Science.gov

Sample records for mu-opioid receptor knockout

  1. Effects of morphine on pentobarbital-induced responses in mu-opioid receptor knockout mice.

    PubMed

    Park, Y; Ho, I K; Jang, C G; Tanaka, S; Ma, T; Loh, H H; Ko, K H

    2001-03-15

    Effects of morphine on the potentiation of pentobarbital-induced responses were investigated using mu-opioid receptor knockout mice. The duration of loss of righting reflex, hypothermia, and loss of motor coordination induced by pentobarbital were measured after pretreatment with either morphine or saline. Morphine pretreatment failed to show potentiation of both pentobarbital-induced loss of righting reflex and hypothermia in mu-opioid receptor knockout mice, while it significantly potentiated these responses in the wild-type controls. For motor incoordination test, morphine potentiated pentobarbital-induced motor incoordination in the wild-type mice. However, morphine may have opposite effects in the mu-opioid receptor knockout mice. These results demonstrate that synergism between morphine and pentobarbital is not detected in mu-opioid receptor knockout mice and that potentiation of pentobarbital-induced loss of righting reflex and hypothermia by morphine is mediated through mu-opioid receptor. It was interesting to note that pentobarbital-induced decrease in body temperature was less severe in mu-opioid receptor knockout mice than in wild-type mice.

  2. Naloxone fails to produce conditioned place aversion in mu-opioid receptor knock-out mice.

    PubMed

    Skoubis, P D; Matthes, H W; Walwyn, W M; Kieffer, B L; Maidment, N T

    2001-01-01

    There is growing evidence that tonic activity of the opioid system may be important in the modulation of affective state. Naloxone produces a conditioned place aversion in rodents, an effect that is centrally mediated. Previous pharmacological data using antagonists with preferential actions at mu-, delta-, and kappa-opioid receptors indicate the importance of the mu-opioid receptor in mediating this effect. We sought to test the mu-opioid receptor selectivity of naloxone aversion using mu-opioid receptor knock-out mice. mu-Opioid receptor knock-out and wild-type mice were tested for naloxone (10 mg/kg, s.c.) aversion using a place conditioning paradigm. As a positive control for associative learning, knock-out mice were tested for conditioned place aversion to a kappa agonist, U50,488H (2 mg/kg, s.c.). Naloxone produced a significant place aversion in wild-type mice, but failed to have any effect in mu-opioid receptor knock-out mice. On the other hand, both knock-out and wild-type mice treated with U50,488H spent significantly less time in the drug-paired chamber compared to their respective vehicle controls. We conclude that the mu-opioid receptor is crucial for the acquisition of naloxone-induced conditioned place aversion. Furthermore, in a separate experiment using C57BL/6 mice, the delta-selective antagonist naltrindole (10 or 30 mg/kg, s.c.) failed to produce conditioned place aversion.Taken together, these data further support the notion that naloxone produces aversion by antagonizing tonic opioid activity at the mu-opioid receptor.

  3. The mu-opioid receptor gene-dose dependent reductions in G-protein activation in the pons/medulla and antinociception induced by endomorphins in mu-opioid receptor knockout mice.

    PubMed

    Mizoguchi, H; Narita, M; Oji, D E; Suganuma, C; Nagase, H; Sora, I; Uhl, G R; Cheng, E Y; Tseng, L F

    1999-01-01

    There appear to be different relationships between mu-opioid receptor densities and the acute and neuroadaptive mu-opioid agonist-induced responses of the multiple opioid neuronal systems, including important pons/medulla circuits. The recent success in creating mu-opioid receptor knockout mice allows studies of mu-opioid agonist-induced pharmacological and physiological effects in animals that express no, one or two copies of the mu-opioid receptor gene. We now report that the binding of mu-opioid receptor ligand, [3H][D-Ala2,NHPhe4,Gly-ol]enkephalin to membrane preparations of the pons/medulla was reduced by half in heterozygous mu-opioid receptor knockout mice and eliminated in homozygous mu-opioid receptor knockout mice. The endogenous mu-opioid agonist peptides endomorphin-1 and -2 activate G-proteins in the pons/medulla from wild-type mice in a concentration-dependent fashion, as assessed using [35S]guanosine-5'-o-(3-thio)triphosphate binding. This stimulation was reduced to half of the wild-type levels in heterozygous mice and eliminated in homozygous knockout mice. The intracerebroventricular injection of either endomorphin-1 or endomorphin-2 produced marked antinociception in the hot-plate and tail-flick tests in wild-type mice. These antinociceptive actions were significantly reduced in heterozygous mu-opioid receptor knockout mice, and virtually abolished in homozygous knockout mice. The mu-opioid receptors are the principal molecular targets for endomorphin-induced G-protein activation in the pons/medulla and the antinociception caused by the intracerebroventricular administration of mu-opioid agonists. These data support the notion that there are limited physiological mu-opioid receptor reserves for inducing G-protein activation in the pons/medulla and for the nociceptive modulation induced by the central administration of endomorphin-1 and -2.

  4. Functional coupling, desensitization and internalization of virally expressed mu opioid receptors in cultured dorsal root ganglion neurons from mu opioid receptor knockout mice.

    PubMed

    Walwyn, W M; Keith, D E; Wei, W; Tan, A M; Xie, C W; Evans, C J; Kieffer, B L; Maidment, N T

    2004-01-01

    Although mu opioid receptors desensitize in various cell lines in vitro, the relationship of this change in signaling efficacy to the development of tolerance in vivo remains uncertain. It is clear that a system is needed in which functional mu opioid receptor expression is obtained in appropriate neurons so that desensitization can be measured, manipulated, and mutated receptors expressed in this environment. We have developed a recombinant system in which expression of a flag-tagged mu opioid receptor is returned to dorsal root ganglia neurons from mu opioid receptor knockout mice in vitro. Flow cytometry analysis showed that adenoviral-mediated expression of the amino-terminal flag-tagged mu opioid receptor in neurons resulted in approximately 1.3x10(6) receptors/cell. Many mu opioid receptor cell lines express a similar density of receptors but this is approximately 7x greater than the number of endogenous receptors expressed by matched wild-type neurons. Inhibition of the high voltage-activated calcium currents in dorsal root ganglia neurons by the mu agonist, D-Ala(2), N-MePhe(4), Gly(5)-ol-enkephalin (DAMGO), was not different between the endogenous and flag-tagged receptor at several concentrations of DAMGO used. Both receptors desensitized equally over the first 6 h of DAMGO pre-incubation, but after 24 h the response of the endogenous receptor to DAMGO had desensitized further than the flag- tagged receptor (71+/-3 vs 29+/-7% respectively; P<0.002), indicating less desensitization in neurons expressing a higher density of receptor. Using flow cytometry to quantify the percentage of receptors remaining on the neuronal cell surface, the flag-tagged receptor internalized by 17+/-1% after 20 min and 55+/-2% after 24 h of DAMGO. These data indicate that this return of function model in neurons recapitulates many of the characteristics of endogenous mu opioid receptor function previously identified in non-neuronal cell lines.

  5. mu-Opioid receptor knockout mice are insensitive to methamphetamine-induced behavioral sensitization.

    PubMed

    Shen, Xine; Purser, Chris; Tien, Lu-Tai; Chiu, Chi-Tso; Paul, Ian A; Baker, Rodney; Loh, Horace H; Ho, Ing K; Ma, Tangeng

    2010-08-01

    Repeated administration of psychostimulants to rodents can lead to behavioral sensitization. Previous studies, using nonspecific opioid receptor (OR) antagonists, revealed that ORs were involved in modulation of behavioral sensitization to methamphetamine (METH). However, the contribution of OR subtypes remains unclear. In the present study, using mu-OR knockout mice, we examined the role of mu-OR in the development of METH sensitization. Mice received daily intraperitoneal injection of drug or saline for 7 consecutive days to initiate sensitization. To express sensitization, animals received one injection of drug (the same as for initiation) or saline on day 11. Animal locomotor activity and stereotypy were monitored during the periods of initiation and expression of sensitization. Also, the concentrations of METH and its active metabolite amphetamine in the blood were measured after single and repeated administrations of METH. METH promoted significant locomotor hyperactivity at low doses and stereotyped behaviors at relative high doses (2.5 mg/kg and above). Repeated administration of METH led to the initiation and expression of behavioral sensitization in wild-type mice. METH-induced behavioral responses were attenuated in the mu-OR knockout mice. Haloperidol (a dopamine receptor antagonist) showed a more potent effect in counteracting METH-induced stereotypy in the mu-OR knockout mice. Saline did not induce behavioral sensitization in either genotype. No significant difference was observed in disposition of METH and amphetamine between the two genotypes. Our study indicated that the mu-opioid system is involved in modulating the development of behavioral sensitization to METH. (c) 2010 Wiley-Liss, Inc.

  6. Decreased consumption of sweet fluids in mu opioid receptor knockout mice: a microstructural analysis of licking behavior

    PubMed Central

    Ostlund, Sean B.; Kosheleff, Alisa; Maidment, Nigel T.; Murphy, Niall P.

    2013-01-01

    Summary Rationale Evidence suggests that the palatability of food (i.e., the hedonic impact produced by its sensory features) can promote feeding and may underlie compulsive eating, leading to obesity. Pharmacological studies implicate opioid transmission in the hedonic control of feeding, though these studies often rely on agents lacking specificity for particular opioid receptors. Objectives Here, we investigated the role of mu opioid receptors (MORs) specifically in determining hedonic responses to palatable sweet stimuli. Methods In Experiment 1, licking microstructure when consuming sucrose solution (2 to 20 %) was compared in MOR knockout and wildtype mice as a function of sucrose concentration and level of food deprivation. In Experiment 2, a similar examination was conducted using the palatable but calorie-free stimulus sucralose (0.001 to 1%), allowing study of licking behavior independent of homeostatic variables. Results In Experiment 1, MOR knockout mice exhibited several alterations in sucrose licking. Although wildtype mice exhibited a two-fold increase in the burst length when food deprived, relative to the nondeprived test, this aspect of sucrose licking was generally insensitive to manipulations of food deprivation for MOR knockout mice. Furthermore, during concentration testing, their rate of sucrose licking was less than half that of wildtype mice. During sucralose testing (Experiment 2), MOR knockout mice licked at approximately half the wildtype rate, providing more direct evidence that MOR knockout mice were impaired in processing stimulus palatability. Conclusions These results suggest that transmission through MORs mediates hedonic responses to palatable stimuli, and therefore likely contributes to normal and pathological eating. PMID:23568577

  7. Mu Opioid Receptors on Primary Afferent Nav1.8 Neurons Contribute to Opiate-Induced Analgesia: Insight from Conditional Knockout Mice

    PubMed Central

    Karchewski, Laurie; Gardon, Olivier; Matifas, Audrey; Filliol, Dominique; Becker, Jérôme A. J.; Wood, John N.; Kieffer, Brigitte L.; Gaveriaux-Ruff, Claire

    2013-01-01

    Opiates are powerful drugs to treat severe pain, and act via mu opioid receptors distributed throughout the nervous system. Their clinical use is hampered by centrally-mediated adverse effects, including nausea or respiratory depression. Here we used a genetic approach to investigate the potential of peripheral mu opioid receptors as targets for pain treatment. We generated conditional knockout (cKO) mice in which mu opioid receptors are deleted specifically in primary afferent Nav1.8-positive neurons. Mutant animals were compared to controls for acute nociception, inflammatory pain, opiate-induced analgesia and constipation. There was a 76% decrease of mu receptor-positive neurons and a 60% reduction of mu-receptor mRNA in dorsal root ganglia of cKO mice. Mutant mice showed normal responses to heat, mechanical, visceral and chemical stimuli, as well as unchanged morphine antinociception and tolerance to antinociception in models of acute pain. Inflammatory pain developed similarly in cKO and controls mice after Complete Freund’s Adjuvant. In the inflammation model, however, opiate-induced (morphine, fentanyl and loperamide) analgesia was reduced in mutant mice as compared to controls, and abolished at low doses. Morphine-induced constipation remained intact in cKO mice. We therefore genetically demonstrate for the first time that mu opioid receptors partly mediate opiate analgesia at the level of Nav1.8-positive sensory neurons. In our study, this mechanism operates under conditions of inflammatory pain, but not nociception. Previous pharmacology suggests that peripheral opiates may be clinically useful, and our data further demonstrate that Nav1.8 neuron-associated mu opioid receptors are feasible targets to alleviate some forms of persistent pain. PMID:24069332

  8. Mu opioid receptors on primary afferent nav1.8 neurons contribute to opiate-induced analgesia: insight from conditional knockout mice.

    PubMed

    Weibel, Raphaël; Reiss, David; Karchewski, Laurie; Gardon, Olivier; Matifas, Audrey; Filliol, Dominique; Becker, Jérôme A J; Wood, John N; Kieffer, Brigitte L; Gaveriaux-Ruff, Claire

    2013-01-01

    Opiates are powerful drugs to treat severe pain, and act via mu opioid receptors distributed throughout the nervous system. Their clinical use is hampered by centrally-mediated adverse effects, including nausea or respiratory depression. Here we used a genetic approach to investigate the potential of peripheral mu opioid receptors as targets for pain treatment. We generated conditional knockout (cKO) mice in which mu opioid receptors are deleted specifically in primary afferent Nav1.8-positive neurons. Mutant animals were compared to controls for acute nociception, inflammatory pain, opiate-induced analgesia and constipation. There was a 76% decrease of mu receptor-positive neurons and a 60% reduction of mu-receptor mRNA in dorsal root ganglia of cKO mice. Mutant mice showed normal responses to heat, mechanical, visceral and chemical stimuli, as well as unchanged morphine antinociception and tolerance to antinociception in models of acute pain. Inflammatory pain developed similarly in cKO and controls mice after Complete Freund's Adjuvant. In the inflammation model, however, opiate-induced (morphine, fentanyl and loperamide) analgesia was reduced in mutant mice as compared to controls, and abolished at low doses. Morphine-induced constipation remained intact in cKO mice. We therefore genetically demonstrate for the first time that mu opioid receptors partly mediate opiate analgesia at the level of Nav1.8-positive sensory neurons. In our study, this mechanism operates under conditions of inflammatory pain, but not nociception. Previous pharmacology suggests that peripheral opiates may be clinically useful, and our data further demonstrate that Nav1.8 neuron-associated mu opioid receptors are feasible targets to alleviate some forms of persistent pain.

