Partial agonistic effect of 9-hydroxycorynantheidine on mu-opioid receptor in the guinea-pig ileum

Mitragynine is an indole alkaloid isolated from the Thai medicinal plant Mitragyna speciosa that is reported to have opioid agonistic properties. The 9-demethyl analogue of mitragynine, 9-hydroxycorynantheidine, is synthesized from mitragynine. 9-Hydroxycorynantheidine inhibited electrically stimulated guinea-pig ileum contraction, but its maximum inhibition was weaker than that of mitragynine and its effect was antagonized by naloxone, suggesting that 9-hydroxycorynantheidine possesses partial agonist properties on opioid receptors. Receptor binding assays revealed that 9-hydroxycorynantheidine has high affinity for mu-opioid receptors. In an assay of the guinea-pig ileum, naloxone shifted the concentration-response curves for [D-Ala(2), N-MePhe(4), Gly-ol(5)]-enkephalin (DAMGO), (5alpha,7alpha,8beta)-(+)-N-Methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4.5]dec-8-yl]-benzeneacetamide (U69593) and 9-hydroxycorynantheidine to the right in a competitive manner. The pA(2) values of naloxone against 9-hydroxycorynantheidine and DAMGO were very similar, but not that against U69593. As indicated by the two assay systems, the opioid effect of 9-hydroxycorynantheidine is selective for the mu-opioid receptor. 9-Hydroxycorynantheidine shifted the concentration-response curve for DAMGO slightly to the right. Pretreatment with the mu-opioid selective and irreversible antagonist beta-funaltorexamine hydrochloride (beta-FNA) shifted the concentration-response curve for DAMGO to the right without affecting the maximum response. On the other hand, beta-FNA did not affect the curve for 9-hydroxycorynantheidine, but decreased the maximum response because of the lack of spare receptors. These studies suggest that 9-hydroxycorynantheidine has partial agonist properties on mu-opioid receptors in the guinea-pig ileum.     

Post-opioid receptor adaptations to chronic morphine; altered functionality and associations of signaling molecules

Opioid desensitization/tolerance mechanisms have largely focused on adaptations that occur on the level of the mu-opioid receptor (MOR) itself. These include opioid receptor phosphorylation and ensuing trafficking events. Recent research, however, has revealed additional adaptations that occur downstream from the opioid receptor, which involve covalent modification of signaling molecules and altered associations among them. These include augmented isoform-specific synthesis of adenylyl cyclase (AC) and their phosphorylation as well as augmented phosphorylation of the G(beta) subunit of G(beta gamma). The aggregate effect of these changes is to shift mu-opioid receptor-coupled signaling from predominantly G(i alpha) inhibitory to (G(i)-derived) G(beta gamma) stimulatory AC signaling. Most recently, chronic morphine has been shown to enhance the association (interaction) between MOR and G(s), which should provide an additional avenue for offsetting inhibitory MOR signaling sequelae. The unfolding complexity of chronic morphine-induced sequelae demands an evolving broader and more encompassing perspective on opioid tolerance-producing mechanisms. This should facilitate understanding tolerance within the context of physiological plasticity that is activated by chronic exposure to drugs of abuse. Additional research is required to integrate the various tolerance-producing adaptations that have been elucidated to date. Specifically, the relative contribution to opioid tolerance of identified adaptations is still unknown as is the extent to which they vary among different regions of the central nervous system.     

Mu-opioid agonist inhibition of kappa-opioid receptor-stimulated extracellular signal-regulated kinase phosphorylation is dynamin-dependent in C6 glioma cells

In previous studies we found that mu-opioids, acting via mu-opioid receptors, inhibit endothelin-stimulated C6 glioma cell growth. In the preceding article we show that the kappa-selective opioid agonist U69,593 acts as a mitogen with a potency similar to that of endothelin in the same astrocytic model system. Here we report that C6 cell treatment with mu-opioid agonists for 1 h results in the inhibition of kappa-opioid mitogenic signaling. The mu-selective agonist endomorphin-1 attenuates kappa-opioid-stimulated DNA synthesis, phosphoinositide turnover, and extracellular signal-regulated kinase phosphorylation. To investigate the role of receptor endocytosis in signaling, we have examined the effects of dynamin-1 and its GTPase-defective, dominant suppressor mutant (K44A) on opioid modulation of extracellular signal-regulated kinase phosphorylation in C6 cells. Overexpression of dynamin K44A in C6 cells does not affect kappa-opioid phosphorylation of extracellular signal-regulated kinase. However, it does block the inhibitory action on kappa-opioid signaling mediated by the kappa-opioid receptor. Our results are consistent with a growing body of evidence of the opposing actions of mu- and kappa-opioids and provide new insight into the role of opioid receptor trafficking in signaling.     

