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George R Uhl - One of the best experts on this subject based on the ideXlab platform.
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congenic c57bl 6 mu Opiate Receptor mor knockout mice baseline and Opiate effects
Genes Brain and Behavior, 2003Co-Authors: F S Hall, Ichiro Sora, Michelle Goeb, S Roff, H Hoggatt, George R UhlAbstract:Homozygous µ-opioid Receptor (MOR) knockout (KO) mice developed on a chimeric C57B6/129SV background lack morphine-induced antinociception, locomotion and reward. Therefore it appears that MOR largely mediates these morphine actions. However, one factor that could affect the extent of knockout deficits in morphine-induced behavior is the genetic background against which the gene deletion is expressed. To examine the effect of genetic background chimeric C57B6/129SV MOR knockout mice from the 15th generation of those developed in our laboratory were backcrossed for 10 successive generations with C57BL/6 mice, a strain which is more sensitive to many of the properties of morphine, to produce congenic MOR (conMOR) KO mice. Heterozygote conMOR KO mice display attenuated morphine locomotion and reduced morphine analgesia compared to wild-type mice. Homozygote conMOR KO mice display baseline hyperalgesia, no morphine place preference, no morphine analgesia and no morphine locomotion. These results are not qualitatively different from those observed in the MOR KO strain with a chimeric C57B6/129SV background, and suggest that although the strain has separate influences on these functions, it does not substantially interact with deletion of the µ Opiate Receptor gene.
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ethanol consumption and reward are decreased in mu Opiate Receptor knockout mice
Psychopharmacology, 2001Co-Authors: F S Hall, Ichiro Sora, George R UhlAbstract:Rationale: Differences in µ-Opiate Receptor (MOR) gene expression may modulate the rewarding effects of ethanol. Objective: The effects of MOR gene knockout (KO) were examined in wild-type (+/+), heterozygote MOR KO (+/–), and homozygote MOR KO (–/–) mice on voluntary ethanol consumption, conditioned place preference produced by ethanol, and locomotor responses to ethanol in separate groups of mice. Methods: Voluntary ethanol consumption (2–32% v/v) was examined in a two-bottle home-cage consumption test. The conditioned place preference paradigm was a biased design. Mice received four pairings of ethanol (2.0 g/kg IP) on the initially preferred side and four pairings on the initially non-preferred side with saline. The difference in time spent on the initially non-preferred side (pre- versus post-conditioning) was the measure of drug-induced preference. After habituation to a novel locomotor test chamber mice were tested, on subsequent sessions, for ethanol induced locomotion (0.0, 0.5, 1.0, and 2.0 g/kg IP). Results: Heterozygous and homozygous MOR KO mice consumed less ethanol than wild-type mice. These effects appeared to be greater in female KO mice than in male KO mice. MOR KO mice, especially females, exhibited less ethanol reward in a conditioned place preference paradigm. These effects on ethanol reward were produced by reductions in MOR expression levels as small as 50%. MOR KO mice exhibited less ethanol-stimulated locomotion than did wild-type mice, an effect that was also largest in females. Conclusions: These data fit with the reported therapeutic efficacy of MOR antagonists in the treatment of human alcoholism. Allelic variants that confer differing levels of MOR expression could provide different degrees of risk for alcoholism.
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Opiate Receptor knockout mice define μ Receptor roles in endogenous nociceptive responses and morphine induced analgesia
Proceedings of the National Academy of Sciences of the United States of America, 1997Co-Authors: Ichiro Sora, Nobuyuki Takahashi, Masahiko Funada, Hiroshi Ujike, Randal S Revay, David M Donovan, Lucinda L Miner, George R UhlAbstract:Morphine produces analgesia at Opiate Receptors expressed in nociceptive circuits. μ, δ, and κ Opiate Receptor subtypes are expressed in circuits that can modulate nociception and receive inputs from endogenous opioid neuropeptide ligands. The roles played by each Receptor subtype in nociceptive processing in drug-free and morphine-treated states have not been clear, however. We produced homologous, recombinant μ, Opiate Receptor, heterozygous and homozygous knockout animals that displayed ≈54% and 0% of wild-type levels of μ Receptor expression, respectively. These mice expressed κ Receptors and δ Receptors at near wild-type levels. Untreated knockout mice displayed shorter latencies on tail flick and hot plate tests for spinal and supraspinal nociceptive responses than wild-type mice. These findings support a significant role for endogenous opioid–peptide interactions with μ Opiate Receptors in normal nociceptive processing. Morphine failed to significantly reduce nociceptive responses in hot plate or tail flick tests of homozygous μ Receptor knockout mice, and heterozygote mice displayed right and downward shifts in morphine analgesia dose–effect relationships. These results implicate endogenous opioid–peptide actions at μ Opiate Receptors in several tests of nociceptive responsiveness and support μ Receptor mediation of morphine-induced analgesia in tests of spinal and supraspinal analgesia.