  9. The mu opioid receptor: A new target for cancer therapy?

    PubMed

    Singleton, Patrick A; Moss, Jonathan; Karp, Daniel D; Atkins, Johnique T; Janku, Filip

    2015-08-15

    Mu opioids are among the most widely used drugs for patients with cancer with both acute and chronic pain as well as in the perioperative period. Several retrospective studies have suggested that opioid use might promote tumor progression and as a result negatively impact survival in patients with advanced cancer; however, in the absence of appropriate prospective validation, any changes in recommendations for opioid use are not warranted. In this review, the authors present preclinical and clinical data that support their hypothesis that the mu opioid receptor is a potential target for cancer therapy because of its plausible role in tumor progression. The authors also propose the hypothesis that peripheral opioid antagonists such as methylnaltrexone, which reverses the peripheral effects of mu opioids but maintains centrally mediated analgesia and is approved by the US Food and Drug Administration for the treatment of opioid-induced constipation, can be used to target the mu opioid receptor. © 2015 American Cancer Society.

  10. Endomorphins fully activate a cloned human mu opioid receptor.

    PubMed

    Gong, J; Strong, J A; Zhang, S; Yue, X; DeHaven, R N; Daubert, J D; Cassel, J A; Yu, G; Mansson, E; Yu, L

    1998-11-13

    Endomorphins were recently identified as endogenous ligands with high selectivity for mu opioid receptors. We have characterized the ability of endomorphins to bind to and functionally activate the cloned human mu opioid receptor. Both endomorphin-1 and endomorphin-2 exhibited binding selectivity for the mu opioid receptor over the delta and kappa opioid receptors. Both agonists inhibited forskolin-stimulated increase of cAMP in a dose-dependent fashion. When the mu opioid receptor was coexpressed in Xenopus oocytes with G protein-activated K+ channels, application of either endomorphin activated an inward K+ current. This activation was dose-dependent and blocked by naloxone. Both endomorphins acted as full agonists with efficacy similar to that of [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin (DAMGO). These data indicate that endomorphins act as full agonists at the human mu opioid receptor, capable of stimulating the receptor to inhibit the cAMP/adenylyl cyclase pathway and activate G-protein-activated inwardly rectifying potassium (GIRK) channels.

  11. Mu opioid receptor binding sites in human brain

    SciTech Connect

    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 (/sup 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 (/sup 3/H)DAGO binding sites clearly reveals the discrete lamination (layers I and III-IV) of mu sites in cortical areas.

  12. Synergy between mu opioid ligands: evidence for functional interactions among mu opioid receptor subtypes.

    PubMed

    Bolan, Elizabeth A; Tallarida, Ronald J; Pasternak, Gavril W

    2002-11-01

    Pharmacological differences among mu opioid drugs have been observed in in vitro and in vivo preclinical models, as well as clinically, implying that all mu opioids may not be working through the same mechanism of action. Here we demonstrate analgesic synergy between L-methadone and several mu opioid ligands. Of the compounds examined, L-methadone selectively synergizes with morphine, morphine-6beta-glucuronide, codeine, and the active metabolite of heroin, 6-acetylmorphine. Morphine synergizes only with L-methadone. In analgesic assays, D-methadone was inactive alone and did not enhance morphine analgesia when the two were given together, confirming that L-methadone was not acting through N-methyl-D-aspartate mechanisms. Both L-methadone and morphine displayed only additive effects when paired with oxymorphone, oxycodone, fentanyl, alfentanyl, or meperidine. Although it displays synergy in analgesic assays, the L-methadone/morphine combination does not exhibit synergy in the gastrointestinal transit assay. This analgesic synergy of L-methadone with selective mu opioid drugs and the differences in opioid-mediated actions suggest that these drugs may be acting via different mechanisms. These findings provide further evidence for the complexity of the pharmacology of mu opioids.

  13. Splice variation of the mu-opioid receptor and its effect on the action of opioids.

    PubMed

    Gretton, Sophy K; Droney, Joanne

    2014-11-01

    An individual's response to opioids is influenced by a complex combination of genetic, molecular and phenotypic factors.Intra- and inter-individual variations in response to mu opioids have led to the suggestion that mu-opioid receptor subtypes exist.Scientists have now proven that mu-opioid receptor subtypes exist and that they occur through a mechanism promoting protein diversity, called alternative splicing.The ability of mu opioids to differentially activate splice variants may explain some of the clinical differences observed between mu opioids.This article examines how differential activation of splice variants by mu opioids occurs through alternative mu-opioid receptor binding, through differential receptor activation, and as a result of the distinct distribution of variants located regionally and at the cellular level.

  14. Fluoxetine alters mu opioid receptor expression in obese Zucker rat extrahypothalamic regions.

    PubMed

    Churruca, Itziar; Portillo, María P; Zumalabe, José María; Macarulla, María T; Sáenz Del Burgo, Laura; Zarate, Jon; Echevarría, Enrique

    2006-03-01

    The aim of this article was to describe the effects of chronic fluoxetine on mu opioid receptor expression in obese Zucker rat extrahypothalamic regions. Male obese Zucker (fa/fa) rats were administered with fluoxetine (10 mg/kg; i.p.) daily for two weeks. Brain regional immunostaining for mu opioid receptor was carried out. An increase in the numbers of neural cells immunostained for mu opioid receptor in caudatus-putamen, dentate gyrus, lateral septum, amygdala, and frontal, parietal, and piriform cortices was observed. Increased mu opioid receptor expression in the central amygdaloid nuclei suggests a decreased opioidergic tone at this level that could be involved in fluoxetine anorectic action.

  15. Differential opioid agonist regulation of the mouse mu opioid receptor.

    PubMed

    Blake, A D; Bot, G; Freeman, J C; Reisine, T

    1997-01-10

    Mu opioid receptors mediate the analgesia induced by morphine. Prolonged use of morphine causes tolerance development and dependence. To investigate the molecular basis of tolerance and dependence, the cloned mouse mu opioid receptor with an amino-terminal epitope tag was stably expressed in human embryonic kidney (HEK) 293 cells, and the effects of prolonged opioid agonist treatment on receptor regulation were examined. In HEK 293 cells the expressed mu receptor showed high affinity, specific, saturable binding of radioligands and a pertussis toxin-sensitive inhibition of adenylyl cyclase. Pretreatment (1 h, 3 h, or overnight) of cells with 1 microM morphine or [D-Ala2MePhe4,Gly(ol)5]enkephalin (DAMGO) resulted in no apparent receptor desensitization, as assessed by opioid inhibition of forskolin-stimulated cAMP levels. In contrast, the morphine and DAMGO pretreatments (3 h) resulted in a 3-4-fold compensatory increase in forskolin-stimulated cAMP accumulation. The opioid agonists methadone and buprenorphine are used in the treatment of addiction because of a markedly lower abuse potential. Pretreatment of mu receptor-expressing HEK 293 cells with methadone or buprenorphine abolished the ability of opioids to inhibit adenylyl cyclase. No compensatory increase in forskolin-stimulated cAMP accumulation was found with methadone or buprenorphine; these opioids blocked the compensatory effects observed with morphine and DAMGO. Taken together, these results indicate that methadone and buprenorphine interact differently with the mouse mu receptor than either morphine or DAMGO. The ability of methadone and buprenorphine to desensitize the mu receptor and block the compensatory rise in forskolin-stimulated cAMP accumulation may be an underlying mechanism by which these agents are effective in the treatment of morphine addiction.

  16. Casein kinase 1-epsilon deletion increases mu opioid receptor-dependent behaviors and binge eating1.

    PubMed

    Goldberg, L R; Kirkpatrick, S L; Yazdani, N; Luttik, K P; Lacki, O A; Keith Babbs, R; Jenkins, D F; Evan Johnson, W; Bryant, C D

    2017-09-01

    Genetic and pharmacological studies indicate that casein kinase 1 epsilon (Csnk1e) contributes to psychostimulant, opioid, and ethanol motivated behaviors. We previously used pharmacological inhibition to demonstrate that Csnk1e negatively regulates the locomotor stimulant properties of opioids and psychostimulants. Here, we tested the hypothesis that Csnk1e negatively regulates opioid and psychostimulant reward using genetic inhibition and the conditioned place preference assay in Csnk1e knockout mice. Similar to pharmacological inhibition, Csnk1e knockout mice showed enhanced opioid-induced locomotor activity with the mu opioid receptor agonist fentanyl (0.2 mg/kg i.p.) as well as enhanced sensitivity to low-dose fentanyl reward (0.05 mg/kg). Interestingly, female knockout mice also showed a markedly greater escalation in consumption of sweetened palatable food - a behavioral pattern consistent with binge eating that also depends on mu opioid receptor activation. No difference was observed in fentanyl analgesia in the 52.5°C hot plate assay (0-0.4 mg/kg), naloxone conditioned place aversion (4 mg/kg), or methamphetamine conditioned place preference (0-4 mg/kg). To identify molecular adaptations associated with increased drug and food behaviors in knockout mice, we completed transcriptome analysis via mRNA sequencing of the striatum. Enrichment analysis identified terms associated with myelination and axon guidance and pathway analysis identified a differentially expressed gene set predicted to be regulated by the Wnt signaling transcription factor, Tcf7l2. To summarize, Csnk1e deletion increased mu opioid receptor-dependent behaviors, supporting previous studies indicating an endogenous negative regulatory role of Csnk1e in opioid behavior. © 2017 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

  17. [Endomorphins--endogenous ligands of the mu-opioid receptor].

    PubMed

    Perlikowska, Renata; Fichna, Jakub; Janecka, Anna

    2009-01-01

    Two endogenous opioid peptides with extremely high mu-opioid receptor affinity and selectivity, endomorphin-1 and endomorphin-2, were: discovered and isolated from the mammalian brain in 1997. Endomorphins are amidated tetrapeptides, structurally different from so called typical opioids: enkephalins, dynorphins and endorphins. A protein precursor of endomorphins and a gene encoding their sequence remain unknown. Endomorphins are unable to cross the blood-brain barrier because of their low hydrophobicity. In animal models, these peptides turned out to be very potent in relieving neuropathic and inflammatory pain. In comparison with morphine, a prototype opioid receptor ligand, endomorphins produces less undesired side effects. In this article we describe the discovery of endomorphins, their cellular localization and functions in the organism, as well as their structure-activity relationships and biodegradation pathways.

  18. BOLD Imaging in Awake Wild-Type and Mu-Opioid Receptor Knock-Out Mice Reveals On-Target Activation Maps in Response to Oxycodone

    PubMed Central

    Moore, Kelsey; Madularu, Dan; Iriah, Sade; Yee, Jason R.; Kulkarni, Praveen; Darcq, Emmanuel; Kieffer, Brigitte L.; Ferris, Craig F.

    2016-01-01

    Blood oxygen level dependent (BOLD) imaging in awake mice was used to identify differences in brain activity between wild-type, and Mu (μ) opioid receptor knock-outs (MuKO) in response to oxycodone (OXY). Using a segmented, annotated MRI mouse atlas and computational analysis, patterns of integrated positive and negative BOLD activity were identified across 122 brain areas. The pattern of positive BOLD showed enhanced activation across the brain in WT mice within 15 min of intraperitoneal administration of 2.5 mg of OXY. BOLD activation was detected in 72 regions out of 122, and was most prominent in areas of high μ opioid receptor density (thalamus, ventral tegmental area, substantia nigra, caudate putamen, basal amygdala, and hypothalamus), and focus on pain circuits indicated strong activation in major pain processing centers (central amygdala, solitary tract, parabrachial area, insular cortex, gigantocellularis area, ventral thalamus primary sensory cortex, and prelimbic cortex). Importantly, the OXY-induced positive BOLD was eliminated in MuKO mice in most regions, with few exceptions (some cerebellar nuclei, CA3 of the hippocampus, medial amygdala, and preoptic areas). This result indicates that most effects of OXY on positive BOLD are mediated by the μ opioid receptor (on-target effects). OXY also caused an increase in negative BOLD in WT mice in few regions (16 out of 122) and, unlike the positive BOLD response the negative BOLD was only partially eliminated in the MuKO mice (cerebellum), and in some case intensified (hippocampus). Negative BOLD analysis therefore shows activation and deactivation events in the absence of the μ receptor for some areas where receptor expression is normally extremely low or absent (off-target effects). Together, our approach permits establishing opioid-induced BOLD activation maps in awake mice. In addition, comparison of WT and MuKO mutant mice reveals both on-target and off-target activation events, and set an OXY brain

  19. BOLD Imaging in Awake Wild-Type and Mu-Opioid Receptor Knock-Out Mice Reveals On-Target Activation Maps in Response to Oxycodone.

    PubMed

    Moore, Kelsey; Madularu, Dan; Iriah, Sade; Yee, Jason R; Kulkarni, Praveen; Darcq, Emmanuel; Kieffer, Brigitte L; Ferris, Craig F

    2016-01-01

    Blood oxygen level dependent (BOLD) imaging in awake mice was used to identify differences in brain activity between wild-type, and Mu (μ) opioid receptor knock-outs (MuKO) in response to oxycodone (OXY). Using a segmented, annotated MRI mouse atlas and computational analysis, patterns of integrated positive and negative BOLD activity were identified across 122 brain areas. The pattern of positive BOLD showed enhanced activation across the brain in WT mice within 15 min of intraperitoneal administration of 2.5 mg of OXY. BOLD activation was detected in 72 regions out of 122, and was most prominent in areas of high μ opioid receptor density (thalamus, ventral tegmental area, substantia nigra, caudate putamen, basal amygdala, and hypothalamus), and focus on pain circuits indicated strong activation in major pain processing centers (central amygdala, solitary tract, parabrachial area, insular cortex, gigantocellularis area, ventral thalamus primary sensory cortex, and prelimbic cortex). Importantly, the OXY-induced positive BOLD was eliminated in MuKO mice in most regions, with few exceptions (some cerebellar nuclei, CA3 of the hippocampus, medial amygdala, and preoptic areas). This result indicates that most effects of OXY on positive BOLD are mediated by the μ opioid receptor (on-target effects). OXY also caused an increase in negative BOLD in WT mice in few regions (16 out of 122) and, unlike the positive BOLD response the negative BOLD was only partially eliminated in the MuKO mice (cerebellum), and in some case intensified (hippocampus). Negative BOLD analysis therefore shows activation and deactivation events in the absence of the μ receptor for some areas where receptor expression is normally extremely low or absent (off-target effects). Together, our approach permits establishing opioid-induced BOLD activation maps in awake mice. In addition, comparison of WT and MuKO mutant mice reveals both on-target and off-target activation events, and set an OXY brain

  20. Mu Opioid Receptor Actions in the Lateral Habenula

    PubMed Central

    Margolis, Elyssa B.; Fields, Howard L.

