane markers of Rab family\

    Brejchová, Jana; Vošahlíková, Miroslava; Roubalová, Lenka; Parenti, M.; Mauri, M.; Chernyavskiy, Oleksandr; Svoboda, Petr

    2016-01-01

    Roč. 48, č. 4 (2016), s. 375-396 ISSN 0145-479X R&D Projects: GA ČR(CZ) GAP207/12/0919 Institutional support: RVO:67985823 Keywords : cholesterol * plasma membrane * delta-opioid receptor * internalization * Rab proteins Subject RIV: CE - Biochemistry Impact factor: 2.576, year: 2016

  14. High Efficacy but Low Potency of delta-Opioid Receptor-G Protein Coupling in Brij-58-Treated, Low-Density Plasma Membrane Fragments

    Roubalová, Lenka; Vošahlíková, Miroslava; Brejchová, Jana; Sýkora, Jan; Rudajev, Vladimír; Svoboda, Petr

    2015-01-01

    Roč. 10, č. 8 (2015), e0135664 E-ISSN 1932-6203 R&D Projects: GA ČR(CZ) GAP207/12/0919 Institutional support: RVO:67985823 ; RVO:61388955 Keywords : delta - opioid receptor * G protein coupling * detergent * efficacy * potency Subject RIV: CE - Biochemistry; CF - Physical ; Theoretical Chemistry (UFCH-W) Impact factor: 3.057, year: 2015

  15. The role of TAM family receptors and ligands in the nervous system: From development to pathobiology.

    Shafit-Zagardo, Bridget; Gruber, Ross C; DuBois, Juwen C

    2018-03-04

    Tyro3, Axl, and Mertk, referred to as the TAM family of receptor tyrosine kinases, are instrumental in maintaining cell survival and homeostasis in mammals. TAM receptors interact with multiple signaling molecules to regulate cell migration, survival, phagocytosis and clearance of metabolic products and cell debris called efferocytosis. The TAMs also function as rheostats to reduce the expression of proinflammatory molecules and prevent autoimmunity. All three TAM receptors are activated in a concentration-dependent manner by the vitamin K-dependent growth arrest-specific protein 6 (Gas6). Gas6 and the TAMs are abundantly expressed in the nervous system. Gas6, secreted by neurons and endothelial cells, is the sole ligand for Axl. ProteinS1 (ProS1), another vitamin K-dependent protein functions mainly as an anti-coagulant, and independent of this function can activate Tyro3 and Mertk, but not Axl. This review will focus on the role of the TAM receptors and their ligands in the nervous system. We highlight studies that explore the function of TAM signaling in myelination, the visual cortex, neural cancers, and multiple sclerosis (MS) using Gas6 -/- and TAM mutant mice models. Copyright © 2018. Published by Elsevier Inc.

  16. The influences of reproductive status and acute stress on the levels of phosphorylated mu opioid receptor immunoreactivity in rat hippocampus

    Keith L. Gonzales

    2011-08-01

    Full Text Available Opioids play a critical role in hippocampally dependent behavior and plasticity. In the hippocampal formation, mu opioid receptors (MOR are prominent in parvalbumin (PARV containing interneurons. Previously we found that gonadal hormones modulate the trafficking of MORs in PARV interneurons. Although sex differences in response to stress are well documented, the point at which opioids, sex and stress interact to influence hippocampal function remains elusive. Thus, we used quantitative immunocytochemistry in combination with light and electron microscopy for the phosphorylated MOR at the SER375 carboxy-terminal residue (pMOR in male and female rats to assess these interactions. In both sexes, pMOR-immunoreactivity (ir was prominent in axons and terminals and in a few neuronal somata and dendrites, some of which contained PARV in the mossy fiber pathway region of the dentate gyrus (DG hilus and CA3 stratum lucidum. In unstressed rats, the levels of pMOR-ir in the DG or CA3 were not affected by sex or estrous cycle stage. However, immediately following 30 minutes of acute immobilization stress (AIS, males had higher levels of pMOR-ir whereas females at proestrus and estrus (high estrogen stages had lower levels of pMOR-ir within the DG. In contrast, the number and types of neuronal profiles with pMOR-ir were not altered by AIS in either males or proestrus females. These data demonstrate that although gonadal steroids do not affect pMOR levels at resting conditions, they are differentially activated both pre- and post-synaptic MORs following stress. These interactions may contribute to the reported sex differences in hippocampally dependent behaviors in stressed animals.