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differential Opiate Receptor phosphorylation and desensitization induced by agonists and phorbol esters
Journal of Biological Chemistry, 1996Co-Authors: Li Zhang, Jia Bei Wang, George R Uhl, Seamus Mackin, Forrest F WeightAbstract:Abstract μ Opiate Receptors, the principal sites for Opiate analgesia and reward, can display compensatory responses to Opiate agonist drug administration. Agonist-induced K channel responses mediated by these Receptors desensitize when examined in Xenopus oocyte expression systems. Mechanisms underlying such processes could include phosphorylation events similar to those reported to desensitize other G-protein-linked Receptors. We used C-terminally directed anti-μ Receptor antibodies to immunoprecipitate a phosphoprotein with size appropriate for the μ Receptor from stably expressing Chinese hamster ovary cells. Phosphorylation of this μ Opiate Receptor protein was enhanced approximately 5-fold by treatment with the μ agonist morphine. The time course and dose-response relationships between μ Receptor phosphorylation and agonist-induced desensitization display interesting parallels. Phosphorylation of μ Opiate Receptor protein is also enhanced 5-fold by treatment with the protein kinase C activator phorbol 12-myristate 13-acetate. The protein kinase inhibitor staurosporine blocked the effect of phorbol 12-myristate 13-acetate on μ Receptor phosphorylation. However, staurosporine failed to block morphine-induced phosphorylation. These observations suggest that several biochemical pathways can lead to μ Receptor phosphorylation events that may include mechanisms involved in μ Receptor desensitization.
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cdna cloning of an orphan Opiate Receptor gene family member and its splice variant
FEBS Letters, 1994Co-Authors: Jia Bei Wang, Peter S. Johnson, Antonio M Persico, Yasuo Imai, Bradley A Ozenberger, Mark C Eppler, George R UhlAbstract:Radioligand binding and cDNA homology studies have suggested the existence of Opiate Receptors distinct from the recently-cloned, μ, δ and κ Receptors. XOR1S, a rat brain cDNA whose predicted translation product displays 67–72% homology with those encoded by μ1, δ1 and κ1 Opiate Receptor cDNAs, was constructed from two partial cDNAs identified through cDNA homology approaches. A longer XOR1L variant of this cDNA was also identified by polymerase chain reaction studies using genomic DNA and cDNA from brain and peripheral tissues. XOR1 mRNA is most highly expressed in hypothalamus. COS cell expression of both clones confers neither robust binding of Opiate ligands nor reproducible Opiate inhibition of forskolin-stimulated adenylate cyclase. These studies identify an orphan clone that helps to define features of the Opiate Receptor gene family, including apparent differential splicing and expression in peripheral tissues.
George B. Stefano - One of the best experts on this subject based on the ideXlab platform.
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Biological indications of a novel "short" µ Opiate Receptor in domestic chicken.