    2016-01-01

    Increased activity of lateral habenula (LHb) neurons is correlated with aversive states including pain, opioid abstinence, rodent models of depression, and failure to receive a predicted reward. Agonists at the mu opioid receptor (MOR) are among the most powerful rewarding and pain relieving drugs. Injection of the MOR agonist morphine directly into the habenula produces analgesia, raising the possibility that MOR acts locally within the LHb. Consequently, we examined the synaptic actions of MOR agonists in the LHb using whole cell patch clamp recording. We found that the MOR selective agonist DAMGO inhibits a subset of LHb neurons both directly and by inhibiting glutamate release onto these cells. Paradoxically, DAMGO also presynaptically inhibited GABA release onto most LHb neurons. The behavioral effect of MOR activation will thus depend upon both the level of intrinsic neuronal activity in the LHb and the balance of activity in glutamate and GABA inputs to different LHb neuronal populations. PMID:27427945

  1. Mu opioid receptor polymorphism, early social adversity, and social traits.

    PubMed

    Carver, Charles S; Johnson, Sheri L; Kim, Youngmee

    2016-10-01

    A polymorphism in the mu opioid receptor gene OPRM1 (rs1799971) has been investigated for its role in sensitivity to social contexts. Evidence suggests that the G allele of this polymorphism is associated with higher levels of sensitivity. This study tested for main effects of the polymorphism and its interaction with a self-report measure of childhood adversity as an index of negative environment. Outcomes were several personality measures relevant to social connection. Significant interactions were obtained, such that the negative impact of childhood adversity on personality was greater among G carriers than among A homozygotes on measures of agreeableness, interdependence, anger proneness, hostility, authentic pride, life engagement, and an index of (mostly negative) feelings coloring one's world view. Findings support the role of OPRM1 in sensitivity to negative environments. Limitations are noted, including the lack of a measure of advantageous social environment to assess sensitivity to positive social contexts.

  2. Alvimopan: a peripherally acting mu-opioid receptor antagonist.

    PubMed

    Leslie, John B

    2007-09-01

    Postoperative ileus (POI), a transient cessation of coordinated bowel motility after surgery, is an important factor in extending the length of hospital stay. The etiology of POI is multifactorial, and related to both the surgical and anesthetic pathways chosen. Additionally, opioids used to manage non-cancer-related and cancer-related chronic pain may also decrease gastrointestinal (GI) motility resulting in opioid-induced bowel dysfunction (OBD). Postoperative ileus has been associated with prolonged hospital stay and readmission, and thus may increase the overall hospital costs per patient with POI. Alvimopan, a peripherally acting mu-opioid receptor antagonist, accelerated time to GI recovery and reduced postoperative hospital length of stay in phase III POI clinical trials and improved symptoms of OBD compared with placebo in phase II/III clinical trials. The U.S. Food and Drug Administration is currently evaluating alvimopan for the management of POI after bowel resection. Alvimopan may provide clinically meaningful benefits to patients and may lower the economic burden of POI to the healthcare system.

  3. Mu opioid receptors are in discrete hippocampal interneuron subpopulations.

    PubMed

    Drake, Carrie T; Milner, Teresa A

    2002-01-01

    In the rat hippocampal formation, application of mu opioid receptor (MOR) agonists disinhibits principal cells, promoting excitation-dependent processes such as epileptogenesis and long-term potentiation. However, the precise location of MORs in particular inhibitory circuits, has not been determined, and the roles of MORs in endogenous functioning are unclear. To address these issues, the distribution of MOR-like immunoreactivity (-li) was examined in several populations of inhibitory hippocampal neurons in the CA1 region using light and electron microscopy. We found that MOR-li was present in many parvalbumin-containing basket cells, but absent from cholecystokinin-labeled basket cells. MOR-li was also commonly in interneurons containing somatostatin-li or neuropeptide Y-li that resembled the "oriens-lacunosum-moleculare" (O-LM) interneurons innervating pyramidal cell distal dendrites. Finally, MOR-li was in some vasoactive intestinal peptide- or calretinin-containing profiles resembling interneurons that primarily innervate other interneurons. These findings indicate that MOR-containing neurons form a neurochemically and functionally heterogeneous subset of hippocampal GABAergic neurons. MORs are most frequently on interneurons that are specialized to inhibit pyramidal cells, and are on a limited number of interneurons that target other interneurons. Moreover, the distribution of MORs to different neuronal types in several laminae, some relatively far from endogenous opioids, suggests normal functional roles that are different from the actions seen with exogenous agonists such as morphine.

  4. Stimulatory effect of stevioside on peripheral mu opioid receptors in animals.

    PubMed

    Yang, Po-Sheng; Lee, Jie-Jen; Tsao, Chiung-Wen; Wu, Hung-Tsung; Cheng, Juei-Tang

    2009-04-17

    Stevioside is a dietary supplement widely used as a sweetener to prevent hyperglycemic disorders. However, the action mechanisms of this substance for glucose homeostasis remain obscure. In the present study, a dose-related plasma glucose reduction was observed in Wistar rats receiving intraperitoneally injections of stevioside. Similar to the regulation of glucose metabolism by the activation of mu opioid receptors, this action of stevioside was reversed by naloxonazine under the blockade of mu opioid receptors. We also found that stevioside increased glycogen synthesis in isolated hepatocytes, which was concentration-dependently blocked by naloxonazine. Stevioside did not modify the plasma beta-endorphin levels in Wistar rats but it directly increased the phosphorylation of mu opioid receptors in Chinese hamster ovary cells transfected with mu opioid receptors. Unlike morphine, chronic administration of stevioside did not induce the withdrawal signs in mice. Furthermore, stevioside by intraperitoneal injections did not influence the feeding behaviors of rats. By contrast, intracerebroventricular injections of stevioside increased the rats' food intake, which was also inhibited by pretreatment with naloxonazine. These results showed that it is difficult for stevioside to enter the brain. Stevioside has the ability to activate peripheral mu opioid receptors for lowering plasma glucose and to increase glycogen synthesis in liver. Thus, the stimulation of peripheral mu opioid receptors is responsible for the action of stevioside in the regulation of glucose homeostasis.

  5. Salvinorin A: allosteric interactions at the mu-opioid receptor.

    PubMed

    Rothman, Richard B; Murphy, Daniel L; Xu, Heng; Godin, Jonathan A; Dersch, Christina M; Partilla, John S; Tidgewell, Kevin; Schmidt, Matthew; Prisinzano, Thomas E

    2007-02-01

    Salvinorin A [(2S,4aR,6aR,7R,9S,10aS,10bR)-9-(acetyloxy)-2-(3-furanyl)-dodecahydro-6a,10b-dimethyl-4,10-dioxo-2h-naphtho[2,1-c]pyran-7-carboxylic acid methyl ester] is a hallucinogenic kappa-opioid receptor agonist that lacks the usual basic nitrogen atom present in other known opioid ligands. Our first published studies indicated that Salvinorin A weakly inhibited mu-receptor binding, and subsequent experiments revealed that Salvinorin A partially inhibited mu-receptor binding. Therefore, we hypothesized that Salvinorin A allosterically modulates mu-receptor binding. To test this hypothesis, we used Chinese hamster ovary cells expressing the cloned human opioid receptor. Salvinorin A partially inhibited [(3)H]Tyr-D-Ala-Gly-N-Me-Phe-Gly-ol (DAMGO) (0.5, 2.0, and 8.0 nM) binding with E(MAX) values of 78.6, 72.1, and 45.7%, respectively, and EC(50) values of 955, 1124, and 4527 nM, respectively. Salvinorin A also partially inhibited [(3)H]diprenorphine (0.02, 0.1, and 0.5 nM) binding with E(MAX) values of 86.2, 64, and 33.6%, respectively, and EC(50) values of 1231, 866, and 3078 nM, respectively. Saturation binding studies with [(3)H]DAMGO showed that Salvinorin A (10 and 30 microM) decreased the mu-receptor B(max) and increased the K(d) in a dose-dependent nonlinear manner. Saturation binding studies with [(3)H]diprenorphine showed that Salvinorin A (10 and 40 microM) decreased the mu-receptor B(max) and increased the K(d) in a dose-dependent nonlinear manner. Similar findings were observed in rat brain with [(3)H]DAMGO. Kinetic experiments demonstrated that Salvinorin A altered the dissociation kinetics of both [(3)H]DAMGO and [(3)H]diprenorphine binding to mu receptors. Furthermore, Salvinorin A acted as an uncompetitive inhibitor of DAMGO-stimulated guanosine 5'-O-(3-[(35)S]thio)-triphosphate binding. Viewed collectively, these data support the hypothesis that Salvinorin A allosterically modulates the mu-opioid receptor.

  6. Ligand-Specific Regulation of the Endogenous Mu-Opioid Receptor by Chronic Treatment with Mu-Opioid Peptide Agonists

    PubMed Central

    Murányi, Marianna; Cinar, Resat; Kékesi, Orsolya; Birkás, Erika; Fábián, Gabriella; Bozó, Beáta; Zentai, András; Tóth, Géza; Kicsi, Emese Gabriella; Mácsai, Mónika; Szabó, Gyula; Szücs, Mária

    2013-01-01

    Since the discovery of the endomorphins (EM), the postulated endogenous peptide agonists of the mu-opioid receptors, several analogues have been synthesized to improve their binding and pharmacological profiles. We have shown previously that a new analogue, cis-1S,2R-aminocyclohexanecarboxylic acid2-endomorphin-2 (ACHC-EM2), had elevated mu-receptor affinity, selectivity, and proteolytic stability over the parent compound. In the present work, we have studied its antinociceptive effects and receptor regulatory processes. ACHC-EM2 displayed a somewhat higher (60%) acute antinociceptive response than the parent peptide, EM2 (45%), which peaked at 10 min after intracerebroventricular (icv) administration in the rat tail-flick test. Analgesic tolerance developed to the antinociceptive effect of ACHC-EM2 upon its repeated icv injection that was complete by a 10-day treatment. This was accompanied by attenuated coupling of mu-sites to G-proteins in subcellular fractions of rat brain. Also, the density of mu-receptors was upregulated by about 40% in the light membrane fraction, with no detectable changes in surface binding. Distinct receptor regulatory processes were noted in subcellular fractions of rat brains made tolerant by the prototypic full mu-agonist peptide, DAMGO, and its chloromethyl ketone derivative, DAMCK. These results are discussed in light of the recently discovered phenomenon, that is, the “so-called biased agonism” or “functional selectivity”. PMID:24350273

  7. Ligand-specific regulation of the endogenous mu-opioid receptor by chronic treatment with mu-opioid peptide agonists.

    PubMed

    Murányi, Marianna; Cinar, Resat; Kékesi, Orsolya; Birkás, Erika; Fábián, Gabriella; Bozó, Beáta; Zentai, András; Tóth, Géza; Kicsi, Emese Gabriella; Mácsai, Mónika; Dochnal, Roberta; Szabó, Gyula; Szücs, Mária

    2013-01-01

    Since the discovery of the endomorphins (EM), the postulated endogenous peptide agonists of the mu-opioid receptors, several analogues have been synthesized to improve their binding and pharmacological profiles. We have shown previously that a new analogue, cis-1S,2R-aminocyclohexanecarboxylic acid(2)-endomorphin-2 (ACHC-EM2), had elevated mu-receptor affinity, selectivity, and proteolytic stability over the parent compound. In the present work, we have studied its antinociceptive effects and receptor regulatory processes. ACHC-EM2 displayed a somewhat higher (60%) acute antinociceptive response than the parent peptide, EM2 (45%), which peaked at 10 min after intracerebroventricular (icv) administration in the rat tail-flick test. Analgesic tolerance developed to the antinociceptive effect of ACHC-EM2 upon its repeated icv injection that was complete by a 10-day treatment. This was accompanied by attenuated coupling of mu-sites to G-proteins in subcellular fractions of rat brain. Also, the density of mu-receptors was upregulated by about 40% in the light membrane fraction, with no detectable changes in surface binding. Distinct receptor regulatory processes were noted in subcellular fractions of rat brains made tolerant by the prototypic full mu-agonist peptide, DAMGO, and its chloromethyl ketone derivative, DAMCK. These results are discussed in light of the recently discovered phenomenon, that is, the "so-called biased agonism" or "functional selectivity".

  8. Ca2+ channel inhibition by endomorphins via the cloned mu-opioid receptor expressed in NG108-15 cells.

    PubMed

    Mima, H; Morikawa, H; Fukuda, K; Kato, S; Shoda, T; Mori, K

    1997-12-11

    Endomorphin-1 and -2, recently isolated endogenous peptides specific for the mu-opioid receptor, inhibited Ca2+ channel currents with EC50 of 6 and 9 nM, respectively, in NG108-15 cells transformed to express the cloned rat mu-opioid receptor. On the other hand, they elicited no response in nontransfected NG108-15 cells. It is concluded that endomorphin-1 and -2 induce Ca2+ channel inhibition by selectively activating the mu-opioid receptor.