  17. Data for amino acid alignment of Japanese stingray melanocortin receptors with other gnathostome melanocortin receptor sequences, and the ligand selectivity of Japanese stingray melanocortin receptors

    Akiyoshi Takahashi

    2016-06-01

    Full Text Available This article contains structure and pharmacological characteristics of melanocortin receptors (MCRs related to research published in “Characterization of melanocortin receptors from stingray Dasyatis akajei, a cartilaginous fish” (Takahashi et al., 2016 [1]. The amino acid sequences of the stingray, D. akajei, MC1R, MC2R, MC3R, MC4R, and MC5R were aligned with the corresponding melanocortin receptor sequences from the elephant shark, Callorhinchus milii, the dogfish, Squalus acanthias, the goldfish, Carassius auratus, and the mouse, Mus musculus. These alignments provide the basis for phylogenetic analysis of these gnathostome melanocortin receptor sequences. In addition, the Japanese stingray melanocortin receptors were separately expressed in Chinese Hamster Ovary cells, and stimulated with stingray ACTH, α-MSH, β-MSH, γ-MSH, δ-MSH, and β-endorphin. The dose response curves reveal the order of ligand selectivity for each stingray MCR.

  18. Opioid Antagonists and the A118G Polymorphism in the μ-Opioid Receptor Gene: Effects of GSK1521498 and Naltrexone in Healthy Drinkers Stratified by OPRM1 Genotype

    Ziauddeen, Hisham; Nestor, Liam J; Subramaniam, Naresh; Dodds, Chris; Nathan, Pradeep J; Miller, Sam R; Sarai, Bhopinder K; Maltby, Kay; Fernando, Disala; Warren, Liling; Hosking, Louise K; Waterworth, Dawn; Korzeniowska, Anna; Win, Beta; Richards, Duncan B; Vasist Johnson, Lakshmi; Fletcher, Paul C; Bullmore, Edward T

    2016-01-01

    The A118G single-nucleotide polymorphism (SNP rs1799971) in the μ-opioid receptor gene, OPRM1, has been much studied in relation to alcohol use disorders. The reported effects of allelic variation at this SNP on alcohol-related behaviors, and on opioid receptor antagonist treatments, have been inconsistent. We investigated the pharmacogenetic interaction between A118G variation and the effects of two μ-opioid receptor antagonists in a clinical lab setting. Fifty-six overweight and moderate–heavy drinkers were prospectively stratified by genotype (29 AA homozygotes, 27 carriers of at least 1 G allele) in a double-blind placebo-controlled, three-period crossover design with naltrexone (NTX; 25 mg OD for 2 days, then 50 mg OD for 3 days) and GSK1521498 (10 mg OD for 5 days). The primary end point was regional brain activation by the contrast between alcohol and neutral tastes measured using functional magnetic resonance imaging (fMRI). Secondary end points included other fMRI contrasts, subjective responses to intravenous alcohol challenge, and food intake. GSK1521498 (but not NTX) significantly attenuated fMRI activation by appetitive tastes in the midbrain and amygdala. GSK1521498 (and NTX to a lesser extent) significantly affected self-reported responses to alcohol infusion. Both drugs reduced food intake. Across all end points, there was less robust evidence for significant effects of OPRM1 allelic variation, or for pharmacogenetic interactions between genotype and drug treatment. These results do not support strong modulatory effects of OPRM1 genetic variation on opioid receptor antagonist attenuation of alcohol- and food-related behaviors. However, they do support further investigation of GSK1521498 as a potential therapeutic for alcohol use and eating disorders. PMID:27109624