Archives of medical science : AMS, 2010Co-Authors: Melinda Sheehan, Richard M Kream, George B. StefanoAbstract:Previous work from our laboratory has established that cellular signaling processes of endogenous morphine are mediated by cognate G protein coupled Receptor (GPCR) proteins, designated µ3 and µ4 Opiate Receptors. µ3 and µ4 Opiate Receptors are structurally unique “short” 6 transmembrane helical (TMH) domain GPCRs that are selectively responsive to endogenous morphine, not to families of endogenous opioid peptides, and are uniquely coupled to activation of constitutive nitric oxide synthase (cNOS). Based on high resolution predictive measures, it appears likely that domestic poultry express a µ Opiate Receptor mRNA encoding potentially two novel GPCRs with similar biochemical characteristics as described for µ3 and µ4 Opiate Receptors as well as traditional µ1 opioid Receptors. The biological indications of these novel µ Opiate Receptors are discussed within the context of this short review.
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a functionally coupled μ3 like Opiate Receptor nitric oxide regulatory pathway in human multi lineage progenitor cells
Journal of Immunology, 2007Co-Authors: Patrick Cadet, Kirk J. Mantione, Wei Zhu, Richard M Kream, Melinda Sheehan, George B. StefanoAbstract:Ongoing studies from our group support the existence and biological importance of a distinct cellular signaling pathway involving endogenously synthesized, chemically authentic, l-morphine, its cognate mu(3) Opiate Receptor subtype, and constitutive NO synthase. Based on prior studies indicating evolutionary conservation and adaptation of morphinergic/NO-coupled signaling to mediate autocrine/paracrine control of cellular functions, our goal was to determine whether a functionally competent mu(3) Opiate Receptor/NO-coupled regulatory pathway exists in human multilineage progenitor cells (MLPC) prepared from umbilical cord blood. Real-time PCR analysis indicated significant expression of mu(3) Opiate Receptor-encoding RNA by undifferentiated human MLPC, in the absence of traditional mu(1) opioid Receptor-encoding RNA expression. Unpredictably, confirmatory RT-PCR analyses indicated cellular expression of a splice variant of the previously characterized mu(3) Opiate Receptor-encoding mRNA. Pharmacological analyses provided critical validating evidence of functional mu(3)-like Opiate Receptor/NO-coupled signaling within primary cultures of undifferentiated human MLPC via morphine-evoke real-time release of NO. Control analyses indicated that morphine-stimulated NO release was markedly inhibited by prior treatment with the Opiate antagonist l-naloxone or the constitutive NO synthase inhibitor N(G)-nitro-l-arginine methyl ester and unresponsive to stimulation by the opioid peptide methionine enkephalin. Complementary microarray analysis demonstrated that traditional mu(1), delta, and kappa opioid Receptor gene expression is not detected in both undifferentiated and differentiated MLPC. Chemical differentiation of MLPC into neuronal progenitor cells effected significant phenotypic expression of a variety of neurally-associated genes. Our data provide compelling evidence in support of both the evolutionary primacy and primordial regulatory role of mu(3)-like Opiate Receptor/NO signaling in embryogenesis.
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molecular identification and functional expression of μ3 a novel alternatively spliced variant of the human μ Opiate Receptor gene
Journal of Immunology, 2003Co-Authors: Patrick Cadet, Kirk J. Mantione, George B. StefanoAbstract:Studies from our laboratory have revealed a novel μ Opiate Receptor, μ 3 , which is expressed in both vascular tissues and leukocytes. The μ 3 Receptor is selective for Opiate alkaloids and is insensitive to opioid peptides. We now identify the μ 3 Receptor at the molecular level using a 441-bp conserved region of the μ 1 Receptor. Sequence analysis of the isolated cDNA suggests that it is a novel, alternatively spliced variant of the μ Opiate Receptor gene. To determine whether protein expressed from this cDNA exhibits the biochemical characteristics expected of the μ 3 Receptor, the cDNA clone was expressed in a heterologous system. At the functional level, COS-1 cells transfected with the μ 3 Receptor cDNA exhibited dose-dependent release of NO following treatment with morphine, but not opioid peptides (i.e., Met-enkephalin). Naloxone was able to block the effect of morphine on COS-1 transfected cells. Nontransfected COS-1 cells did not produce NO in the presence of morphine or the opioid peptides at similar concentrations. Receptor binding analysis with [ 3 H]dihydromorphine further supports the Opiate alkaloid selectivity and opioid peptide insensitivity of this Receptor. These data suggest that this new μ Opiate Receptor cDNA encodes the μ 3 Opiate Receptor, since it exhibits biochemical characteristics known to be unique to this Receptor (Opiate alkaloid selective and opioid peptide insensitive). Furthermore, using Northern blot, RT-PCR, and sequence analysis, we have demonstrated the expression of this new μ variant in human vascular tissue, mononuclear cells, polymorphonuclear cells, and human neuroblastoma cells.
