Binding properties of C-truncated delta opioid receptors.
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AIM: To study the role of C-terminal delta opioid receptor involved in ligand binding affinity and selectivity. METHODS: The 31 amino acid residues of C-terminal truncated delta opioid receptors and the wild-type were expressed stably in Chinese hamster ovary (CHO) cells, respectively. Then the ligand binding properties of the products were studied by receptor binding assay. RESULTS: A typical mutated receptor clone CHO-T and a wild-type receptor clone CHO-W were obtained. The Kd values of [3H] diprenorphine (Dip) and [3H]leucine-2-alanine enkephalin (DADLE) bound to CHO-T were similar to CHO-W. Both the specific [3H]Dip bindings of CHO-T and CHO-W were strongly inhibited by delta selective agonists with similar Ki, but neither by mu nor kappa selective agonists. CONCLUSION: The C-terminal of the delta opioid receptor is not involved in the ligands binding affinity and selectivity. (+info)
Molecular modeling of interaction between delta opioid receptor and 3-methylfentanylisothiocyanate.
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AIM: To construct a 3D structural model of delta opioid receptor (delta OR) and study its interaction with 3-methylfentanylisothiocyanate (SuperFIT). METHODS: Using the bacteriohodopsin as a template, the 3D structure of delta OR was modeled; SuperFIT was docked into its inside. RESULTS: The interaction model between delta OR and (3R, 4S)-SuperFIT was achieved, in which the important binding sites possibly were Asp128, Ser106, Phe104, Tyr308, and Pro315. Asp128 formed the electrostatic and hydrogen-binding interactions with the protonated nitrogen on piperidine of the ligand. Ser106 formed the electrostatic interaction with the N atom of isothiocyano group of the ligand; whereas Phe104, Tyr308, and Pro315 formed the hydrophobic interactions with the S atom of isothiocyano group. In addition, there were some other interactions between delta OR and the ligand. CONCLUSION: The residues Phe104, Tyr308, Pro315, and Ser106 of delta OR are crucial to the delta selectivity of the ligand, which is beneficial for designing novel delta-selective ligand. (+info)
Absence of G-protein activation by mu-opioid receptor agonists in the spinal cord of mu-opioid receptor knockout mice.
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1. The ability of mu-opioid receptor agonists to activate G-proteins in the spinal cord of mu-opioid receptor knockout mice was examined by monitoring the binding to membranes of the non-hydrolyzable analogue of GTP, guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS). 2. In the receptor binding study, Scatchard analysis of [3H][D-Ala2,NHPhe4,Gly-ol]enkephalin ([3H]DAMGO; mu-opioid receptor ligand) binding revealed that the heterozygous mu-knockout mice displayed approximately 40% reduction in the number of mu-receptors as compared to the wild-type mice. The homozygous mu-knockout mice showed no detectable mu-binding sites. 3. The newly isolated mu-opioid peptides endomorphin-1 and -2, the synthetic selective mu-opioid receptor agonist DAMGO and the prototype of mu-opioid receptor agonist morphine each produced concentration-dependent increases in [35S]GTPgammaS binding in wild-type mice. This stimulation was reduced by 55-70% of the wild-type level in heterozygous, and virtually eliminated in homozygous knockout mice. 4. No differences in the [35S]GTPgammaS binding stimulated by specific delta1- ([D-Pen2,5]enkephalin), delta2-([D-Ala2]deltorphin II) or kappa1-(U50,488H) opioid receptor agonists were noted in mice of any of the three genotypes. 5. The data clearly indicate that mu-opioid receptor gene products play a key role in G-protein activation by endomorphins, DAMGO and morphine in the mouse spinal cord. They support the idea that mu-opioid receptor densities could be rate-limiting steps in the G-protein activation by mu-opioid receptor agonists in the spinal cord. These thus indicate a limited physiological mu-receptor reserve. Furthermore, little change in delta1-, delta2- or kappa1-opioid receptor-G-protein complex appears to accompany mu-opioid receptor gene deletions in this region. (+info)
Loperamide (ADL 2-1294), an opioid antihyperalgesic agent with peripheral selectivity.
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The antihyperalgesic properties of the opiate antidiarrheal agent loperamide (ADL 2-1294) were investigated in a variety of inflammatory pain models in rodents. Loperamide exhibited potent affinity and selectivity for the cloned micro (Ki = 3 nM) compared with the delta (Ki = 48 nM) and kappa (Ki = 1156 nM) human opioid receptors. Loperamide potently stimulated [35S]guanosine-5'-O-(3-thio)triphosphate binding (EC50 = 56 nM), and inhibited forskolin-stimulated cAMP accumulation (IC50 = 25 nM) in Chinese hamster ovary cells transfected with the human mu opioid receptor. The injection of 0.3 mg of loperamide into the intra-articular space of the inflamed rat knee joint resulted in potent antinociception to knee compression that was antagonized by naloxone, whereas injection into the contralateral knee joint or via the i.m. route failed to inhibit compression-induced changes in blood pressure. Loperamide potently inhibited late-phase formalin-induced flinching after intrapaw injection (A50 = 6 microgram) but was ineffective against early-phase flinching or after injection into the paw contralateral to the formalin-treated paw. Local injection of loperamide also produced antinociception against Freund's adjuvant- (ED50 = 21 microgram) or tape stripping- (ED50 = 71 microgram) induced hyperalgesia as demonstrated by increased paw pressure thresholds in the inflamed paw. In all animal models examined, the potency of loperamide after local administration was comparable to or better than that of morphine. Loperamide has potential therapeutic use as a peripherally selective opiate antihyperalgesic agent that lacks many of the side effects generally associated with administration of centrally acting opiates. (+info)
Expression of delta, kappa and mu human opioid receptors in Escherichia coli and reconstitution of the high-affinity state for agonist with heterotrimeric G proteins.