Differential Effects of Mg2+ and Other Divalent Cations on the Binding of Tritiated Opioid Ligands

The effects of MgCl2 on the binding of tritiated ligands to opioid binding sites in homogenates of guinea-pig brain in HEPES buffer have been studied. The binding of tritiated mu-, delta-, and kappa-opioid agonists was promoted in a concentration-dependent manner over a range of MgCl2 concentrations from 0.1 mM to 10 mM, as was binding of the nonselective antagonists [3H]diprenorphine and [3H]naloxone. At concentrations of MgCl2 above 10 mM reversal of this effect was observed. The effects of MgCl2 on binding parameters differed at each site. The promoting effects of MgCl2 were mimicked by MnCl2, CaCl2, and MgSO4, but CoCl2 and ZnCl2 were inhibitory. Following treatment of guinea-pig brain synaptosomes at pH 11.5 to eliminate G proteins, the binding of the mu-opioid agonist [3H][D-Ala2, MePhe4, Gly-ol5]enkephalin and [3H]naloxone was much reduced but binding of [3H]diprenorphine was unaffected. Under these conditions MgCl2 still promoted binding of [3H]diprenorphine. The results suggest that Mg2+ ions promote binding by an action at the opioid receptor, even in the absence of G protein, and that opioid antagonists may differ in their recognition of opioid receptor binding sites.     

The role of opioid receptor phosphorylation and trafficking in adaptations to persistent opioid treatment

Mu-opioid receptor activation underpins clinical analgesia and is the central event in the abuse of narcotics. Continued opioid use produces tolerance to the acute effects of the drug and adaptations that lead to physical and psychological dependence. Continued mu-receptor signaling provides the engine for these adaptations, with most evidence suggesting that chronic agonist treatment produces only limited alterations in primary mu-opioid receptor signaling. Here we examine agonist regulation of mu-opioid receptor function, and whether this is altered by chronic treatment. Receptor phosphorylation is thought to be the key initial event in agonist regulation of the mu-opioid receptor, providing a signal for acute receptor desensitization and also subsequent receptor resensitization. Morphine appears to produce qualitatively and quantitatively different mu-receptor phosphorylation than other agonists, but the consequences of this remain obscure, at least in neurons. There is no evidence that agonist-induced mu-opioid receptor phosphorylation changes in chronically morphine-treated animals, although receptor regulation appears to be altered. Thus, as receptor phosphorylation and resensitization appear to maintain continued signaling through the mu-opioid receptor, these two events are crucial in facilitating adaptations to chronic opioid treatment, and the possibility that agonist-specific phosphorylation can contribute to the development of different adaptations remains open.     

Antagonism of phosphoramidon-induced antinociception in mice by mu- but not kappa-opioid receptor blockers

Intracerebroventricular (i.c.v.) administration of the neutral endopeptidase 24.11-inhibitor phosphoramidon evoked a dose-dependent antinociceptive effect in the mouse acetic acid abdominal constriction test. The present study was conducted to identify the opioid receptor subtype(s) that mediate phosphoramidon antinociception in this paradigm. Mice were pretreated with different opioid antagonists prior to being challenged with phosphoramidon, i.c.v., the mu-opioid agonist sufentanil, s.c., or the kappa-opioid agonist U-50,488H, s.c. Naltrexone significantly attenuated phosphoramidon-induced antinociception at an i.c.v. dose that also blocked both sufentanil and U-50,488H. The mu-opioid antagonist beta-funaltrexamine (beta-FNA) blocked phosphoramidon and sufentanil at an i.c.v. dose that did not block U-50,488H. The kappa-opioid antagonist nor-binaltorphimine (nor-BNI) produced dose-related effects. A low dose (10 microg) of nor-BNI had no effect on either phosphoramidon or sufentanil but did reduce U-50,488H antinociception. A higher dose (30 microg) of nor-BNI blocked phosphoramidon, sufentanil, and U-50,488H, suggesting a loss of kappa-opioid receptor selectivity at this dose. These findings suggest that mu- but not kappa-opioid receptors mediate phosphoramidon-induced antinociception in the abdominal constriction test.     