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Morphine 6 glucuronide stimulates nitric oxide release in mussel neural tissues: evidence for a morphine 6 glucuronide Opiate Receptor subtype
Cellular and Molecular Life Sciences, 2002Co-Authors: Kirk J. Mantione, Christos M. Rialas, Frederico Casares, A. L. Franklin, Patrick Cadet, Jan Tønnesen, George B. StefanoAbstract:We have previously demonstrated that Mytilus edulis pedal ganglia contain Opiate alkaloids, i.e., morphine and morphine 6 glucuronide (M6G), as well as mu Opiate Receptor subtype fragments exhibiting high sequence similarity to those found in mammals. Now we demonstrate that M6G stimulates pedal ganglia constitutive nitric oxide (NO) synthase (cNOS)-derived NO release at identical concentrations and to similar peak levels as morphine. However, the classic Opiate antagonist, naloxone, only blocked the ability of morphine to stimulate cNOS-derived NO release and not that of M6G. CTOP, a mu-specific antagonist, blocked the ability of M6G to induce cNOS-derived NO release as well as that of morphine, suggesting that a novel mu Opiate Receptor was present and selective toward M6G. In examining a Receptor displacement analysis, both Opiate alkaloids displaced [3H]-dihydromorphine binding to the mu Opiate Receptor subtype. However, morphine exhibited a twofold higher affinity, again suggesting that a novel mu Opiate Receptor may be present.
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morphine suppresses complement Receptor expression phagocytosis and respiratory burst in neutrophils by a nitric oxide and μ3 Opiate Receptor dependent mechanism
Journal of Neuroimmunology, 2000Co-Authors: I Welters, A Menzebach, Yannick Goumon, T W Langefeld, H Teschemacher, G Hempelmann, George B. StefanoAbstract:We investigated whether morphine and fentanyl influence surface Receptor expression, phagocytic activity and superoxide anion generation of neutrophils in a whole blood flow cytometric assay. Morphine suppressed complement and Fcgamma Receptor expression and neutrophil function in a concentration- and time-dependent manner. Morphine-induced changes were similar to those caused by the nitric oxide (NO) donor S-nitroso-N-acetyl-penicillamine and were abolished by preincubation with the NO synthase inhibitor N-nitro-L-arginine as well as naloxone. Fentanyl had no immunosuppressive effects. These results suggest that these neutrophil functions are inhibited by morphine-stimulated NO release mediated by the mu(3) Opiate Receptor subtype found on immunocytes.
Jia Bei Wang - One of the best experts on this subject based on the ideXlab platform.
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differential Opiate Receptor phosphorylation and desensitization induced by agonists and phorbol esters
Journal of Biological Chemistry, 1996Co-Authors: Li Zhang, Jia Bei Wang, George R Uhl, Seamus Mackin, Forrest F WeightAbstract:Abstract μ Opiate Receptors, the principal sites for Opiate analgesia and reward, can display compensatory responses to Opiate agonist drug administration. Agonist-induced K channel responses mediated by these Receptors desensitize when examined in Xenopus oocyte expression systems. Mechanisms underlying such processes could include phosphorylation events similar to those reported to desensitize other G-protein-linked Receptors. We used C-terminally directed anti-μ Receptor antibodies to immunoprecipitate a phosphoprotein with size appropriate for the μ Receptor from stably expressing Chinese hamster ovary cells. Phosphorylation of this μ Opiate Receptor protein was enhanced approximately 5-fold by treatment with the μ agonist morphine. The time course and dose-response relationships between μ Receptor phosphorylation and agonist-induced desensitization display interesting parallels. Phosphorylation of μ Opiate Receptor protein is also enhanced 5-fold by treatment with the protein kinase C activator phorbol 12-myristate 13-acetate. The protein kinase inhibitor staurosporine blocked the effect of phorbol 12-myristate 13-acetate on μ Receptor phosphorylation. However, staurosporine failed to block morphine-induced phosphorylation. These observations suggest that several biochemical pathways can lead to μ Receptor phosphorylation events that may include mechanisms involved in μ Receptor desensitization.