  9. The presence of the mu-opioid receptor in the isthmus of mare oviduct.

    PubMed

    Desantis, S; Albrizio, M; Ventriglia, G; Deflorio, M; Guaricci, A C; Minoia, R; De Metrio, G

    2008-05-01

    The presence of the mu-opioid receptor and the type of glycosylation in the third extra-cellular loop of this receptor was investigated in the isthmus of mare oviduct during oestrus by means of immunoblotting and immunohistochemistry combined with enzymatic (N-glycosidase F and O-glycosidase) and chemical (beta-elimination) treatments. Immunoblotting analysis showed that the mu-opioid receptor consists of two peptides with molecular weights of around 65 and 50 kDa. After N-deglycosylation with N-glycosidase F an additional immunoreactive peptide was observed at around 30 KDa. The cleavage of O-glycans by O-glycosidase failed in immunoblotting as well as in immunohistochemistry investigations, revealing that the third extra-cellular loop of the mu-opioid receptor expressed in mare isthmus oviduct contains some modifications of the Galbeta(1-3)GalNAc core binding to serine or threonine. Immunohistochemistry revealed the mu-opioid receptor in the mucosal epithelium, some stromal cells, muscle cells and blood vessels. In ciliated cells the mu-opioid receptor showed N-linked glycans, since the immunoreactivity was abolished after N-glycosidase F treatment, whereas it was preserved in the apical region after beta-elimination. Most non-ciliated cells expressed the mu-opioid receptor with both N- and O-linked oligosaccharides, as revealed by the abolition of immunostaining after N-glycosidase F and beta-elimination. Stromal cells, endothelial and muscle cells of blood vessels expressed the mu-opioid receptor containing both N- and O-linked oligosaccharides. Myosalpinx myocytes expressed the mu-opioid receptor with O-linked oligosaccharides. The immunopositive myocytes formed a circular coat in the intrinsic musculature, whereas they were arranged in some isolated, oblique bundles in the extrinsic musculature. In conclusion, the mu-opioid receptor could have a role in the production and the movement of isthmus lumen content that contributes to ensuring the effective

  10. Anatomical and functional correlation of the endomorphins with mu opioid receptor splice variants.

    PubMed

    Abbadie, C; Rossi, G C; Orciuolo, A; Zadina, J E; Pasternak, G W

    2002-09-01

    The present study characterizes the relationship between the endogenous mu opioid peptides endomorphin-1 (EM-1) and endomorphin-2 (EM-2) and several splice variants of the cloned mu opioid receptor (MOR-1) encoded by the mu opioid receptor gene (Oprm). Confocal laser microscopy revealed that fibers containing EM-2-like immunoreactivity (-LI) were distributed in close apposition to fibers showing MOR-1-LI (exon 4-LI) and to MOR-1C-LI (exons 7/8/9-LI) in the superficial laminae of the lumbar spinal cord. We also observed colocalization of EM-2-LI and MOR-1-LI in a few fibers of lamina II, and colocalization of EM-2-LI and MOR-1C-LI in laminae I-II, and V-VI. To assess the functional relevance of the MOR-1 variants in endomorphin analgesia, we examined the effects of antisense treatments that targeted individual exons within the Oprm1 gene on EM-1 and EM-2 analgesia in the tail flick test. This antisense mapping study implied mu opioid receptor mechanisms for the endomorphins are distinct from those of morphine or morphine-6beta-glucuronide (M6G).

  11. Genomic variations and transcriptional regulation of the human mu-opioid receptor gene.

    PubMed

    Bayerer, Bettina; Stamer, Ulrike; Hoeft, Andreas; Stüber, Frank

    2007-05-01

    The mu-opioid receptor (MOR1) is a target of endogenous and exogenous opioids and plays a pivotal role for anesthesia and analgesia. Variations in the 5' flanking sequence of the mu-opioid receptor gene may influence transcriptional regulation and ultimately alter protein expression of MOR1. In the present study we investigated the influence of eight single nucleotide polymorphisms (SNP) within the mu-opioid receptor promoter on promoter activity and evaluated the frequencies of the relevant SNPs in 700 patients under opioid medication. Reporter-gene-constructs were created by means of PCR and site directed mutagenesis, testing eight SNPs previously described. The neuroblastoma cell line SHSY5Y was used for transfection and promoter activity was estimated by luciferase activity. Of the eight reporter gene constructs employed to test genomic variations, two produced a significant change in luciferase activity when compared to wild-type constructs. The G-554A variation located within a known NFkB binding element resulted in a decreased activity whereas the A/G base exchange at position -1320 showed an increased luciferase activity. This particular variant generated a myeloid zinc finger (MZF1) cis-acting element known to impact transcription. The allele frequency of the -1320G variant was 0.21% in 700 Caucasian patients under opioid medication in contrast to 9.1% reported previously in drug addicted African Americans. Because of this unexpected low frequency an association analysis to opioid requirements and effects of mu-opioid receptor agonists was not feasible. In conclusion, transcriptional regulation of MOR1 is modified by two genetic variations at positions -554 and -1320 of the mu-opioid receptor promoter. Individuals presenting these variations may have an altered level of MOR expression. A possible association of these genomic variants on efficacy and side effects of opioid treatment in different ethnic groups has to be elucidated.

  12. Potent Dmt-Tic pharmacophoric delta- and mu-opioid receptor antagonists.

    PubMed

    Li, Tingyou; Fujita, Yoshio; Shiotani, Kimitaka; Miyazaki, Anna; Tsuda, Yuko; Ambo, Akihiro; Sasaki, Yusuke; Jinsmaa, Yunden; Marczak, Ewa; Bryant, Sharon D; Salvadori, Severo; Lazarus, Lawrence H; Okada, Yoshio

    2005-12-15

    A series of dimeric Dmt-Tic (2',6'-dimethyl-L-tyrosyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) analogues (8-14, 18-22) were covalently linked through diaminoalkane and symmetric or asymmetric 3,6-diaminoalkyl-2(1H)-pyrazinone moieties. All the compounds exhibited high affinity for both delta-opioid receptors [Ki(delta) = 0.06-1.53 nM] and mu-opioid receptors [Ki(mu) = 1.37-5.72 nM], resulting in moderate delta-receptor selectivity [Ki(mu)/Ki(delta) = 3-46]. Regardless of the type of linker between the Dmt-Tic pharmacophores, delta-opioid-mediated antagonism was extraordinarily high in all analogues (pA2 = 10.42-11.28), while in vitro agonism (MVD and GPI bioassays) was essentially absent (ca. 3 to >10 microM). While an unmodified N-terminus (9, 13, 18) revealed weak mu-opioid antagonism (pA2 = 6.78-6.99), N,N'-dimethylation (21, 22), which negatively impacts on mu-opioid-associated agonism (Balboni et al., Bioorg. Med. Chem. 2003, 11, 5435-5441), markedly enhanced mu-opioid antagonism (pA2 = 8.34 and 7.71 for 21 and 22, respectively) without affecting delta-opioid activity. These data are the first evidence that a single dimeric opioid ligand containing the Dmt-Tic pharmacophore exhibits highly potent delta- and mu-opioid antagonist activities.

  13. Activation of mu-opioid receptors in the ventrolateral orbital cortex inhibits the GABAergic miniature inhibitory postsynaptic currents in rats.

    PubMed

    Qu, Chao-Ling; Huo, Fu-Quan; Huang, Fen-Sheng; Tang, Jing-Shi

    2015-04-10

    Previous studies have indicated that mu-opioid receptors in the ventrolateral orbital cortex (VLO) are involved in antinociception in tail flick tests and GABAergic neurons or terminals express mu-opioid receptors in the VLO. The current study examined the effect of selective mu-opioid receptor agonist DAMGO on the GABAergic miniature inhibitory postsynaptic currents (mIPSCs) in the VLO in rats using the whole-cell patch clamp. The results demonstrated that 5 μM DAMGO application into the rat VLO slices significantly reduced the GABAergic mIPSCs frequency, without any effect on its amplitude, and this effect of DAMGO was reversed by pretreatment with selective mu-opioid receptor antagonist 1 μM CTOP. Importantly, application of CTOP alone into the VLO slices did not produce any effect on the frequency and amplitude of GABAergic mIPSCs. These results indicate a presynaptic effect of mu-opioid receptor activation on the GABAergic neurons in the VLO. The current data suggests that a presynaptic inhibition of the GABA release may contribute to the mu-opioid receptor mediated effects in the VLO and provides novel electrophysiological evidence for the underlying mechanisms of mu-opioid receptors in the VLO.

  14. Morphine-induced hyperalgesia involves mu opioid receptors and the metabolite morphine-3-glucuronide.

    PubMed

    Roeckel, Laurie-Anne; Utard, Valérie; Reiss, David; Mouheiche, Jinane; Maurin, Hervé; Robé, Anne; Audouard, Emilie; Wood, John N; Goumon, Yannick; Simonin, Frédéric; Gaveriaux-Ruff, Claire

    2017-09-04

    Opiates are potent analgesics but their clinical use is limited by side effects including analgesic tolerance and opioid-induced hyperalgesia (OIH). The Opiates produce analgesia and other adverse effects through activation of the mu opioid receptor (MOR) encoded by the Oprm1 gene. However, MOR and morphine metabolism involvement in OIH have been little explored. Hence, we examined MOR contribution to OIH by comparing morphine-induced hyperalgesia in wild type (WT) and MOR knockout (KO) mice. We found that repeated morphine administration led to analgesic tolerance and hyperalgesia in WT mice but not in MOR KO mice. The absence of OIH in MOR KO mice was found in both sexes, in two KO global mutant lines, and for mechanical, heat and cold pain modalities. In addition, the morphine metabolite morphine-3beta-D-glucuronide (M3G) elicited hyperalgesia in WT but not in MOR KO animals, as well as in both MOR flox and MOR-Nav1.8 sensory neuron conditional KO mice. M3G displayed significant binding to MOR and G-protein activation when using membranes from MOR-transfected cells or WT mice but not from MOR KO mice. Collectively our results show that MOR is involved in hyperalgesia induced by chronic morphine and its metabolite M3G.

  15. Comparison of five benzodiazepine-receptor agonists on buprenorphine-induced mu-opioid receptor regulation.

    PubMed

    Poisnel, Géraldine; Dhilly, Martine; Le Boisselier, Reynald; Barre, Louisa; Debruyne, Danièle

    2009-05-01

    In this study, we compared the effects of five short-, medium-, or long-acting benzodiazepine-receptor agonists (BZDs) [alprazolam (APZ), clonazepam (CLZ), flunitrazepam (FLZ), loprazolam (LPZ), zolpidem (ZLP)], at two distinct doses, 0.2 and 2 mg/kg, on the cell surface regulation of mu-opioid receptor induced by 0.15 mg/kg buprenorphine (BPN) in specific regions of the rat brain. Using 0.312 - 5 nM [(3)H]-DAMGO concentrations and Scatchard plot analysis, B(max) (maximal receptor density) and K(d) (dissociation constant) were determined at different brain regions of interest (amygdala, cortex, hippocampus, hypothalamus, thalamus). Acute BPN induced an expected down-regulation and addition of each of the BZDs to BPN induced less down-regulation than did BPN alone, sometimes while altering affinity. Some significant differences in the intensity of these effects were observed between BZDs. FLZ that is widely abused and enlarges BPN toxicity appeared the most potent to increase mu-cell surface receptor density at the lowest dose of 0.2 mg/kg. Besides, LPZ for which the effect on mu-opioid-receptor regulation appeared lower is considered to have a low risk of dependence in the epidemiological data banks. CLZ and ZLP (2 mg/kg) induced the strongest modification on mu-opioid-receptor density, but a substantial decrease in affinity could minimize the functional consequences. The reported changes were maximal in the amygdala, hippocampus, and thalamus. Among people using BPN and BZDs, the effects described here are likely to influence addictive behaviors and induce toxic effects that could be quantitatively different due to the quality of the BZD.

  16. Involvement of peripheral mu opioid receptors in scratching behavior in mice.

    PubMed

    Yamamoto, Atsuki; Sugimoto, Yukio

    2010-12-15

    Pruritus is a common adverse effect of opioid treatment. However, the mechanism by which pruritus is induced by opioid administration is unclear. In this study, we examined the effects of the intradermal injection of loperamide, a peripherally restricted opioid receptor agonist, on the itch sensation. When injected intradermally into the rostral part of the back in mice, loperamide elicited scratching behavior. We also examined the effects of the selective mu opioid receptor agonist [d-Ala², N-Me-Phe⁴, Gly⁵-ol]-enkephalin acetate (DAMGO), the selective delta opioid receptor agonist [d-Pen(2,5)]-enkephalin (DPDPE), and the selective kappa opioid receptor agonist U-50488H on scratching behavior in mice in order to determine which subtype is involved in opioid-induced pruritus. Following intradermal injection into the rostral part of the back in mice, DAMGO elicited scratching behavior, while DPDPE and U-50488H did not. This suggests that peripheral mu opioid activation elicits the itch sensation. Next, we focused on the treatment of opioid-induced itch sensation without central adverse effects. Naloxone methiodide is a peripherally restricted opioid receptor antagonist. In the present study, naloxone methiodide significantly suppressed scratching behavior induced by loperamide and DAMGO. These findings suggest that mu opioid receptors play a primary role in peripheral pruritus and that naloxone methiodide may represent a possible remedy for opioid-induced itching.

  17. Endomorphins 1 and 2, endogenous mu-opioid receptor agonists, impair passive avoidance learning in mice.

    PubMed

    Ukai, M; Watanabe, Y; Kameyama, T

    2001-06-08

    The effects of intracerebroventricular administration of endomorphin-1 and endomorphin-2, endogenous mu-opioid receptor agonists, on passive avoidance learning associated with long-term memory were investigated in mice. Endomorphin-1 (10 and 17.5 microg) and endomorphin-2 (17.5 microg) produced a significant decrease in step-down latency in a passive avoidance learning task. beta-Funaltrexamine (5 microg) almost completely reversed the endomorphin-1 (17.5 microg)- and endomorphin-2 (17.5 microg)-induced shortening of step-down latency, although neither naltrindole (4 ng) nor nor-binaltorphimine (4 microg) produced any significant effects on the effects of endomorphins 1 and 2. These results suggest that endomorphins 1 and 2 impair long-term memory through the mediation of mu-opioid receptors in the brain.

  18. Tetrapeptide Endomorphin Analogs Require Both Full Length and Truncated Splice Variants of the Mu Opioid Receptor Gene Oprm1 for Analgesia.

    PubMed

    Marrone, Gina F; Lu, Zhigang; Rossi, Grace; Narayan, Ankita; Hunkele, Amanda; Marx, Sarah; Xu, Jin; Pintar, John; Majumdar, Susruta; Pan, Ying-Xian; Pasternak, Gavril W

    2016-12-21

    The mu opioid receptor gene undergoes extensive alternative splicing. Mu opioids can be divided into three classes based on the role of different groups of splice variants. Morphine and methadone require only full length seven transmembrane (7TM) variants for analgesia, whereas IBNtxA (3'-iodobenzyol-6β-naltrexamide) needs only truncated 6TM variants. A set of endomorphin analogs fall into a third group that requires both 6TM and 7TM splice variants. Unlike morphine, endomorphin 1 and 2, DAPP (Dmt,d-Ala-Phe-Phe-NH2), and IDAPP (3'-iodo-Dmt-d-Ala-Phe-Phe-NH2) analgesia was lost in an exon 11 knockout mouse lacking 6TM variants. Restoring 6TM variant expression in a knockout mouse lacking both 6TM and 7TM variants failed to rescue DAPP or IDAPP analgesia. However, re-establishing 6TM expression in an exon 11 knockout mouse that still expressed 7TM variants did rescue the response, consistent with the need for both 6TM and 7TM variants. In receptor binding assays, (125)I-IDAPP labeled more sites (Bmax) than (3)H-DAMGO ([d-Ala(2),N-MePhe(4),Gly(ol)(5)]-enkephalin) in wild-type mice. In exon 11 knockout mice, (125)I-IDAPP binding was lowered to levels similar to (3)H-DAMGO, which remained relatively unchanged compared to wild-type mice. (125)I-IDAPP binding was totally lost in an exon 1/exon 11 knockout model lacking all Oprm1 variant expression, confirming that the drug was not cross labeling non-mu opioid receptors. These findings suggested that (125)I-IDAPP labeled two populations of mu binding sites in wild-type mice, one corresponding to 7TM variants and the second dependent upon 6TM variants. Together, these data indicate that endomorphin analogs represent a unique, genetically defined, and distinct class of mu opioid analgesic.