Endogenous opiates and behavior: 2003

This paper is the 26th consecutive installment of the annual review of research concerning the endogenous opioid system, now spanning over a quarter-century of research. It summarizes papers published during 2003 that studied the behavioral effects of molecular, pharmacological and genetic manipulation of opioid peptides, opioid receptors, opioid agonists and opioid antagonists. The particular topics that continue to be covered include the molecular–biochemical effects and neurochemical localization studies of endogenous opioids and their receptors related to behavior (Section 2), and the roles of these opioid peptides and receptors in pain and analgesia (Section 3); stress and social status (Section 4); tolerance and dependence (Section 5); learning and memory (Section 6); eating and drinking (Section 7); alcohol and drugs of abuse (Section 8); sexual activity and hormones, pregnancy, development and endocrinology (Section 9); mental illness and mood (Section 10); seizures and neurologic disorders (Section 11); electrical-related activity and neurophysiology (Section 12); general activity and locomotion (Section 13); gastrointestinal, renal and hepatic functions (Section 14); cardiovascular responses (Section 15); respiration and thermoregulation (Section 16); and immunological responses (Section 17).     

Pyrrolo- and pyridomorphinans: Non-selective opioid antagonists and delta opioid agonists/mu opioid partial agonists

Opioid ligands have found use in a number of therapeutic areas, including for the treatment of pain and opiate addiction (using agonists) and alcohol addiction (using antagonists such as naltrexone and nalmefene). The reaction of imines, derived from the opioid ligands oxymorphone and naltrexone, with Michael acceptors leads to pyridomorphinans with structures similar to known pyrrolo- and indolomorphinans. One of the synthesized compounds, 5e, derived from oxymorphone had substantial agonist activity at delta opioid receptors but not at mu and/or kappa opioid receptors and in that sense profiled as a selective delta opioid receptor agonist. The pyridomorphinans derived from naltrexone and naloxone were all found to be non-selective potent antagonists and as such could have utility as treatments for alcohol abuse.     

背根神经节神经元阿片受体和离子通道的研究进展

阿片及阿片受体与外周神经系统镇痛机制的研究,随着分子生物学技术的发展,已在受体的分子结构、形态学、分子药理学、离子通道和细胞内信号转导系统等方面取得了显著进展。μ、δ、κ阿片受体分子结构上的部分差异决定了它们各自的功能特征。三种受体在初级感觉神经元分布的比例不同,但都能介导细胞Ｃａ＾２＋通道的抑制和Ｋ＾＋电流增加及减少。阿片受体和通道之间由多种第二信使系统偶联。分子药理学研究表明它们还存在亚型受体     

Opioid receptor binding and in vivo antinociceptive activity of position 3-substituted morphiceptin analogs

Analogs of morphiceptin (Tyr-Pro-Phe-Pro-NH2), a mu-selective opioid receptor ligand, with position 3-modifications, including altered size, lipophilicity, and electronic character, while maintaining aromaticity were synthesized. The activity of the new analogs in in vitro assays and in in vivo hot-plate test of analgesia was compared and the results were consistent. [D-1-Nal3]Morphiceptin was the most potent analog of this series with a 26-fold increase in mu-opioid receptor affinity, a 15-fold potency increase in the GPI assay, and a significant potency increase in the hot-plate analgesic test, as compared with morphiceptin. [d-Qal3]Morphiceptin was found to be a weak antagonist in the GPI assay.     