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Human kappa Opiate Receptor second extracellular loop elevates dynorphin's affinity for human mu/kappa chimeras.
Journal of Biological Chemistry, 1994Co-Authors: Jia Bei Wang, Peter S. Johnson, Jun Min Wu, Wen Fei WangAbstract:Abstract To investigate roles of second extracellular loop sequences in peptide and nonpeptide ligand recognition by human Opiate Receptors, we have constructed a chimeric Receptor in which this domain of the human mu Opiate Receptor has been replaced with that of the human kappa Opiate Receptor. The chimeric Opiate Receptor displays dramatically increased affinity for dynorphin peptides. Affinities for dynorphin A-(1-17), dynorphin A-(1-13), and alpha-neoendorphin increase by up to 250-fold when compared with the wild-type human mu Opiate Receptor. The chimera maintains recognition of the mu-selective ligands morphine and [D-Ala2,MePhe4,Gly-ol5]enkephalin and displays no significant changes in affinity for the kappa-selective small molecule ligand U50,488. The chimeric Opiate Receptor displays evidence for effective G-protein coupling; 100 nM dynorphin A-(1-17) is as effective as 100 nM morphine at inhibiting forskolin-stimulated adenyl cyclase activity through actions at the chimeric Receptor. These data suggest that the putative second extracellular loop contributes substantially to the kappa Receptor's selectivity in dynorphin ligand recognition.
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cdna cloning of an orphan Opiate Receptor gene family member and its splice variant
FEBS Letters, 1994Co-Authors: Jia Bei Wang, Peter S. Johnson, Antonio M Persico, Yasuo Imai, Bradley A Ozenberger, Mark C Eppler, George R UhlAbstract:Radioligand binding and cDNA homology studies have suggested the existence of Opiate Receptors distinct from the recently-cloned, μ, δ and κ Receptors. XOR1S, a rat brain cDNA whose predicted translation product displays 67–72% homology with those encoded by μ1, δ1 and κ1 Opiate Receptor cDNAs, was constructed from two partial cDNAs identified through cDNA homology approaches. A longer XOR1L variant of this cDNA was also identified by polymerase chain reaction studies using genomic DNA and cDNA from brain and peripheral tissues. XOR1 mRNA is most highly expressed in hypothalamus. COS cell expression of both clones confers neither robust binding of Opiate ligands nor reproducible Opiate inhibition of forskolin-stimulated adenylate cyclase. These studies identify an orphan clone that helps to define features of the Opiate Receptor gene family, including apparent differential splicing and expression in peripheral tissues.
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human μ Opiate Receptor cdna and genomic clones pharmacologic characterization and chromosomal assignment
FEBS Letters, 1994Co-Authors: Jia Bei Wang, Peter S. Johnson, Antonio M Persico, Anita L Hawkins, Constance A Griffin, George R UhlAbstract:A human μ Opiate Receptor cDNA has been identified from a cerebral cortical cDNA library using sequences from the rat μ Opiate Receptor cDNA. The human μ Opiate Receptor (hμOR1) shares 95% amino acid identity with the rat sequence. The expressed μOR1 recognizes tested Opiate drugs and opioid peptides in a sodium- and GTP-sensitive fashion with affinities virtually identical to those displayed by the rat μ Opiate Receptor. Effects on cyclic AMP are similar to those noted for the rat μ Opiate Receptor. An 18 kb genomic clone hybridizing with the hμOR1 cDNA contains 63 and 489 bp exonic sequences flanked by splice donor/acceptor sequences. Analysis of hybridization to DNA prepared from human rodent hybrid cell lines and chromosomal in situ hybridization studies indicate localization to 6q24–25. An MspI polymorphism, producing a 3.7 kb band, may prove useful in assessing this gene's involvement in neuropsychiatric disorders involving Opiatergic systems.