  19. Quantification of brain mu-opioid receptors with [11C]carfentanil: reference-tissue methods.

    PubMed

    Endres, Christopher J; Bencherif, Badreddine; Hilton, John; Madar, Igal; Frost, J James

    2003-02-01

    [(11)C]Carfentanil (CFN) is a mu-opioid agonist used for in vivo positron emission tomography (PET) studies of mu-opioid receptors. Previously, a tissue-ratio method was validated for the quantification of CFN binding. However, since that initial validation, several other blood independent (reference-tissue) methods have become available. To evaluate these methods, CFN PET studies with arterial blood sampling were acquired in six healthy male control subjects. Specific binding estimates obtained from reference-tissue methods were compared to those obtained with a more rigorous blood input modeling technique. It was determined that both a graphical method, and a simplified reference tissue model, were more accurate than the tissue-ratio method for quantification of CFN binding.

  20. [Morphofunctional manifestations of cardioprotective effect of mu-opioid receptor stimulation in stress].

    PubMed

    Maslov, L N; Revinskaia, Iu G; Ryzhov, A I; Naryzhnaia, N V

    2001-01-01

    Activation of peripheral mu-opioid receptors contributes to an increase in stability of cardiomyocytes to stress damage manifesting with decreased accumulation of Tc-99m pyrophosphate in the heart muscle and contractures of the myocardium. As a principal mechanism of mu-receptor-dependent increase in resistance of the heart to stress damage, modulated influence of opioids on adrenergic pathogenetic links of heart stress damage is considered. In realization of the discovered cardioprotective effect associated with mu-receptor activation, opioidergic limitation of histamine release from mast cells in the myocardium and also mu-receptor-dependent intensification of coronary bloodstream in stressed animals may play a definite role.

  1. Mu Opioids and Their Receptors: Evolution of a Concept

    PubMed Central

    Pan, Ying-Xian

    2013-01-01

    Opiates are among the oldest medications available to manage a number of medical problems. Although pain is the current focus, early use initially focused upon the treatment of dysentery. Opium contains high concentrations of both morphine and codeine, along with thebaine, which is used in the synthesis of a number of semisynthetic opioid analgesics. Thus, it is not surprising that new agents were initially based upon the morphine scaffold. The concept of multiple opioid receptors was first suggested almost 50 years ago (Martin, 1967), opening the possibility of new classes of drugs, but the morphine-like agents have remained the mainstay in the medical management of pain. Termed mu, our understanding of these morphine-like agents and their receptors has undergone an evolution in thinking over the past 35 years. Early pharmacological studies identified three major classes of receptors, helped by the discovery of endogenous opioid peptides and receptor subtypes—primarily through the synthesis of novel agents. These chemical biologic approaches were then eclipsed by the molecular biology revolution, which now reveals a complexity of the morphine-like agents and their receptors that had not been previously appreciated. PMID:24076545

  2. Mu-opioid receptors modulate the stability of dendritic spines

    PubMed Central

    Liao, Dezhi; Lin, Hang; Law, Ping Yee; Loh, Horace H.

    2005-01-01

    Opioids classically regulate the excitability of neurons by suppressing synaptic GABA release from inhibitory neurons. Here, we report a role for opioids in modulating excitatory synaptic transmission. By activating ubiquitously clustered μ-opioid receptor (MOR) in excitatory synapses, morphine caused collapse of preexisting dendritic spines and decreased synaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors. Meanwhile, the opioid antagonist naloxone increased the density of spines. Chronic treatment with morphine decreased the density of dendritic spines even in the presence of Tetrodotoxin, a sodium channel blocker, indicating that the morphine's effect was not caused by altered activity in neural network through suppression of GABA release. The effect of morphine on dendritic spines was absent in transgenic mice lacking MORs and was blocked by CTOP (D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-ThrNH2), a μ-receptor antagonist. These data together with others suggest that endogenous opioids and/or constitutive activity of MORs participate in maintaining normal morphology and function of spines, challenging the classical model of opioids. Abnormal alteration of spines may occur in drug addiction when opioid receptors are overactivated by exogenous opiates. PMID:15659552

  3. Enkephalin release promotes homeostatic increases in constitutively active mu opioid receptors during morphine withdrawal.

    PubMed

    Shoblock, J R; Maidment, N T

    2007-11-09

    We previously demonstrated that naloxone administration produces a robust conditioned place aversion (CPA) in opiate-naive rodents by blocking the action of enkephalins at mu opioid receptors (MORs). The aversive response to naloxone is potentiated by prior exposure to morphine. Morphine-induced MOR constitutive activity is hypothesized to underlie this enhanced effect of naloxone, an inverse agonist at the MOR. We sought additional evidence for the role of constitutively active MORs in this morphine-induced enhancement using the pro-enkephalin knockout (pENK(-)/(-)) mouse, which is devoid of naloxone CPA in the morphine-naive state. Naloxone, but not the neutral antagonist, 6-beta-naloxol, produced CPA and physical withdrawal signs in pENK(-)/(-) mice when administered 2 h, but not 20 h, after morphine administration. Naloxone-precipitated physical withdrawal signs were attenuated in the pENK(-)/(-) mice relative to wild-type (WT) animals. In both WT and pENK(-)/(-) mice, naloxone-precipitated withdrawal jumping was greatest when naloxone was administered 2 h after morphine treatment and diminished at 3 h, in agreement with previous estimates of the time course for morphine-induced MOR constitutive activity in vitro. However, naloxone regained an ability to precipitate physical withdrawal in the WT, but not the pENK(-)/(-) mice when administered 4.5 h after morphine administration. Taken together, the data suggest that a compensatory increase in enkephalin release during spontaneous morphine withdrawal promotes a second period of MOR constitutive activity in WT mice that is responsible for the enhanced naloxone aversion observed in such animals even when naloxone is administered 20 h after morphine. The endogenous enkephalin system and MOR constitutive activity may therefore play vital roles in hedonic homeostatic dysregulation following chronic opiate administration.

  4. Mu opioid receptor-effector coupling and trafficking in dorsal root ganglia neurons.

    PubMed

    Walwyn, W M; Wei, W; Xie, C-W; Chiu, K; Kieffer, B L; Evans, C J; Maidment, N T

    2006-10-13

    Morphine induces profound analgesic tolerance in vivo despite inducing little internalization of the mu opioid receptor (muOR). Previously proposed explanations suggest that this lack of internalization could either lead to prolonged signaling and associated compensatory changes in downstream signaling systems, or that the receptor is unable to recycle and resensitize and so loses efficacy, either mechanism resulting in tolerance. We therefore examined, in cultured neurons, the relationship between muOR internalization and desensitization in response to two agonists, D-Ala2, N-MePhe4, Gly5-ol-enkephalin (DAMGO) and morphine. In addition, we studied the chimeric mu/delta opioid receptor (mu/ partial differentialOR) which could affect internalization and desensitization in neurons. Dorsal root ganglia neurons from muOR knockout mice were transduced with an adenovirus expressing either receptor and their respective internalization, desensitization and trafficking profiles determined. Both receptors desensitized equally, measured by Ca2+ current inhibition, during the first 5 min of agonist exposure to DAMGO or morphine treatment, although the mu/partial differentialOR desensitized more extensively. Such rapid desensitization was unrelated to internalization as DAMGO, but not morphine, internalized both receptors after 20 min. In response to DAMGO the mu/partial differentialOR internalized more rapidly than the muOR and was trafficked through Rab4-positive endosomes and lysosomal-associated membrane protein-1-labeled lysosomes whereas the muOR was trafficked through Rab4 and Rab11-positive endosomes. Chronic desensitization of the Ca2+ current response, after 24 h of morphine or DAMGO incubation, was seen in the DAMGO, but not morphine-treated, muOR-expressing cells. Such persistence of signaling after chronic morphine treatment suggests that compensation of downstream signaling systems, rather than loss of efficacy due to poor receptor recycling, is a more likely

  5. mu-Opioid receptor-independent fashion of the suppression of sodium currents by mu-opioid analgesics in thalamic neurons.

    PubMed

    Hashimoto, Keisuke; Amano, Taku; Kasakura, Akiko; Uhl, George R; Sora, Ichiro; Sakai, Norio; Kuzumaki, Naoko; Suzuki, Tsutomu; Narita, Minoru

    2009-03-27

    Most reports in the literature have shown that the effects of opioid analgesics are primarily mediated by mu-opioid receptor (MOR), whereas other potential targets of opioid analgesics have not been thoroughly characterized. In this study, we found that extracellular application of morphine, fentanyl or oxycodone, which are all considered to be MOR agonists, at relatively high concentrations, but not endogenous mu-opioid peptides, produced a concentration-dependent suppression of sodium currents in cultured thalamic neurons. These effects of opioids were not affected by either a MOR antagonist naloxone or a deletion of MOR gene. Among these opioids, fentanyl strongly suppressed sodium currents to the same degree as lidocaine, and both morphine and oxycodone slightly but significantly reduced sodium currents when they were present extracellularly. In contrast, the intracellular application of morphine, but not oxycodone, fentanyl or lidocaine, reduced sodium currents. These results suggest that morphine, fentanyl and oxycodone each produce the MOR-independent suppression of sodium currents by distinct mechanisms in thalamic neurons.

  6. Bioorthogonal click chemistry to assay mu-opioid receptor palmitoylation using 15-hexadecynoic acid and immunoprecipitation

    PubMed Central

    Ebersole, Brittany; Petko, Jessica; Levenson, Robert

    2014-01-01

    We have developed a modification of bioorthogonal click chemistry to assay the palmitoylation of cellular proteins. This assay utilizes 15-hexadecynoic acid (15-HDYA) as a chemical probe in combination with protein immunoprecipitation using magnetic beads in order to detect S-palmitoylation of proteins of interest. Here we demonstrate the utility of this approach for the mu-opioid receptor (MOR), a GPCR responsible for mediating the analgesic and addictive properties of most clinically relevant opioid agonist drugs. This technique provides a rapid, non-isotopic, and efficient method to assay the palmitoylation status of a variety of cellular proteins including most GPCRs. PMID:24463015

  7. Activation of mu opioid receptors in the striatum differentially augments methamphetamine-induced gene expression and enhances stereotypic behavior.

    PubMed

    Horner, Kristen A; Hebbard, John C; Logan, Anna S; Vanchipurakel, Golda A; Gilbert, Yamiece E

    2012-03-01

    Mu opioid receptors are densely expressed in the patch compartment of striatum and contribute to methamphetamine-induced patch-enhanced gene expression and stereotypy. To further elucidate the role of mu opioid receptor activation in these phenomena, we examined whether activation of mu opioid receptors would enhance methamphetamine-induced stereotypy and prodynorphin, c-fos, arc and zif/268 expression in the patch and/or matrix compartments of striatum, as well as the impact of mu opioid receptor activation on the relationship between patch-enhanced gene expression and stereotypy. Male Sprague-Dawley rats were intrastriatally infused with d-Ala(2)-N-Me-Phe(4),Gly(5)-ol]enkephalin (DAMGO; 1 μg/μL), treated with methamphetamine (0.5 mg/kg) and killed at 45 min or 2 h later. DAMGO augmented methamphetamine-induced zif/268 mRNA expression in the patch and matrix compartments, while prodynorphin expression was increased in the dorsolateral patch compartment. DAMGO pre-treatment did not affect methamphetamine-induced arc and c-fos expression. DAMGO enhanced methamphetamine-induced stereotypy and resulted in greater patch versus matrix expression of prodynorphin in the dorsolateral striatum, leading to a negative correlation between the two. These findings indicate that mu opioid receptors contribute to methamphetamine-induced stereotypy, but can differentially influence the genomic responses to methamphetamine. These data also suggest that prodynorphin may offset the overstimulation of striatal neurons by methamphetamine.

  8. ACTIVATION OF MU OPIOID RECEPTORS IN THE STRIATUM DIFFERENTIALLY AUGMENTS METHAMPHETAMINE-INDUCED GENE EXPRESSION AND ENHANCES STEREOTYPIC BEHAVIOR

    PubMed Central

    Horner, Kristen A.; Hebbard, John C.; Logan, Anna S.; Vanchipurakel, Golda A.; Gilbert, Yamiece E.

    2013-01-01

    Mu opioid receptors are densely expressed in the patch compartment of striatum and contribute to methamphetamine-induced patch-enhanced gene expression and stereotypy. In order to further elucidate the role of mu opioid receptor activation in these phenomena, we examined whether activation of mu opioid receptors would enhance methamphetamine-induced stereotypy and prodynorphin, c-fos, arc and zif/268 expression in the patch and/or matrix compartments of striatum, as well as the impact of mu opioid receptor activation on the relationship between patch-enhanced gene expression and stereotypy. Male Sprague-Dawley rats were intrastriatally infused with D-Ala(2)-N-Me-Phe(4),Gly(5)-ol]enkephalin (DAMGO; 1 μg/μl), treated with methamphetamine (0.5 mg/kg) and sacrificed at 45 minutes or 2 hours later. DAMGO augmented methamphetamine-induced zif/268 mRNA expression in the patch and matrix compartments, while prodynorphin expression was increased in the dorsolateral patch compartment. DAMGO pretreatment did not affect methamphetamine-induced arc and c-fos expression. DAMGO enhanced methamphetamine-induced stereotypy and resulted in greater patch versus matrix expression of prodynorphin in the dorsolateral striatum, leading to a negative correlation between the two. These findings indicate that mu opioid receptors contribute to methamphetamine-induced stereotypy, but can differentially influence the genomic responses to methamphetamine. These data also suggest that prodynorphin may offset the overstimulation of striatal neurons by methamphetamine. PMID:22150526

  9. Human Mu Opioid Receptor (OPRM1 A118G) polymorphism is associated with brain mu-opioid receptor binding potential in smokers

    PubMed Central

    Ray, Riju; Ruparel, Kosha; Newberg, Andrew; Wileyto, E. Paul; Loughead, James W.; Divgi, Chaitanya; Blendy, Julie A.; Logan, Jean; Zubieta, Jon-Kar; Lerman, Caryn

    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 (BPND or receptor availability). Twenty-two smokers prescreened for genotype (12 A/A, 10 */G) completed two [11C]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 BPND 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 BPND 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. PMID:21576462

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

    SciTech Connect

    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.