Activation of the endothelin receptor inhibits the G protein-coupled inwardly rectifying potassium channel by a phospholipase A2-mediated mechanism

To develop a malleable system to model the well-described, physiological interactions between Gq/11 - coupled receptor and Gi/o-coupled receptor signaling, we coexpressed the endothelin A receptor, the mu-opioid receptor, and the G protein-coupled inwardly rectifying potassium channel (Kir 3) heteromultimers in Xenopus laevis oocytes. Activation of the Gi/o-coupled mu-opioid receptor strongly increased Kir 3 channel current, whereas activation of the Gq/11-coupled endothelin A receptor inhibited the Kir 3 response evoked by mu-opioid receptor activation. The magnitude of the inhibition of Kir 3 was channel subtype specific; heteromultimers composed of Kir 3.1 and Kir 3.2 or Kir 3.1 and Kir 3.4 were significantly more sensitive to the effects of endothelin-1 than heteromultimers composed of Kir 3.1 and Kir 3.5. The difference in sensitivity of the heteromultimers suggests that the endothelin-induced inhibition of the opioid- activated current was caused by an effect at the channel rather than at the opioid receptor. The endothelin-1-mediated inhibition was mimicked by arachidonic acid and blocked by the phospholipase A2 inhibitor arachidonoyl trifluoromethyl ketone. Consistent with a possible phospholipase A2-mediated mechanism, the endothelin-1 effect was blocked by calcium chelation with BAPTA-AM and was not affected by kinase inhibition by either staurosporine or genistein. The data suggest the hypothesis that Gq/11-coupled receptor activation may interfere with Gi/o-coupled receptor signaling by the activation of phospholipase A2 and subsequent inhibition of effector function by a direct effect of an eicosanoid on the channel.     

Changes in the expression of G protein-coupled receptor kinases and beta-arrestin 2 in rat brain during opioid tolerance and supersensitivity

We previously demonstrated that chronic treatment of rats with the mu-opioid receptor agonist sufentanil induced pharmacological tolerance associated with mu-opioid receptor desensitization and down-regulation. Administration of the calcium channel blocker nimodipine during chronic treatment with sufentanil prevented mu-opioid receptor down-regulation, induced down-stream supersensitization, and produced supersensitivity to the opioid effects. The focus of the present study was to determine a role for G protein-coupled receptor kinases (GRKs) and beta-arrestin 2 in agonist-induced mu-opioid receptor signalling modulation during chronic opioid tolerance and supersensitivity. Tolerance was induced by 7-day chronic infusion of sufentanil (2 microgram/h). Supersensitivity was induced by concurrent infusion of sufentanil (2 microgram/h) and nimodipine (1 microgram/h) for 7 days. Antinociception was evaluated by the tail-flick test. GRK2, GRK3, GRK6 and beta-arrestin 2 immunoreactivity levels were determined by western blot in brain cortices. Acute and chronic treatment with sufentanil induced analgesic tolerance, associated with up-regulation of GRK2, GRK6, and beta-arrestin 2. GRK3 expression only was increased in the acutely treated group. When nimodipine was associated to the chronic opioid treatment, tolerance expression was prevented, and immunoreactivity levels of GRK2, GRK6 and beta-arrestin 2 recovered the control values. These data indicate that GRK2, GRK3, GRK6 and beta-arrestin 2 are involved in the short- and long-term adaptive changes in mu-opioid receptor activity, contributing to tolerance development in living animals. These observations also suggest that GRKs and beta-arrestin 2 could constitute pharmacological targets to prevent opioid tolerance development, and to improve the analgesic efficacy of opioid drugs.     

Evidence for the Implication of Phosphoinositol Signal Transduction in μ-Opioid Inhibition of DNA Synthesis

An opioid receptor agonist, [D-Ala2,Me-Phe4,Glyol5]enkephalin (DAMGE), decreased [3H]thymidine incorporation into DNA of fetal rat brain cell aggregates. This action proved to depend on the dose of this enkephalin analog and the interval the aggregates were maintained in culture. The opioid antagonist naltrexone and the mu-specific antagonist cyclic D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr amide (CTOP) reversed the DAMGE effect, arguing for a receptor-mediated mechanism. The mu-opioid nature of this receptor was further established by inhibiting DNA synthesis with the highly mu-selective agonist morphiceptin and blocking its action with CTOP. Several other opioids, pertussis toxin, and LiCl also diminished DNA synthesis, whereas cholera toxin elicited a modest increase. Naltrexone completely reversed the inhibition elicited by the combination of DAMGE and low doses of LiCl but not by that of high levels of LiCl alone. The enkephalin analog also reduced basal [3H]inositol trisphosphate and glutamate-stimulated [3H]inositol monophosphate and [3H]inositol bisphosphate accumulation in the aggregates. These DAMGE effects were reversed by naltrexone and were temporally correlated with the inhibition of DNA synthesis. A selective protein kinase C inhibitor, chelerythrine, also inhibited thymidine incorporation dose-dependently. The effect of DAMGE was not additive in the presence of chelerythrine but appeared to be consistent with their actions being mediated via a common signaling pathway. These results suggest the involvement of the phosphoinositol signal transduction system in the modulation of thymidine incorporation into DNA by DAMGE.     