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mu Opiate Receptor: cDNA cloning and expression
Proceedings of the National Academy of Sciences of the United States of America, 1993Co-Authors: Jia Bei Wang, Y. Imai, C. M. Eppler, P. Gregor, C. E. Spivak, George R UhlAbstract:Abstract mu Opiate Receptors recognize morphine with high affinity. A 2.1-kb rat brain cDNA whose predicted translation product displays 63% identity with recently described delta and kappa Opiate Receptor sequences was identified through polymerase chain reaction and cDNA homology approaches. This cDNA recognizes a 10.5-kb mRNA that is expressed in thalamic neurons. COS-cell expression confers naloxonazine-, Na(+)-, and GTP-sensitive binding of mu but not delta or kappa opioid ligands. Expressing cells bind morphine, [D-Ala2,N-methyl-Phe4,glyol5]enkephalin (DAMGO), and [D-Ala2,D-Leu5]enkephalin (DADLE) with nanomolar or subnanomolar affinities, defining a mu Opiate Receptor that avidly recognizes analgesic and euphoric Opiate drugs and opioid peptides.
Ichiro Sora - One of the best experts on this subject based on the ideXlab platform.
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congenic c57bl 6 mu Opiate Receptor mor knockout mice baseline and Opiate effects
Genes Brain and Behavior, 2003Co-Authors: F S Hall, Ichiro Sora, Michelle Goeb, S Roff, H Hoggatt, George R UhlAbstract:Homozygous µ-opioid Receptor (MOR) knockout (KO) mice developed on a chimeric C57B6/129SV background lack morphine-induced antinociception, locomotion and reward. Therefore it appears that MOR largely mediates these morphine actions. However, one factor that could affect the extent of knockout deficits in morphine-induced behavior is the genetic background against which the gene deletion is expressed. To examine the effect of genetic background chimeric C57B6/129SV MOR knockout mice from the 15th generation of those developed in our laboratory were backcrossed for 10 successive generations with C57BL/6 mice, a strain which is more sensitive to many of the properties of morphine, to produce congenic MOR (conMOR) KO mice. Heterozygote conMOR KO mice display attenuated morphine locomotion and reduced morphine analgesia compared to wild-type mice. Homozygote conMOR KO mice display baseline hyperalgesia, no morphine place preference, no morphine analgesia and no morphine locomotion. These results are not qualitatively different from those observed in the MOR KO strain with a chimeric C57B6/129SV background, and suggest that although the strain has separate influences on these functions, it does not substantially interact with deletion of the µ Opiate Receptor gene.
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ethanol consumption and reward are decreased in mu Opiate Receptor knockout mice
Psychopharmacology, 2001Co-Authors: F S Hall, Ichiro Sora, George R UhlAbstract:Rationale: Differences in µ-Opiate Receptor (MOR) gene expression may modulate the rewarding effects of ethanol. Objective: The effects of MOR gene knockout (KO) were examined in wild-type (+/+), heterozygote MOR KO (+/–), and homozygote MOR KO (–/–) mice on voluntary ethanol consumption, conditioned place preference produced by ethanol, and locomotor responses to ethanol in separate groups of mice. Methods: Voluntary ethanol consumption (2–32% v/v) was examined in a two-bottle home-cage consumption test. The conditioned place preference paradigm was a biased design. Mice received four pairings of ethanol (2.0 g/kg IP) on the initially preferred side and four pairings on the initially non-preferred side with saline. The difference in time spent on the initially non-preferred side (pre- versus post-conditioning) was the measure of drug-induced preference. After habituation to a novel locomotor test chamber mice were tested, on subsequent sessions, for ethanol induced locomotion (0.0, 0.5, 1.0, and 2.0 g/kg IP). Results: Heterozygous and homozygous MOR KO mice consumed less ethanol than wild-type mice. These effects appeared to be greater in female KO mice than in male KO mice. MOR KO mice, especially females, exhibited less ethanol reward in a conditioned place preference paradigm. These effects on ethanol reward were produced by reductions in MOR expression levels as small as 50%. MOR KO mice exhibited less ethanol-stimulated locomotion than did wild-type mice, an effect that was also largest in females. Conclusions: These data fit with the reported therapeutic efficacy of MOR antagonists in the treatment of human alcoholism. Allelic variants that confer differing levels of MOR expression could provide different degrees of risk for alcoholism.