  11. Immunocytochemical characterization of Delta-opioid and Mu-opioid receptor protein in the bovine pineal gland.

    PubMed

    Phansuwan-Pujito, Pansiri; Ebadi, Manuchair; Govitrapong, Piyarat

    2006-01-01

    Opioidergic innervation has been identified in the mammalian pineal gland. Recently, opioid receptors in bovine pineal glands have been characterized; the activation of these receptors leads to the stimulation of melatonin synthesis. In this study, the precise localization of opioid receptors in bovine pineal glands was determined by an immunohistochemical technique using antibodies raised against delta-opioid and mu-opioid receptors. Immunoreactivity of these two receptors was present at a moderate level in pinealocytes. A double-labeling study has shown that delta-opioid receptors are localized predominantly with mu-opioid receptors in the same pinealocytes. These immunopositive pinealocytes are often located in a group; however, some of them are dispersed individually. In addition, both types of receptors were found in glial cells and processes. A small number of delta-receptor-immunoreactive nerve fibers were observed in the perivascular space and intraparenchyma of the pineal gland. Mu-opioid receptor immunoreactivity was found in a number of nerve fibers throughout the gland, and in terminal-like dots on pinealocytes. There was immunocolocalization between delta-opioid receptors or mu-opioid receptors and leu-enkephalin in some nerve fibers. The results of this study indicate that the modulatory effect of the opioid system on melatonin secretion in pineal glands might act via opioid receptors on pinealocytes, whereas receptors located on nerve fibers might modulate the release of opioid peptides.

  12. Partial purification of the mu opioid receptor irreversibly labeled with (/sup 3/H)b-funaltrexamine

    SciTech Connect

    Liu-Chen, L.Y.; Phillips, C.A.; Tam, S.W.

    1986-03-01

    The mu opioid receptor in bovine striatal membranes was specifically and irreversibly labeled by incubation with 5 nM (/sup 3/H)..beta..-funaltrexamine (approx.-FNA) at 37/sup 0/C for 90 min in the presence of 100 mM NaCl. The specific and irreversible binding of (/sup 3/H)..beta..-FNA as defined by that blocked by 1 /sup +/M naloxone was about 60% of total irreversible binding. The specific irreversible binding was saturable, stereospecific, time-, temperature, and tissue-dependent. Mu opioid ligands were much more potent than delta or kappa ligands in inhibiting the specific irreversible labeling. SDS polyacrylamide gel electrophoresis of solubilized membranes in the presence of 2-mercaptoethanol yielded a major radiolabeled broad band of MW 68-97K daltons, characteristic of a glycoprotein band. This band was not observed in membranes labeled in the presence of excess unlabeled naloxone. The glycoprotein nature of the (/sup 3/H)..beta..-FNA-labeled opioid receptor was confirmed by its binding to a wheat germ agglutinin-Sepharose column and its elution with N-acetylglucosamine.

  13. Curvilinear relationships between mu-opioid receptor labeling and undirected song in male European starlings (Sturnus vulgaris).

    PubMed

    Kelm-Nelson, Cynthia A; Riters, Lauren V

    2013-08-21

    Female-directed communication in male songbirds has been reasonably well studied; yet, relatively little is known about communication in other social contexts. Songbirds also produce song that is not clearly directed towards another individual (undirected song) when alone or in flocks. Although the precise functions of undirected song may differ across species, this type of song is considered important for flock maintenance, song learning or practice. Past studies show that undirected song is tightly coupled to analgesia and positive affective state, which are both mediated by opioid activity. Furthermore, labeling for the opioid met-enkephalin in the medial preoptic nucleus (POM) correlates positively with undirected song production. We propose that undirected song is facilitated and maintained by opioid receptor activity in the POM and other brain regions involved in affective state, analgesia, and social behavior. To provide insight into this hypothesis, we used immunohistochemistry to examine relationships between undirected song and mu-opioid receptors in male starlings. Polynomial regression analyses revealed significant inverted-U shaped relationships between measures of undirected song and mu-opioid receptor labeling in the POM, medial bed nucleus of the stria terminalis (BSTm), and periaqueductal gray (PAG). These results suggest that low rates of undirected song may stimulate and/or be maintained by mu-opioid receptor activity; however, it may be that sustained levels of mu-opioid receptor activity associated with high rates of undirected song cause mu-opioid receptor down-regulation. The results indicate that mu-opioid receptor activity in POM, BSTm, and PAG may underlie previous links identified between undirected song, analgesia, and affective state. Copyright © 2013 Elsevier B.V. All rights reserved.

  14. Quantitative autoradiography of (/sup 3/H)CTOP binding to mu opioid receptors in rat brain

    SciTech Connect

    Hawkins, K.N.; Knapp, R.J.; Gehlert, D.R.; Lui, G.K.; Yamamura, M.S.; Roeske, L.C.; Hruby, V.J.; Yamamura, H.I.

    1988-01-01

    (/sup 3/H)H-D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 ((/sup 3/H)CTOP), a potent and highly selective mu opioid antagonist, was used to localize the mu receptors in rat brain by light microscopic autoradiography. Radioligand binding studies with (/sup 3/H)CTOP using slide-mounted tissue sections of rat brain produced a Kd value of 1.1 nM with a Bmax value of 79.1 fmol/mg protein. Mu opioid agonists and antagonists inhibited (/sup 3/H)CTOP binding with high affinity (IC50 values of 0.2-2.4nM), while the delta agonist DPDPE, delta antagonist ICI 174,864, and kappa agonist U 69,593 were very weak inhibitors of (/sup 3/H)CTOP binding. Light microscopic autoradiography of (/sup 3/H)CTOP binding sites revealed regions of high density and regions of moderate labeling. The cerebral cortex showed a low density of (/sup 3/H)CTOP binding.

  15. Molecular signatures of mu opioid receptor and somatostatin receptor 2 in pancreatic cancer.

    PubMed

    Jorand, Raphael; Biswas, Sunetra; Wakefield, Devin L; Tobin, Steven J; Golfetto, Ottavia; Hilton, Kelsey; Ko, Michelle; Ramos, Joe W; Small, Alexander R; Chu, Peiguo; Singh, Gagandeep; Jovanovic-Talisman, Tijana

    2016-11-07

    Pancreatic ductal adenocarcinoma (PDAC), a particularly aggressive malignancy, has been linked to atypical levels, certain mutations, and aberrant signaling of G-protein-coupled receptors (GPCRs). GPCRs have been challenging to target in cancer because they organize into complex networks in tumor cells. To dissect such networks with nanometer-scale precision, here we combine traditional biochemical approaches with superresolution microscopy methods. A novel interaction specific to PDAC is identified between mu opioid receptor (MOR) and somatostatin receptor 2 (SSTR2). Although MOR and SSTR2 did not colocalize in healthy pancreatic cells or matching healthy patient tissues, the pair did significantly colocalize in pancreatic cancer cells, multicellular tumor spheroids, and cancerous patient tissues. Moreover, this association in pancreatic cancer cells correlated with functional cross-talk and increased metastatic potential of cells. Coactivation of MOR and SSTR2 in PDAC cells led to increased expression of mesenchymal markers and decreased expression of an epithelial marker. Together these results suggest that the MOR-SSTR2 heteromer may constitute a novel therapeutic target for PDAC.

  16. Exploration of Bivalent Ligands Targeting Putative Mu Opioid Receptor and Chemokine Receptor CCR5 Dimerization

    PubMed Central

    Arnatt, Christopher K.; Falls, Bethany A.; Yuan, Yunyun; Raborg, Thomas J.; Masvekar, Ruturaj R.; El-Hage, Nazira; Selley, Dana E.; Nicola, Anthony V.; Knapp, Pamela E.; Hauser, Kurt F.; Zhang, Yan

    2016-01-01

    Modern antiretroviral therapies have provided HIV-1 infected patients longer lifespans and better quality of life. However, several neurological complications are now being seen in these patients due to HIV-1 associated injury of neurons by infected microglia and astrocytes. In addition, these effects can be further exacerbated with opiate use and abuse. One possible mechanism for such potentiation effects of opiates is the interaction of the mu opioid receptor (MOR) with the chemokine receptor CCR5 (CCR5), a known HIV-1 co-receptor, to form MOR-CCR5 heterodimer. In an attempt to understand this putative interaction and its relevance to neuroAIDS, we designed and synthesized a series of bivalent ligands targeting the putative CCR5-MOR heterodimer. To understand how these bivalent ligands may interact with the heterodimer, biological studies including calcium mobilization inhibition, binding affinity, HIV-1 invasion, and cell fusion assays were applied. In particular, HIV-1 infection assays using human peripheral blood mononuclear cells, macrophages, and astrocytes revealed a notable synergy in activity for one particular bivalent ligand. Further, a molecular model of the putative CCR5-MOR heterodimer was constructed, docked with the bivalent ligand, and molecular dynamics simulations of the complex was performed in a membrane-water system to help understand the biological observation. PMID:27720326

  17. Molecular signatures of mu opioid receptor and somatostatin receptor 2 in pancreatic cancer

    PubMed Central

    Jorand, Raphael; Biswas, Sunetra; Wakefield, Devin L.; Tobin, Steven J.; Golfetto, Ottavia; Hilton, Kelsey; Ko, Michelle; Ramos, Joe W.; Small, Alexander R.; Chu, Peiguo; Singh, Gagandeep; Jovanovic-Talisman, Tijana

    2016-01-01

    Pancreatic ductal adenocarcinoma (PDAC), a particularly aggressive malignancy, has been linked to atypical levels, certain mutations, and aberrant signaling of G-protein–coupled receptors (GPCRs). GPCRs have been challenging to target in cancer because they organize into complex networks in tumor cells. To dissect such networks with nanometer-scale precision, here we combine traditional biochemical approaches with superresolution microscopy methods. A novel interaction specific to PDAC is identified between mu opioid receptor (MOR) and somatostatin receptor 2 (SSTR2). Although MOR and SSTR2 did not colocalize in healthy pancreatic cells or matching healthy patient tissues, the pair did significantly colocalize in pancreatic cancer cells, multicellular tumor spheroids, and cancerous patient tissues. Moreover, this association in pancreatic cancer cells correlated with functional cross-talk and increased metastatic potential of cells. Coactivation of MOR and SSTR2 in PDAC cells led to increased expression of mesenchymal markers and decreased expression of an epithelial marker. Together these results suggest that the MOR-SSTR2 heteromer may constitute a novel therapeutic target for PDAC. PMID:27682590

  18. Recovery from Mu-opioid Receptor Desensitization following Chronic Treatment with Morphine and Methadone

    PubMed Central

    Quillinan, Nidia; Lau, Elaine; Virk, Michael; von Zastrow, Mark; Williams, John T

    2011-01-01

    Chronic treatment with morphine results in a decrease in mu-opioid receptor sensitivity, an increase in acute desensitization and a reduction in the recovery from acute desensitization in locus coeruleus neurons. With acute administration, morphine is unlike many other opioid agonists in that it does not mediate robust acute desensitization or induce receptor trafficking. This study compares mu-opioid receptor desensitization and trafficking in brain slices taken from rats treated for 6–7 days with a range of doses of morphine (60, 30, 15 mg/kg/day) and methadone (60, 30, 5 mg/kg/day) applied by subcutaneous implantation of osmotic mini pumps. Mice were treated with 45 mg/kg/day. In morphine treated animals, recovery from acute [Met]5enkephalin-induced desensitization and receptor recycling was diminished. In contrast, recovery and recycling were unchanged in slices from methadone treated animals. Remarkably the reduced recovery from desensitization and receptor recycling found in slices from morphine treated animals were not observed in animals lacking β-arrestin2. Further, pharmacological inhibition of GRK2, while not affecting the ability of [Met]5enkephalin to induce desensitization, acutely reversed the delay in recovery from desensitization produced by chronic morphine treatment. These results characterize a previously unidentified function of the GRK/arrestin system in mediating opioid regulation in response to chronic morphine administration. They also suggest that the GRK/arrestin system, rather then serving as a primary mediator of acute desensitization, controls recovery from desensitization by regulating receptor reinsertion to the plasma membrane after chronic treatment with morphine. The sustained GRK/arrestin dependent desensitization is another way in which morphine and methadone are distinguished. PMID:21430144

  19. Fourteen. beta. -(bromoacetamido)morphine irreversibly labels. mu. opioid receptors in rat brain membranes

    SciTech Connect

    Bidlack, J.M.; Frey, D.K.; Seyed-Mozaffari, A.; Archer, S. )

    1989-05-16

    The binding properties of 14{beta}-(bromoacetamido)morphine (BAM) and the ability of BAM to irreversibly inhibit opioid binding to rat brain membranes were examined to characterize the affinity and selectivity of BAM as an irreversible affinity ligand for opioid receptors. BAM had the same receptor selectivity as morphine, with a 3-5-fold decrease in affinity for the different types of opioid receptors. When brain membranes were incubated with BAM, followed by extensive washing, opioid binding was restored to control levels. However, when membranes were incubated with dithiothreitol (DTT), followed by BAM, and subsequently washed, 90% of the 0.25 nM ({sup 3}H)(D-Ala{sup 2},(Me)Phe{sup 4},Gly(ol){sup 5})enkephalin (DAGO) binding was irreversibly inhibited as a result of the specific alkylation of a sulfhydryl group at the {mu} binding site. This inhibition was dependent on the concentrations of both DTT and BAM. The {mu} receptor specificity of BAM alkylation was demonstrated by the ability of BAM alkylated membranes to still bind the {delta}-selective peptide ({sup 3}H)(D-penicillamine{sup 2},D-penicillamine{sup 5})enkephalin (DPDPE) and (-)-({sup 3}H)bremazocine in the presence of {mu} and {delta} blockers, selective for {kappa} binding sites. Morphine and naloxone partially protected the binding site from alkylation with BAM, while ligands that did not bind to the {mu}s site did not afford protection. These studies have demonstrated that when a disulfide bond at or near {mu} opioid binding sites was reduced, BAM could then alkylate this site, resulting in the specific irreversible labeling of {mu} opioid receptors.