Bioinformatic analysis of the origin, sequence and diversification of mu opioid receptors in vertebrates

Opioid receptors are a class of G protein-coupled receptors that mediate the effects of the different families of endogenous opioid peptides and natural alkaloid drugs such as morphine and its synthetic derivatives. In particular, the μ opioid receptor (MOR) represents the principal molecular target for morphine and it plays key roles in opioid analgesia and addiction. In this work, new putative MORs from different vertebrate species were identified in silico and their gene organization and predicted protein products are compared with the previously characterized MORs. Also, for the first time a new genomic organization in euteleleostei teleosts has been identified. Moreover, we suggest that MORs may be specific to craniate lineage. The analysis of functional mapping of MORs we present is an important contribution to the identification of their evolutionarily conserved regions.     

Stress responsivity, addiction, and a functional variant of the human mu-opioid receptor gene

Discovery and characterization of the functional A118G mu-(mu)-opioid receptor variant led to hypotheses, now in part proven, about its role in alterations of endogenous human physiology and in responses to opioid antagonist administration. Differences in cellular expression levels, ligand binding, and signal transduction for variant receptors have been documented in vitro. Human genetic studies also indicate that individuals carrying one or two copies of the 118G allele may have increased risk for opiate and alcohol addictions and that this polymorphism may also explain some of the variability in success of opioid antagonist treatment for alcoholism. Future research will further define the role of the A118G variant in addictive diseases and their treatment, in pain perception and opioid analgesia, and for a myriad of other responses mediated by the mu-opioid receptor.     

Are mu-opioid receptor polymorphisms important for clinical opioid therapy?

Mutations in the mu-opioid receptor--the primary site of action of opioid analgesics--are candidates for the variability of clinical opioid effects. This has been substantiated by recent advances in genetic research. A common mu-opioid receptor polymorphism was associated with higher demands for alfentanil or morphine for pain relief. It also decreased the potency of morphine for pupil constriction and experimental analgesia, but its molecular mechanisms are unclear. Another opioid receptor mutation greatly impaired receptor signalling in vitro, but is very rare. The accumulated evidence provides a solid basis for continuing research that should address the underlying molecular mechanisms and the role and benefits of OPRM1 genotyping for clinical pain therapy.     

C-terminal splice variants of the mouse mu-opioid receptor differ in morphine-induced internalization and receptor resensitization

The main analgesic effects of the opioid alkaloid morphine are mediated by the mu-opioid receptor. In contrast to endogenous opioid peptides, morphine activates the mu-opioid receptor without causing its rapid endocytosis. Recently, three novel C-terminal splice variants (MOR1C, MOR1D, and MOR1E) of the mouse mu-opioid receptor (MOR1) have been identified. In the present study, we show that these receptors differ substantially in their agonist-selective membrane trafficking. MOR1 and MOR1C stably expressed in human embryonic kidney 293 cells exhibited phosphorylation, internalization, and down-regulation in the presence of the opioid peptide [d-Ala(2),Me-Phe(4),Gly(5)-ol]enkephalin (DAMGO) but not in response to morphine. In contrast, MOR1D and MOR1E exhibited robust phosphorylation, internalization, and down-regulation in response to both DAMGO and morphine. DAMGO elicited a similar desensitization (during an 8-h exposure) and resensitization (during a 50-min drug-free interval) of all four mu-receptor splice variants. After morphine treatment, however, MOR1 and MOR1C showed a faster desensitization and no resensitization as compared with MOR1D and MOR1E. These results strongly reinforce the hypothesis that receptor phosphorylation and internalization are required for opioid receptor reactivation thus counteracting agonist-induced desensitization. Our findings also suggest a mechanism by which cell- and tissue-specific C-terminal splicing of the mu-opioid receptor may significantly modulate the development of tolerance to the various effects of morphine.     