  19. Endogenous hallucinogens as ligands of the trace amine receptors: a possible role in sensory perception.

    Wallach, J V

    2009-01-01

    While the endogenous hallucinogens, N,N-dimethyltryptamine, 5-hydroxy-N,N-dimethyl-tryptamine and 5-methoxy-N,N-dimethyltryptamine, have been acknowledged as naturally occurring components of the mammalian body for decades, their biological function remains as elusive now as it was at the time of their discovery. The recent discovery of the trace amine associated receptors and the activity of DMT and other hallucinogenic compounds at these receptor sites leads to the hypothesis that the endogenous hallucinogens act as neurotransmitters of a subclass of these trace amine receptors. Additionally, while activity at the serotonin 5-HT2A receptor has been proposed as being responsible for the hallucinogenic affects of administered hallucinogens, in their natural setting the 5-HT2A receptor may not interact with the endogenous hallucinogens at all. Additionally 5-HT2A agonist activity is unable to account for the visual altering effects of many of the administered hallucinogens; these effects may be mediated by one of the endogenous hallucinogen trace amine receptors rather than the serotonin 5-HT2A receptor. Therefore, activity at the trace amine receptors, in addition to serotonin receptors, may play a large role in the sensory altering effects of administered hallucinogens and the trace amine receptors along with their endogenous hallucinogen ligands may serve an endogenous role in mediating sensory perception in the mammalian central nervous system. Thus the theory proposed states that these compounds act as true endogenous hallucinogenic transmitters acting in regions of the central nervous system involved in sensory perception.

  20. NOpiates: Novel Dual Action Neuronal Nitric Oxide Synthase Inhibitors with μ-Opioid Agonist Activity.

    Renton, Paul; Green, Brenda; Maddaford, Shawn; Rakhit, Suman; Andrews, John S

    2012-03-08

    A novel series of benzimidazole designed multiple ligands (DMLs) with activity at the neuronal nitric oxide synthase (nNOS) enzyme and the μ-opioid receptor was developed. Targeting of the structurally dissimilar heme-containing enzyme and the μ-opioid GPCR was predicated on the modulatory role of nitric oxide on μ-opioid receptor function. Structure-activity relationship studies yielded lead compound 24 with excellent nNOS inhibitory activity (IC50 = 0.44 μM), selectivity over both endothelial nitric oxide synthase (10-fold) and inducible nitric oxide synthase (125-fold), and potent μ-opioid binding affinity, K i = 5.4 nM. The functional activity as measured in the cyclic adenosine monosphospate secondary messenger assay resulted in full agonist activity (EC50 = 0.34 μM). This work represents a novel approach in the development of new analgesics for the treatment of pain.

  1. Social touch modulates endogenous μ-opioid system activity in humans.

    Nummenmaa, Lauri; Tuominen, Lauri; Dunbar, Robin; Hirvonen, Jussi; Manninen, Sandra; Arponen, Eveliina; Machin, Anna; Hari, Riitta; Jääskeläinen, Iiro P; Sams, Mikko

    2016-09-01

    In non-human primates, opioid-receptor blockade increases social grooming, and the endogenous opioid system has therefore been hypothesized to support maintenance of long-term relationships in humans as well. Here we tested whether social touch modulates opioidergic activation in humans using in vivo positron emission tomography (PET). Eighteen male participants underwent two PET scans with [11C]carfentanil, a ligand specific to μ-opioid receptors (MOR). During the social touch scan, the participants lay in the scanner while their partners caressed their bodies in a non-sexual fashion. In the baseline scan, participants lay alone in the scanner. Social touch triggered pleasurable sensations and increased MOR availability in the thalamus, striatum, and frontal, cingulate, and insular cortices. Modulation of activity of the opioid system by social touching might provide a neurochemical mechanism reinforcing social bonds between humans. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Estrogen receptor determination in endometrial carcinoma: ligand binding assay versus enzyme immunoassay

    Nyholm, H C; Nielsen, Anette Lynge; Lyndrup, J

    1995-01-01

    We compared concentrations of cytosolic estrogen receptors (ERc) measured in 35 postmenopausal endometrial carcinomas by ligand binding method (LBA) (dextran-coated charcoal assay) and enzyme immunoassay (EIA). Correlations between ERc, nuclear estrogen receptors (ERn) determined by EIA......, and cytosolic progesterone receptors (PR) measured by LBA were also studied. While ERc concentrations determined by LBA and EIA were highly correlated (r: 0.94), ERc values detected by LBA were approximately twice those found by EIA (median values of ERc: 155 vs. 64 fmol/mg cytosol protein, DCC vs. EIA......). The percentages of ERc positive tumors were 89% by LBA and 77% by EIA. The median fraction of total ER present as ERn was 63%. PR levels correlated positively with ERn concentrations (r: 0.73). We explore possible reasons why greater concentrations of ERc are determined by estradiol binding than by the ER-EIA kit...