  20. Biased mu-opioid receptor ligands: a promising new generation of pain therapeutics.

    PubMed

    Siuda, Edward R; Carr, Richard; Rominger, David H; Violin, Jonathan D

    2016-12-06

    Opioid chemistry and biology occupy a pivotal place in the history of pharmacology and medicine. Morphine offers unmatched efficacy in alleviating acute pain, but is also associated with a host of adverse side effects. The advent of biased agonism at G protein-coupled receptors has expanded our understanding of intracellular signaling and highlighted the concept that certain ligands are able to differentially modulate downstream pathways. The ability to target one pathway over another has allowed for the development of biased ligands with robust clinical efficacy and fewer adverse events. In this review we summarize these concepts with an emphasis on biased mu opioid receptor pharmacology and highlight how far opioid pharmacology has evolved.

  1. Alternatively spliced mu opioid receptor C termini impact the diverse actions of morphine.

    PubMed

    Xu, Jin; Lu, Zhigang; Narayan, Ankita; Le Rouzic, Valerie P; Xu, Mingming; Hunkele, Amanda; Brown, Taylor G; Hoefer, William F; Rossi, Grace C; Rice, Richard C; Martínez-Rivera, Arlene; Rajadhyaksha, Anjali M; Cartegni, Luca; Bassoni, Daniel L; Pasternak, Gavril W; Pan, Ying-Xian

    2017-04-03

    Extensive 3' alternative splicing of the mu opioid receptor gene OPRM1 creates multiple C-terminal splice variants. However, their behavioral relevance remains unknown. The present study generated 3 mutant mouse models with truncated C termini in 2 different mouse strains, C57BL/6J (B6) and 129/SvEv (129). One mouse truncated all C termini downstream of Oprm1 exon 3 (mE3M mice), while the other two selectively truncated C-terminal tails encoded by either exon 4 (mE4M mice) or exon 7 (mE7M mice). Studies of these mice revealed divergent roles for the C termini in morphine-induced behaviors, highlighting the importance of C-terminal variants in complex morphine actions. In mE7M-B6 mice, the exon 7-associated truncation diminished morphine tolerance and reward without altering physical dependence, whereas the exon 4-associated truncation in mE4M-B6 mice facilitated morphine tolerance and reduced morphine dependence without affecting morphine reward. mE7M-B6 mutant mice lost morphine-induced receptor desensitization in the brain stem and hypothalamus, consistent with exon 7 involvement in morphine tolerance. In cell-based studies, exon 7-associated variants shifted the bias of several mu opioids toward β-arrestin 2 over G protein activation compared with the exon 4-associated variant, suggesting an interaction of exon 7-associated C-terminal tails with β-arrestin 2 in morphine-induced desensitization and tolerance. Together, the differential effects of C-terminal truncation illustrate the pharmacological importance of OPRM1 3' alternative splicing.

  2. The potential for mu-opioid receptor agonists to be anti-emetic in humans: a review of clinical data.

    PubMed

    Johnston, Kevin D

    2010-02-01

    In animal models of vomiting, mu-opioid (MOP, OP(3)) receptors mediate both emesis and anti-emesis. mu-receptors within the blood-brain barrier, mediating anti-emesis, are more rapidly accessible to lipid-soluble mu-opioid receptor agonists such as fentanyl than to morphine, and fentanyl has broad-spectrum anti-emetic effects in a number of species. Whether a similar situation exists in humans is not known. A search was performed for clinical studies comparing the emetic side effects of opioids administered peri-operatively in an attempt to identify differences between morphine and more lipid-soluble mu-receptor-selective agonists such as fentanyl. Overall, the evidence appears to suggest that fentanyl and other phenylpiperidines are associated with less nausea and vomiting than morphine, but not all studies support this, and fentanyl-like drugs are associated with nausea and vomiting per se. Good evidence, however, exists to show that fentanyl and alfentanil do not cause more nausea and vomiting than the ultra fast-acting remifentanil. Because remifentanil is cleared rapidly post-operatively, such trials suggest that the emetic side effects of fentanyl and alfentanil are minimal. The clinical evidence, although limited, is at least consistent with the possibility that central mu-opioid receptors may mediate anti-emesis in humans. It is possible that the role of mu-opioid agonists in anti-emesis may become clearer in the future as a result of the use of peripheral mu-opioid receptor antagonists.

  3. Autoradiographic mapping of mu-opioid receptors during opiate tolerance and supersensitivity in the rat central nervous system.

    PubMed

    Díaz, A; Pazos, A; Flórez, J; Hurlé, M A

    2000-08-01

    In this autoradiographic study we have analysed the regional changes in the density of mu-opioid receptors produced by the chronic administration of sufentanil alone and after concurrent administration with nimodipine. mu-Opioid receptors in the central nervous system (CNS) of rats were labelled using 5 nM [3H]DAMGO. Sufentanil, a high-efficacy agonist, was administered for 7 days by chronic infusion (2 microg/h). Another group of animals received a simultaneous infusion of sufentanil (2 microg/h) and nimodipine (1 microg/h) for 7 days. These two drug regimes have been previously shown to induce tolerance and supersensitivity to the analgesic effect of the opioid, respectively. Our results clearly demonstrate that opioid tolerance is associated with a generalised down-regulation of mu-opioid binding sites throughout the brain and the spinal cord. Compared with the findings in tolerant animals, the CNS of animals supersensitive to sufentanil showed less down-regulation of mu-opioid receptors, to the extent that, particularly in brain areas related to nociception, such as the somatosensory cortex, central grey, raphe magnus nucleus and dorsal horn of the spinal cord, no down-regulation occurred. These neurochemical findings may contribute to the functional interaction between nimodipine and sufentanil that we have previously observed in analgesic studies.

  4. Characterization of the antihyperalgesic action of a novel peripheral mu-opioid receptor agonist--loperamide.

    PubMed

    Nozaki-Taguchi, N; Yaksh, T L

    1999-01-01

    Preclinical and clinical evidence indicates that locally administered opioid agonists produce an antihyperalgesic effect through peripheral opioid receptors in inflamed tissue. Loperamide, a mu opioid agonist, does not cross the blood-brain barrier and therefore lacks central effects after systemic administration. The authors defined the effects of topical loperamide on a thermal injury-induced hyperalgesia. In halothane-anesthetized rats, thermal injury was induced by placing the plantar surface of a hindpaw on a hot plate (52.0+/-1 degrees C) for 45 s. Loperamide was prepared in a cream emulsion (ADL 2-1294B, 0.5%, 1.7%, and 5.0%). The drug was applied as follows: before or after injury on the injured paw and on a normal paw and after injury on the injured paw of morphine-tolerant rats. Paw withdrawal latency to a radiant heat source was measured to determine the nociceptive threshold. A pharmacokinetic study was performed with the use of 14C-labeled drug. Thermal injury yielded a significant thermal hyperalgesia. Loperamide, but not the vehicle, posttreatment on the injured paw resulted in a dose-dependent antihyperalgesic effect, which was reversible with naloxone (1 mg/kg given intraperitoneally). Treatment with loperamide on the normal paw produced short-lasting hypoalgesia, but the effect was not reversible with naloxone. Pretreatment at 1 and 2 but not 4 h with loperamide was effective. A rightward shift of the dose-response curve was observed in rats made tolerant to systemic morphine with subcutaneous morphine pellets. No rats with drug treatment displayed any evident behavior changes (eg., loss of corneal or pinna reflexes or change in ambulation). Drug activity in the tissue revealed an elimination half life of 2.3 h and negligible concentration in the blood. Loperamide, a peripherally acting mu opioid agonist, applied topically at the site of inflammation possesses a significant antihyperalgesic action without any systemic side effects.

  5. Isolation and characterization of new exon 11-associated N-terminal splice variants of the human mu opioid receptor gene.

    PubMed

    Xu, Jin; Xu, Mingming; Hurd, Yasmin L; Pasternak, Gavril W; Pan, Ying-Xian

    2009-02-01

    Alternative splicing of the mu opioid receptor genes to create multiple mu receptor subtypes has been demonstrated in animals and humans. Previously, we identified a number of C-terminal variants in mice, rats and human, followed by several N-terminal variants associated with a new upstream exon in mice (exon 11). Behavioral studies in exon 11 knockout mice suggest an important role for the exon 11 variants in the analgesic actions of heroin and morphine-6beta-glucuronide, but not morphine or methadone. We now have identified a homologous human exon 11 and three similar human exon 11-associated variants, suggesting conservation of exon 11 and its associated variants across species. hMOR-1i has an additional 93 amino acids at the tip of the N-terminus but is otherwise identical to hMOR-1. When expressed in Chinese hamster ovary cells, the additional 93 amino acids in hMOR-1i had little effect on opioid binding, but significantly altered agonist-induced G-protein activation. hMOR-1G1 and hMOR-1G2 predicted six transmembrane domain variants, similar to those seen in mice. The regional expression of these exon 11-associated variants, as determined by RT-PCR, varied markedly, implying region-specific alternative splicing. The presence of exon 11-associated variants in humans raises questions regarding their potential role in heroin and morphine-6beta-glucuronide actions in people as they do in mice.

  6. Chronic heroin self-administration desensitizes mu opioid receptor-activated G-proteins in specific regions of rat brain.

    PubMed

    Sim-Selley, L J; Selley, D E; Vogt, L J; Childers, S R; Martin, T J

    2000-06-15

    In previous studies from our laboratory, chronic noncontingent morphine administration decreased mu opioid receptor-activated G-proteins in specific brainstem nuclei. In the present study, mu opioid receptor binding and receptor-activated G-proteins were examined after chronic heroin self-administration. Rats were trained to self-administer intravenous heroin for up to 39 d, achieving heroin intake up to 366 mg. kg(-1). d(-1). mu opioid-stimulated [(35)S]GTPgammaS and [(3)H]naloxone autoradiography were performed in adjacent brain sections. Agonist-stimulated [(35)S]GTPgammaS autoradiography also examined other G-protein-coupled receptors, including delta opioid, ORL-1, GABA(B), adenosine A(1), cannabinoid, and 5-HT(1A). In brains from heroin self-administering rats, decreased mu opioid-stimulated [(35)S]GTPgammaS binding was observed in periaqueductal gray, locus coeruleus, lateral parabrachial nucleus, and commissural nucleus tractus solitarius, as previously observed in chronic morphine-treated animals. In addition, decreased mu opioid-stimulated [(35)S]GTPgammaS binding was found in thalamus and amygdala after heroin self-administration. Despite this decrease in mu-activated G-proteins, [(3)H]naloxone binding demonstrated increased mu opioid receptor binding in several brain regions after heroin self-administration, and there was a significant decrease in mu receptor G-protein efficiency as expressed as a ratio between agonist-activated G-proteins and mu receptor binding. No effects on agonist-stimulated [(35)S]GTPgammaS binding were found for any other receptor examined. The effect of chronic heroin self-administration to decrease mu-stimulated [(35)S]GTPgammaS binding varied between regions and was highest in brainstem and lowest in the cortex and striatum. These results not only provide potential neuronal mechanisms that may contribute to opioid tolerance and dependence, but also may explain why various chronic effects of opioids develop to different degrees.

  7. Systematic analysis of factors influencing observations of biased agonism at the mu-opioid receptor.

    PubMed

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

    2016-08-01

    Biased agonism describes the ability of distinct G protein-coupled receptor (GPCR) ligands to stabilise distinct receptor conformations leading to the activation of different cell signalling pathways that can deliver different physiologic outcomes. This phenomenon is having a major impact on modern drug discovery as it offers the potential to design ligands that selectively activate or inhibit the signalling pathways linked to therapeutic effects with minimal activation or blockade of signalling pathways that are linked to the development of adverse on-target effects. However, the explosion in studies of biased agonism at multiple GPCR families in recombinant cell lines has revealed a high degree of variability on descriptions of biased ligands at the same GPCR and raised the question of whether biased agonism is a fixed attribute of a ligand in all cell types. The current study addresses this question at the mu-opioid receptor (MOP). Here, we have systematically assessed the impact of differential cellular protein complement (and cellular background), signalling kinetics and receptor species on our previous descriptions of biased agonism at MOP by several opioid peptides and synthetic opioids. Our results show that all these factors need to be carefully determined and reported when considering biased agonism. Nevertheless, our studies also show that, despite changes in overall signalling profiles, ligands that previously showed distinct bias profiles at MOP retained their uniqueness across different cell backgrounds. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Endogenous central amygdala mu-opioid receptor signaling promotes sodium appetite in mice

    PubMed Central

    Smith, Craig M.; Walker, Lesley L.; Leeboonngam, Tanawan; McKinley, Michael J.; Denton, Derek A.; Lawrence, Andrew J.

    2016-01-01

    Due to the importance of dietary sodium and its paucity within many inland environments, terrestrial animals have evolved an instinctive sodium appetite that is commensurate with sodium deficiency. Despite a well-established role for central opioid signaling in sodium appetite, the endogenous influence of specific opioid receptor subtypes within distinct brain regions remains to be elucidated. Using selective pharmacological antagonists of opioid receptor subtypes, we reveal that endogenous mu-opioid receptor (MOR) signaling strongly drives sodium appetite in sodium-depleted mice, whereas a role for kappa (KOR) and delta (DOR) opioid receptor signaling was not detected, at least in sodium-depleted mice. Fos immunohistochemistry revealed discrete regions of the mouse brain displaying an increased number of activated neurons during sodium gratification: the rostral portion of the nucleus of the solitary tract (rNTS), the lateral parabrachial nucleus (LPB), and the central amygdala (CeA). The CeA was subsequently targeted with bilateral infusions of the MOR antagonist naloxonazine, which significantly reduced sodium appetite in mice. The CeA is therefore identified as a key node in the circuit that contributes to sodium appetite. Moreover, endogenous opioids, acting via MOR, within the CeA promote this form of appetitive behavior. PMID:27849613

  9. AM-251 and rimonabant act as direct antagonists at mu-opioid receptors: implications for opioid/cannabinoid interaction studies.