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the μ Opiate Receptor as a candidate gene for pain polymorphisms variations in expression nociception and Opiate responses
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: Ichiro Sora, Zaijie WangAbstract:There are differences between human individuals and between mouse strains in levels of μ Opiate Receptor (μOR) expression, responses to painful stimuli, and responses to Opiate drugs. One of the best candidates for contributing to these differences is variation at the μOR gene locus. Support for this idea comes from analyses of the human and murine μOR genes. Assessments of individual differences in human μOR expression add further support. Studies with mice, including knockout-transgenic, quantitative trait locus, and strain-comparison studies, also strongly support the possibility that μOR gene alleles would be strong candidates for contributing to individual differences in human nociception and Opiate drug responses. This paper reviews current analyses of the murine and human μOR genes, their important variants, and correlations between these variants and Opiate influences on pain.
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Opiate Receptor knockout mice define μ Receptor roles in endogenous nociceptive responses and morphine induced analgesia
Proceedings of the National Academy of Sciences of the United States of America, 1997Co-Authors: Ichiro Sora, Nobuyuki Takahashi, Masahiko Funada, Hiroshi Ujike, Randal S Revay, David M Donovan, Lucinda L Miner, George R UhlAbstract:Morphine produces analgesia at Opiate Receptors expressed in nociceptive circuits. μ, δ, and κ Opiate Receptor subtypes are expressed in circuits that can modulate nociception and receive inputs from endogenous opioid neuropeptide ligands. The roles played by each Receptor subtype in nociceptive processing in drug-free and morphine-treated states have not been clear, however. We produced homologous, recombinant μ, Opiate Receptor, heterozygous and homozygous knockout animals that displayed ≈54% and 0% of wild-type levels of μ Receptor expression, respectively. These mice expressed κ Receptors and δ Receptors at near wild-type levels. Untreated knockout mice displayed shorter latencies on tail flick and hot plate tests for spinal and supraspinal nociceptive responses than wild-type mice. These findings support a significant role for endogenous opioid–peptide interactions with μ Opiate Receptors in normal nociceptive processing. Morphine failed to significantly reduce nociceptive responses in hot plate or tail flick tests of homozygous μ Receptor knockout mice, and heterozygote mice displayed right and downward shifts in morphine analgesia dose–effect relationships. These results implicate endogenous opioid–peptide actions at μ Opiate Receptors in several tests of nociceptive responsiveness and support μ Receptor mediation of morphine-induced analgesia in tests of spinal and supraspinal analgesia.
Patrick Cadet - One of the best experts on this subject based on the ideXlab platform.
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a functionally coupled μ3 like Opiate Receptor nitric oxide regulatory pathway in human multi lineage progenitor cells
Journal of Immunology, 2007Co-Authors: Patrick Cadet, Kirk J. Mantione, Wei Zhu, Richard M Kream, Melinda Sheehan, George B. StefanoAbstract:Ongoing studies from our group support the existence and biological importance of a distinct cellular signaling pathway involving endogenously synthesized, chemically authentic, l-morphine, its cognate mu(3) Opiate Receptor subtype, and constitutive NO synthase. Based on prior studies indicating evolutionary conservation and adaptation of morphinergic/NO-coupled signaling to mediate autocrine/paracrine control of cellular functions, our goal was to determine whether a functionally competent mu(3) Opiate Receptor/NO-coupled regulatory pathway exists in human multilineage progenitor cells (MLPC) prepared from umbilical cord blood. Real-time PCR analysis indicated significant expression of mu(3) Opiate Receptor-encoding RNA by undifferentiated human MLPC, in the absence of traditional mu(1) opioid Receptor-encoding RNA expression. Unpredictably, confirmatory RT-PCR analyses indicated cellular expression of a splice variant of the previously characterized mu(3) Opiate Receptor-encoding mRNA. Pharmacological analyses provided critical validating evidence of functional mu(3)-like Opiate Receptor/NO-coupled signaling within primary cultures of undifferentiated human MLPC via morphine-evoke real-time release of NO. Control analyses indicated that morphine-stimulated NO release was markedly inhibited by prior treatment with the Opiate antagonist l-naloxone or the constitutive NO synthase inhibitor N(G)-nitro-l-arginine methyl ester and unresponsive to stimulation by the opioid peptide methionine enkephalin. Complementary microarray analysis demonstrated that traditional mu(1), delta, and kappa opioid Receptor gene expression is not detected in both undifferentiated and differentiated MLPC. Chemical differentiation of MLPC into neuronal progenitor cells effected significant phenotypic expression of a variety of neurally-associated genes. Our data provide compelling evidence in support of both the evolutionary primacy and primordial regulatory role of mu(3)-like Opiate Receptor/NO signaling in embryogenesis.