  3. ReFlexIn: a flexible receptor protein-ligand docking scheme evaluated on HIV-1 protease.

    Simon Leis

    Full Text Available For many targets of pharmaceutical importance conformational changes of the receptor protein are relevant during the ligand binding process. A new docking approach, ReFlexIn (Receptor Flexibility by Interpolation, that combines receptor flexibility with the computationally efficient potential grid representation of receptor molecules has been evaluated on the retroviral HIV-1 (Human Immunodeficiency Virus 1 protease system. An approximate inclusion of receptor flexibility is achieved by using interpolation between grid representations of individual receptor conformations. For the retroviral protease the method was tested on an ensemble of protease structures crystallized in the presence of different ligands and on a set of structures obtained from morphing between the unbound and a ligand-bound protease structure. Docking was performed on ligands known to bind to the protease and several non-binders. For the binders the ReFlexIn method yielded in almost all cases ligand placements in similar or closer agreement with experiment than docking to any of the ensemble members without degrading the discrimination with respect to non-binders. The improved docking performance compared to docking to rigid receptors allows for systematic virtual screening applications at very small additional computational cost.

  4. Peroxisome proliferator-activated receptor α ligands and modulators from dietary compounds: Types, screening methods and functions.

    Yang, Haixia; Xiao, Lei; Wang, Nanping

    2017-04-01

    Peroxisome proliferator-activated receptor α (PPARα) plays a key role in lipid metabolism and glucose homeostasis and a crucial role in the prevention and treatment of metabolic diseases. Natural dietary compounds, including nutrients and phytochemicals, are PPARα ligands or modulators. High-throughput screening assays have been developed to screen for PPARα ligands and modulators in our diet. In the present review, we discuss recent advances in our knowledge of PPARα, including its structure, function, and ligand and modulator screening assays, and summarize the different types of dietary PPARα ligands and modulators. © 2016 Ruijin Hospital, Shanghai Jiaotong University School of Medicine and John Wiley & Sons Australia, Ltd.

  5. Key structural features of nonsteroidal ligands for binding and activation of the androgen receptor.

    Yin, Donghua; He, Yali; Perera, Minoli A; Hong, Seoung Soo; Marhefka, Craig; Stourman, Nina; Kirkovsky, Leonid; Miller, Duane D; Dalton, James T

    2003-01-01

    The purposes of the present studies were to examine the androgen receptor (AR) binding ability and in vitro functional activity of multiple series of nonsteroidal compounds derived from known antiandrogen pharmacophores and to investigate the structure-activity relationships (SARs) of these nonsteroidal compounds. The AR binding properties of sixty-five nonsteroidal compounds were assessed by a radioligand competitive binding assay with the use of cytosolic AR prepared from rat prostates. The AR agonist and antagonist activities of high-affinity ligands were determined by the ability of the ligand to regulate AR-mediated transcriptional activation in cultured CV-1 cells, using a cotransfection assay. Nonsteroidal compounds with diverse structural features demonstrated a wide range of binding affinity for the AR. Ten compounds, mainly from the bicalutamide-related series, showed a binding affinity superior to the structural pharmacophore from which they were derived. Several SARs regarding nonsteroidal AR binding were revealed from the binding data, including stereoisomeric conformation, steric effect, and electronic effect. The functional activity of high-affinity ligands ranged from antagonist to full agonist for the AR. Several structural features were found to be determinative of agonist and antagonist activities. The nonsteroidal AR agonists identified from the present studies provided a pool of candidates for further development of selective androgen receptor modulators (SARMs) for androgen therapy. Also, these studies uncovered or confirmed numerous important SARs governing AR binding and functional properties by nonsteroidal molecules, which would be valuable in the future structural optimization of SARMs.

  6. Ligand-Receptor Interaction-Mediated Transmembrane Transport of Dendrimer-like Soft Nanoparticles: Mechanisms and Complicated Diffusive Dynamics.