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Human opioid receptors of the delta, mu and kappa subtypes were successfully expressed in Escherichia coli as fusions to the C-terminus of the periplasmic maltose-binding protein, MBP. Expression levels of correctly folded receptor molecules were comparable for the three subtypes and reached an average of 30 receptors.cell-1 or 0.5 pmol.mg-1 membrane protein. Binding of [3H]diprenorphine to intact cells or membrane preparations was saturatable, with a dissociation constant, KD, of 2.5 nM, 0.66 nM and 0.75 nM for human delta, mu and kappa opioid receptors (hDOR, hMOR and hKOR, respectively). Recombinant receptors of the three subtypes retained selectivity and nanomolar affinity for their specific antagonists. Agonist affinities were decreased by one to three orders of magnitude as compared to values measured for receptors expressed in mammalian cells. The effect of sodium on agonist binding to E. coli-expressed receptors was investigated. Receptor high-affinity state for agonists was reconstituted in the presence of heterotrimeric G proteins. We also report affinity values of endomorphins 1 and 2 for mu opioid receptors expressed both in E. coli and in COS cells. Our results confirm that opioid receptors can be expressed in a functional form in bacteria and point out the advantages of E. coli as an expression system for pharmacological studies. (+info)
A molecular mechanism for the cleavage of a disulfide bond as the primary function of agonist binding to G-protein-coupled receptors based on theoretical calculations supported by experiments.
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A model of the binding site of delta-opioids in the extracellular region of the G-protein-coupled opioid receptor based on modelling studies is presented. The distance between Asp288 and the disulfide bridge (Cys121-Cys198) formed between the first and second extracellular loops was found to be short. This model is consistent with site-directed mutagenesis studies. The arrangement of the ligands found in the receptor led to the development of a reaction mechanism for the cleavage of the disulfide bond catalysed by the ligands. Semi-empirical quantum chemical PM3 and AM1 calculations as well as ab initio studies showed that the interaction between the carboxylic acid side chain of aspartic acid and the disulfide bond leads to the polarization of, and withdrawal of a proton from, the protonated nitrogen of the ligand to one of the sulfur atoms. A mixed sulfenic acid and carboxylic acid anhydrate is formed as an intermediate as well as a thiol. The accompanying cleavage of the disulfide bond may produce a conformational change in the extracellular loops such that the pore formed by the seven-helix bundle opens allowing entrance of the ligand, water and ions into the cell. Cleavage of the disulfide bond after opioid administration was demonstrated experimentally by flow-cytometric measurements employing CMTMR and monobromobimane-based analyses of membrane-located thiols. The suggested mechanism may explain, in a consistent way, the action of agonists and antagonists and is assumed to be common for many G-protein coupled receptors. (+info)
Antagonism by acetyl-RYYRIK-NH2 of G protein activation in rat brain preparations and of chronotropic effect on rat cardiomyocytes evoked by nociceptin/orphanin FQ.
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For the further elucidation of the central functions of nociceptin/orphanin FQ (noc/OFQ), the endogenous ligand of the G protein-coupled opioid receptor-like receptor ORL1, centrally acting specific antagonists will be most helpful. In this study it was found that the hexapeptide acetyl-RYYRIK-NH2 (Ac-RYYRIK-NH2), described in literature as partial agonist on ORL1 transfected in CHO cells, antagonizes the stimulation of [35S]-GTPgammaS binding to G proteins by noc/OFQ in membranes and sections of rat brain. The antagonism of the peptide was competitive, of high affinity (Schild constant 6.58 nM), and specific for noc/OFQ in that the stimulation of GTP binding by agonists for the mu-, delta-, and kappa-opioid receptor was not inhibited. The hexapeptide also fully inhibited the chronotropic effect of noc/OFQ on neonatal rat cardiomyocytes. It is suggested that Ac-RYYRIK-NH2 may provide a promising starting point for in vivo tests for antagonism of the action of noc/OFQ and for the further development of highly active and specific antagonists. (+info)
Electrophysiological studies on the postnatal development of the spinal antinociceptive effects of the delta opioid receptor agonist DPDPE in the rat.
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1. The antinociceptive effects of the delta opioid receptor selective agonist, DPDPE [(D-Pen2,D-Pen5)-enkephalin] was studied in rats aged postnatal day (P) 14, P21, P28 and P56. 2. Antinociceptive effects of DPDPE were measured as percentage inhibition of the C-fibre evoked response and post-discharge of dorsal horn neurones evoked by peripheral electrical stimulation. DPDPE was administered by topical application, akin to intrathecal injection. 3. DPDPE (0.1-100 microg) produced dose-related inhibitions at all ages; these inhibitions were reversed by 5 microg of the opioid antagonist naloxone. 4. The dose-response curves for C-fibre evoked response and post-discharge of the neurones were not different in rats aged P14 and P21. DPDPE was significantly more potent at P14 and P21 compared with its inhibitory effects on these responses at P28 and P56. 5. DPDPE produced minor inhibitions of the A-fibre evoked response of the neurones at P14, P21, P28 and P56, suggesting that the inhibitory effects of DPDPE are mediated via presynaptic receptors on the terminals of C-fibre afferents. 6. Since spinal delta opioid receptor density changes little over this period, the increased antinociceptive potency of DPDPE in the rat pups compared with the adult is likely to be due to post-receptor events, or in developmental changes in the actions of other transmitter/receptor systems within the spinal cord. (+info)