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molecular identification and functional expression of μ3 a novel alternatively spliced variant of the human μ Opiate Receptor gene
Journal of Immunology, 2003Co-Authors: Patrick Cadet, Kirk J. Mantione, George B. StefanoAbstract:Studies from our laboratory have revealed a novel μ Opiate Receptor, μ 3 , which is expressed in both vascular tissues and leukocytes. The μ 3 Receptor is selective for Opiate alkaloids and is insensitive to opioid peptides. We now identify the μ 3 Receptor at the molecular level using a 441-bp conserved region of the μ 1 Receptor. Sequence analysis of the isolated cDNA suggests that it is a novel, alternatively spliced variant of the μ Opiate Receptor gene. To determine whether protein expressed from this cDNA exhibits the biochemical characteristics expected of the μ 3 Receptor, the cDNA clone was expressed in a heterologous system. At the functional level, COS-1 cells transfected with the μ 3 Receptor cDNA exhibited dose-dependent release of NO following treatment with morphine, but not opioid peptides (i.e., Met-enkephalin). Naloxone was able to block the effect of morphine on COS-1 transfected cells. Nontransfected COS-1 cells did not produce NO in the presence of morphine or the opioid peptides at similar concentrations. Receptor binding analysis with [ 3 H]dihydromorphine further supports the Opiate alkaloid selectivity and opioid peptide insensitivity of this Receptor. These data suggest that this new μ Opiate Receptor cDNA encodes the μ 3 Opiate Receptor, since it exhibits biochemical characteristics known to be unique to this Receptor (Opiate alkaloid selective and opioid peptide insensitive). Furthermore, using Northern blot, RT-PCR, and sequence analysis, we have demonstrated the expression of this new μ variant in human vascular tissue, mononuclear cells, polymorphonuclear cells, and human neuroblastoma cells.
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Morphine 6 glucuronide stimulates nitric oxide release in mussel neural tissues: evidence for a morphine 6 glucuronide Opiate Receptor subtype
Cellular and Molecular Life Sciences, 2002Co-Authors: Kirk J. Mantione, Christos M. Rialas, Frederico Casares, A. L. Franklin, Patrick Cadet, Jan Tønnesen, George B. StefanoAbstract:We have previously demonstrated that Mytilus edulis pedal ganglia contain Opiate alkaloids, i.e., morphine and morphine 6 glucuronide (M6G), as well as mu Opiate Receptor subtype fragments exhibiting high sequence similarity to those found in mammals. Now we demonstrate that M6G stimulates pedal ganglia constitutive nitric oxide (NO) synthase (cNOS)-derived NO release at identical concentrations and to similar peak levels as morphine. However, the classic Opiate antagonist, naloxone, only blocked the ability of morphine to stimulate cNOS-derived NO release and not that of M6G. CTOP, a mu-specific antagonist, blocked the ability of M6G to induce cNOS-derived NO release as well as that of morphine, suggesting that a novel mu Opiate Receptor was present and selective toward M6G. In examining a Receptor displacement analysis, both Opiate alkaloids displaced [3H]-dihydromorphine binding to the mu Opiate Receptor subtype. However, morphine exhibited a twofold higher affinity, again suggesting that a novel mu Opiate Receptor may be present.