    Liang, Junshi; Chen, Pengyu; Dong, Bojun; Huang, Zihan; Zhao, Kongyin; Yan, Li-Tang

    2016-05-09

    Nearly all nanomedical applications of dendrimer-like soft nanoparticles rely on the functionality of attached ligands. Understanding how the ligands interact with the receptors in cell membrane and its further effect on the cellular uptake of dendrimer-like soft nanoparticles is thereby a key issue for their better application in nanomedicine. However, the essential mechanism and detailed kinetics for the ligand-receptor interaction-mediated transmembrane transport of such unconventional nanoparticles remain poorly elucidated. Here, using coarse-grained simulations, we present the very first study of molecular mechanism and kinetics behaviors for the transmembrane transport of dendrimer-like soft nanoparticles conjugated with ligands. A phase diagram of interaction states is constructed through examining ligand densities and membrane tensions that allows us to identify novel endocytosis mechanisms featured by the direct wrapping and the penetration-extraction vesiculation. The results provide an in-depth insight into the diffusivity of receptors and dendrimer in the membrane plane and demonstrate how the ligand density influences receptor diffusion and uptake kinetics. It is interesting to find that the ligand-conjugated dendrimers present superdiffusive behaviors on a membrane, which is revealed to be driven by the random fluctuation dynamics of the membrane. The findings facilitate our understanding of some recent experimental observations and could establish fundamental principles for the future development of such important nanomaterials for widespread nanomedical applications.

  7. Simple Ligand-Receptor Interaction Descriptor (SILIRID) for alignment-free binding site comparison.

    Chupakhin, Vladimir; Marcou, Gilles; Gaspar, Helena; Varnek, Alexandre

    2014-06-01

    We describe SILIRID (Simple Ligand-Receptor Interaction Descriptor), a novel fixed size descriptor characterizing protein-ligand interactions. SILIRID can be obtained from the binary interaction fingerprints (IFPs) by summing up the bits corresponding to identical amino acids. This results in a vector of 168 integer numbers corresponding to the product of the number of entries (20 amino acids and one cofactor) and 8 interaction types per amino acid (hydrophobic, aromatic face to face, aromatic edge to face, H-bond donated by the protein, H-bond donated by the ligand, ionic bond with protein cation and protein anion, and interaction with metal ion). Efficiency of SILIRID to distinguish different protein binding sites has been examined in similarity search in sc-PDB database, a druggable portion of the Protein Data Bank, using various protein-ligand complexes as queries. The performance of retrieval of structurally and evolutionary related classes of proteins was comparable to that of state-of-the-art approaches (ROC AUC ≈ 0.91). SILIRID can efficiently be used to visualize chemogenomic space covered by sc-PDB using Generative Topographic Mapping (GTM): sc-PDB SILIRID data form clusters corresponding to different protein types.

  8. α2A- and α2C-Adrenoceptors as Potential Targets for Dopamine and Dopamine Receptor Ligands.

    Sánchez-Soto, Marta; Casadó-Anguera, Verònica; Yano, Hideaki; Bender, Brian Joseph; Cai, Ning-Sheng; Moreno, Estefanía; Canela, Enric I; Cortés, Antoni; Meiler, Jens; Casadó, Vicent; Ferré, Sergi

    2018-03-18

    The poor norepinephrine innervation and high density of Gi/o-coupled α 2A - and α 2C -adrenoceptors in the striatum and the dense striatal dopamine innervation have prompted the possibility that dopamine could be an effective adrenoceptor ligand. Nevertheless, the reported adrenoceptor agonistic properties of dopamine are still inconclusive. In this study, we analyzed the binding of norepinephrine, dopamine, and several compounds reported as selective dopamine D 2 -like receptor ligands, such as the D 3 receptor agonist 7-OH-PIPAT and the D 4 receptor agonist RO-105824, to α 2 -adrenoceptors in cortical and striatal tissue, which express α 2A -adrenoceptors and both α 2A - and α 2C -adrenoceptors, respectively. The affinity of dopamine for α 2 -adrenoceptors was found to be similar to that for D 1 -like and D 2 -like receptors. Moreover, the exogenous dopamine receptor ligands also showed high affinity for α 2A - and α 2C -adrenoceptors. Their ability to activate Gi/o proteins through α 2A - and α 2C -adrenoceptors was also analyzed in transfected cells with bioluminescent resonance energy transfer techniques. The relative ligand potencies and efficacies were dependent on the Gi/o protein subtype. Furthermore, dopamine binding to α 2 -adrenoceptors was functional, inducing changes in dynamic mass redistribution, adenylyl cyclase activity, and ERK1/2 phosphorylation. Binding events were further studied with computer modeling of ligand docking. Docking of dopamine at α 2A - and α 2C -adrenoceptors was nearly identical to its binding to the crystallized D 3 receptor. Therefore, we pr