Solubilization, purification, and mass spectrometry analysis of the human mu-opioid receptor expressed in Pichia pastoris

The human mu-opioid receptor was expressed in Pichia pastoris with or without EGFP at the N-terminal end. Expression yields of the recombinant proteins reached several tens of milligram of receptor per liter of culture medium in shacked flasks. Pharmacological studies using specific ligands demonstrated a typical opioid profile for the HuMOR-c-myc-his-tag construct, whereas the GFP-HuMOR-c-myc-his-tag receptor was unable to bind opioid drugs. The hexahistidine epitope-tagged receptors were purified by immobilized-nickel affinity chromatography. The identity of the purified mu-opioid receptor proteins was confirmed by Western blot and mass spectrometry analysis. In conclusion, the expression, solubilization, and purification strategies described herein allow to isolate very high quantities of purified receptor, up to 12 mg/L.     

Neuropeptide FF receptors antagonist, RF9, attenuates opioid-evoked hypothermia in mice

The present study used the endpoint of hypothermia to investigate opioid and neuropeptide FF (NPFF) interactions in conscious animals. Both opioid and NPFF systems played important roles in thermoregulation, which suggested a link between opioid receptors and NPFF receptors in the production of hypothermia. Therefore, we designed a study to investigate the relationship between opioid and NPFF in control of thermoregulation in mice. The selective NPFF receptors antagonist RF9 (30nmol) injected into the third ventricle failed to induce significant effect, but it completely antagonized the hypothermia of NPFF (45 nmol) after cerebral administration in mice. In addition, RF9 (30 nmol) co-injected i.c.v. in the third ventricle reduced the hypothermia induced by morphine (5nmol,) or nociceptin/orphanin FQ (N/OFQ) (2 nmol). Neither the classical opioid receptors antagonist naloxone (10 nmol) nor NOP receptor antagonist [Nphe(1)]NC(1-13)NH(2) (7.5 nmol) reduced the hypothermia induced by the central injection of NPFF at dose of 45 nmol. Co-injected with a low dose of NPFF (5 nmol), the hypothermia of morphine (5 nmol) or N/OFQ (2 nmol) was not modified. These results suggest that NPFF receptors activation is required for opioid to produce hypothermia. In contrast, NPFF-induced hypothermia is mainly mediated by its own receptors, independent of opioid receptors in the mouse brain. This interaction, quantitated in the present study, is the first evidence that NPFF receptors mediate opioid-induced hypothermia in conscious animals.     

Up-regulation of central mu-opioid receptors in a model of hepatic encephalopathy: a potential mechanism for increased sensitivity to morphine in liver failure

Increased GABA-mediated neurotransmission, reported to occur in hepatic encephalopathy (HE), is associated with a decrease in the release of Met-enkephalin and the expression of its coding gene in the brain. Furthermore, patients with cirrhosis and a history of HE exhibit increased sensitivity to the neuroinhibitory effects of morphine. Thus, there is a rationale to study the status of the endogenous opioid system in HE. The aim of this study was to determine whether mu-opioid receptors in the brain are up-regulated in a well characterized model of HE. Binding parameters of mu-opioid receptors were derived by assaying the binding of the opiate agonist [3H]-tyr-D-Ala-Gly-N-Methyl-Phe-Gly-ol (DAMGO) to brain membranes from rats with precisely defined stages of HE and control animals. The mean density of mu-opioid receptor sites (Bmax) in rats with stage II, III, and IV HE was 15, 29, and 33% higher, respectively, than the corresponding control value (p<0.01). In addition, the affinity of mu opioid receptors for the agonist (1/Kd) also increased with progression of HE (mean for stage IV HE vs. corresponding control mean, p<0.01). In conclusion, in liver failure, increased density and affinity of central mu-opioid receptors in the brain may: (i) be the basis for the documented increased sensitivity to opiate agonists; and (ii) occur as a consequence of increased GABAergic tone reducing neuronal synthesis and release of opioid agonist peptides.