    PubMed

    Seely, Kathryn A; Brents, Lisa K; Franks, Lirit N; Rajasekaran, Maheswari; Zimmerman, Sarah M; Fantegrossi, William E; Prather, Paul L

    2012-10-01

    Mu-opioid and CB1-cannabinoid agonists produce analgesia; however, adverse effects limit use of drugs in both classes. Additive or synergistic effects resulting from concurrent administration of low doses of mu- and CB1-agonists may produce analgesia with fewer side effects. Synergism potentially results from interaction between mu-opioid receptors (MORs) and CB1 receptors (CB1Rs). AM-251 and rimonabant are CB1R antagonist/inverse agonists employed to validate opioid-cannabinoid interactions, presumed to act selectively at CB1Rs. Therefore, the potential for direct action of these antagonists at MORs is rarely considered. This study determined if AM-251 and/or rimonabant directly bind and modulate the function of MORs. Surprisingly, AM-251 and rimonabant, but not a third CB1R inverse agonist AM-281, bind with mid-nanomolar affinity to human MORs with a rank order of affinity (K(i)) of AM-251 (251 nM) > rimonabant (652 nM) > AM281 (2135 nM). AM-251 and rimonabant, but not AM-281, also competitively antagonize morphine induced G-protein activation in CHO-hMOR cell homogenates (K(b) = 719 or 1310 nM, respectively). AM-251 and rimonabant block morphine inhibition of cAMP production, while only AM-251 elicits cAMP rebound in CHO-hMOR cells chronically exposed to morphine. AM-251 and rimonabant (10 mg/kg) attenuate morphine analgesia, whereas the same dose of AM-281 produces little effect. Therefore, in addition to high CB1R affinity, AM-251 and rimonabant bind to MORs with mid-nanomolar affinity and at higher doses may affect morphine analgesia via direct antagonism at MORs. Such CB1-independent of these antagonists effects may contribute to reported inconsistencies when CB1/MOR interactions are examined via pharmacological methods in CB1-knockout versus wild-type mice.

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

    SciTech Connect

    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

    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.

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

    SciTech Connect

    Todd, S.L.; Balster, R.L.; Martin, B.R. )

    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.

  12. Ethanol-induced social facilitation in adolescent rats: role of endogenous activity at mu opioid receptors.

    PubMed

    Varlinskaya, Elena I; Spear, Linda P

    2009-06-01

    or with shifts in the biphasic ethanol dose-response curve. Ethanol-induced facilitation of social play behavior seen in adolescent animals is mediated in part through ethanol-induced release of endogenous ligands for the mu-opioid receptor or an ethanol-associated enhancement of sensitivity of these receptors for their endogenous ligands.

  13. Mu Opioid Receptor Binding Correlates with Nicotine Dependence and Reward in Smokers.

    PubMed

    Kuwabara, Hiroto; Heishman, Stephen J; Brasic, James R; Contoreggi, Carlo; Cascella, Nicola; Mackowick, Kristen M; Taylor, Richard; Rousset, Olivier; Willis, William; Huestis, Marilyn A; Concheiro, Marta; Wand, Gary; Wong, Dean F; Volkow, Nora D

    2014-01-01

    The rewarding effects of nicotine are associated with activation of nicotine receptors. However, there is increasing evidence that the endogenous opioid system is involved in nicotine's rewarding effects. We employed PET imaging with [11C]carfentanil to test the hypotheses that acute cigarette smoking increases release of endogenous opioids in the human brain and that smokers have an upregulation of mu opioid receptors (MORs) when compared to nonsmokers. We found no significant changes in binding potential (BPND) of [11C]carfentanil between the placebo and the active cigarette sessions, nor did we observe differences in MOR binding between smokers and nonsmokers. Interestingly, we showed that in smokers MOR availability in bilateral superior temporal cortices during the placebo condition was negatively correlated with scores on the Fagerström Test for Nicotine Dependence (FTND). Also in smokers, smoking-induced decreases in [11C]carfentanil binding in frontal cortical regions were associated with self-reports of cigarette liking and wanting. Although we did not show differences between smokers and nonsmokers, the negative correlation with FTND corroborates the role of MORs in superior temporal cortices in nicotine addiction and provides preliminary evidence of a role of endogenous opioid signaling in frontal cortex in nicotine reward.

  14. Mu Opioid Receptor Binding Correlates with Nicotine Dependence and Reward in Smokers

    PubMed Central

    Brasic, James R.; Contoreggi, Carlo; Cascella, Nicola; Mackowick, Kristen M.; Taylor, Richard; Rousset, Olivier; Willis, William; Huestis, Marilyn A.; Concheiro, Marta; Wand, Gary; Wong, Dean F.; Volkow, Nora D.

    2014-01-01

    The rewarding effects of nicotine are associated with activation of nicotine receptors. However, there is increasing evidence that the endogenous opioid system is involved in nicotine's rewarding effects. We employed PET imaging with [11C]carfentanil to test the hypotheses that acute cigarette smoking increases release of endogenous opioids in the human brain and that smokers have an upregulation of mu opioid receptors (MORs) when compared to nonsmokers. We found no significant changes in binding potential (BPND) of [11C]carfentanil between the placebo and the active cigarette sessions, nor did we observe differences in MOR binding between smokers and nonsmokers. Interestingly, we showed that in smokers MOR availability in bilateral superior temporal cortices during the placebo condition was negatively correlated with scores on the Fagerström Test for Nicotine Dependence (FTND). Also in smokers, smoking-induced decreases in [11C]carfentanil binding in frontal cortical regions were associated with self-reports of cigarette liking and wanting. Although we did not show differences between smokers and nonsmokers, the negative correlation with FTND corroborates the role of MORs in superior temporal cortices in nicotine addiction and provides preliminary evidence of a role of endogenous opioid signaling in frontal cortex in nicotine reward. PMID:25493427

  15. Mu-opioid receptor (MOR) expression in the human spiral ganglia

    PubMed Central

    Nguyen, Kimanh D.; Mowlds, Donald; Lopez, Ivan A.; Hosokawa, Seiji; Ishiyama, Akira; Ishiyama, Gail

    2015-01-01

    Opioid peptides and their receptors have been localized to the inner ear of the rat and guinea pig mammalian models. The expression of mu opioid receptor (MOR) in the human and mouse cochlea is not yet known. We present MOR protein localization by immunohistochemistry and mRNA expression by in situ hybridization in the human and mouse spiral ganglia (SG) and organ of Corti. In the human most of the (SG) neurons were immunoreactive; a subset was non-immunoreactive. In situ hybridization revealed a similar labeling pattern across the neurons of the SG. A similar distribution MOR pattern was demonstrated in the mouse SG. In the mouse organ of Corti MOR was expressed in inner and outer hair cells. Fibers underneath the inner hair cells were also MOR immunoreactive. These results are consistent with a role of MOR in neuro-modulation of the auditory periphery. The present results show that the expression of MORs is well-conserved across multiple mammalian species, indicative of an important role in auditory processing. PMID:25278190

  16. Remifentanil produces cross-desensitization and tolerance with morphine on the mu-opioid receptor.

    PubMed

    Nowoczyn, M; Marie, N; Coulbault, L; Hervault, M; Davis, A; Hanouz, J L; Allouche, S

    2013-10-01

    Remifentanil is a powerful mu-opioid (MOP) receptor agonist used in anaesthesia with a very short half-life. However, per-operative perfusion of remifentanil was shown to increase morphine consumption during post-operative period to relieve pain. In the present study, we aimed to describe the cellular mechanisms responsible for this apparent reduction of morphine efficacy. For this purpose, we first examined the pharmacological properties of both remifentanil and morphine at the MOP receptor, endogenously expressed in the human neuroblastoma SH-SY5Y cell line, to regulate adenylyl cyclase and the MAP kinase ERK1/2 pathway, their potency to promote MOP receptor phosphorylation, arrestin 3-CFP (cyan fluorescent protein) recruitment and receptor trafficking during acute and sustained exposure. In the second part of this work, we studied the effects of a prior exposure of remifentanil on morphine-induced inhibition of cAMP accumulation, activation of ERK1/2 and analgesia. We showed that sustained exposure to remifentanil promoted a rapid desensitization of opioid receptors on both signalling pathways and a pretreatment with this agonist reduced signal transduction produced by a second challenge with morphine. While both opioid agonists promoted Ser(375) phosphorylation on MOP receptor, remifentanil induced a rapid internalization of opioid receptors compared to morphine but without detectable arrestin 3-CFP translocation to the plasma membrane in our experimental conditions. Lastly, a cross-tolerance between remifentanil and morphine was observed in mice using the hot plate test. Our in vitro and in vivo data thus demonstrated that remifentanil produced a rapid desensitization and internalization of the MOP receptor that would reduce the anti-nociceptive effects of morphine.

  17. Autistic-like syndrome in mu opioid receptor null mice is relieved by facilitated mGluR4 activity.

    PubMed

    Becker, Jérôme A J; Clesse, Daniel; Spiegelhalter, Coralie; Schwab, Yannick; Le Merrer, Julie; Kieffer, Brigitte L

    2014-08-01

    The etiology of Autism Spectrum Disorders (ASDs) remains largely unknown. Identifying vulnerability genes for autism represents a major challenge in the field and allows the development of animal models for translational research. Mice lacking the mu opioid receptor gene (Oprm1(-/-)) were recently proposed as a monogenic mouse model of autism, based on severe deficits in social behavior and communication skills. We confirm this hypothesis by showing that adult Oprm1(-/-) animals recapitulate core and multiple comorbid behavioral symptoms of autism and also display anatomical, neurochemical, and genetic landmarks of the disease. Chronic facilitation of mGluR4 signaling, which we identified as a novel pharmacological target in ASDs in these mice, was more efficient in alleviating behavioral deficits than the reference molecule risperidone. Altogether, our data provide first evidence that disrupted mu opioid receptor signaling is sufficient to trigger a comprehensive autistic syndrome, maybe through blunted social reward processes, and this mouse model opens promising avenues for therapeutic innovation.

  18. Involvement of mu opioid receptors of periaqueductal gary (PAG) in acupuncture inhibition of noxious blood pressure response in rabbits.

    PubMed

    Gao, M; Xu, W; Chen, W; He, L

    1994-01-01

    Strong electric shock stimulation of the rabbit front paw elicited a pressor blood pressure response regarded as noxious response. Ligands of mu opioid receptors were microinjected into the PAG to observe their effects on acupunture inhibition of the pressor response. (1) Ohmefentanyl (OMF), a mu agonist, significantly attenuated the pressor response. Mu antagonist TCTAP greatly enhanced the pressor response. (2) Electroacupuncture (EA) significantly inhibited the pressor response, the inhibition being readily reversed by TCTAP. The response after TCTAP was significantly greater than that of the control before EA. The results suggest that noxious stimulation is able to activate the mu opioid receptor of the PAG to modulate the noxious response and EA is able to enhance the activation.

  19. Effects of endomorphins-1 and -2, endogenous mu-opioid receptor agonists, on spontaneous alternation performance in mice.

    PubMed

    Ukai, M; Watanabe, Y; Kameyama, T

    2000-05-03

    The effects of intracerebroventricular (i.c.v.) administration of endomorphins-1 and -2, endogenous mu-opioid receptor agonists, on the spontaneous alternation performance associated with spatial working memory were investigated in mice. Endomorphin-1 (10 and 17.5 microg) and endomorphin-2 (10 microg) produced a significant decrease in percent alternation without affecting total arm entries. beta-Funaltrexamine (5 microg) almost completely reversed the endomorphin-1 (10 microg)- and endomorphin-2 (10 microg)-induced decrease in percent alternation, although neither naltrindole (4 ng) nor nor-binaltorphimine (4 microg) produced any significant effects on alternation performance. These results suggest that endomorphins impair spatial working memory through the mediation of mu-opioid receptors.

  20. Graphene decorated with mu-opioid receptor: the ionic screening effect and detection of enkephalin

    NASA Astrophysics Data System (ADS)

    Ping, Jinglei; Johnson, A. T. Charlie; Liu, Renyu; A. T. Charlie Johnson Team; Renyu Liu Collaboration

    2015-03-01

    We investigated the properties of graphene field effect transistors (GFETs) decorated with a computaionally redesigned, water-soluble variant of the human mu-opioid receptor (wsMOR) in physiological buffer solution. The shift of the Fermi level in the GFETs is quantitatively described by chemical-gating effect of charges on the wsMOR that are screened by the ionic solution. Our results suggest that sensitivity to the molecular target is lost when the Debye screening length of the solution is shorter than the distance from the graphene to the wsMOR; thus de-salting may be necessary when wsMOR decorated GFETs are used as biosensors in solution. We used this insight to detect DAMGO, a synthetic analog to the endogenous opioid peptide encephalin, at a concentration of 10 pM (5.1 pg/mL) in artificial cerebrospinal fluid (aCSF) diluted to 5% of its normal salt concentration. When the sensors were measured in a dry state, the limit of detection for DAGMO was 1 pM (0.5 pg/mL), one-third of the baseline in human body.Funding for this work was provided by DARPA.

  1. Novel endomorphin analogues with antagonist activity at the mu-opioid receptor in the gastrointestinal tract.

    PubMed

    Fichna, Jakub; Gach, Katarzyna; Perlikowska, Renata; Cravezic, Aurore; Bonnet, Jean Jacques; do-Rego, Jean-Claude; Janecka, Anna; Storr, Martin A

    2010-06-08

    Opioid bowel dysfunction (OBD) summarizes common adverse side effects of opiate-based management of pain. A promising therapeutic approach to prevent OBD and other opioid-related disorders of the gastrointestinal (GI) tract is the co-administration of opiates with peripherally-restricted mu-opioid receptor (MOR)-selective antagonists. The aim of this study was to investigate the selectivity and efficacy of three novel peptide antagonists: antanal-1, antanal-2, and antanal-2A at MOR in the GI tract in vitro and in vivo. The effects of the antanals on GI motility were studied in vitro, using isolated preparations of mouse ileum and colon and in vivo, by measuring colonic propulsion in mice. Additionally, in vitro stability against enzymatic degradation and blood-brain barrier (BBB) permeability using the hot plate test in mice were examined. The antanals significantly reduced the inhibitory effect of the MOR agonists endomorphin-2, morphine, and loperamide on mouse ileum and colon contractions in vitro and blocked morphine-induced decrease of colonic bead expulsion in vivo. The hot plate test in mice showed that the antagonist activity of all antanals was restricted to the periphery. Antanal-1, antanal-2, and antanal-2A are promising MOR antagonists with limited BBB permeability, which may be developed into future therapeutics of opioid-related GI dysfunction.

  2. Naloxegol: First oral peripherally acting mu opioid receptor antagonists for opioid-induced constipation

    PubMed Central

    Anantharamu, Tejus; Sharma, Sushil; Gupta, Ajay Kumar; Dahiya, Navdeep; Singh Brashier, Dick B.; Sharma, Ashok Kumar

    2015-01-01

    Opioid-induced constipation (OIC) is one of the most troublesome and the most common effects of opioid use leading to deterioration in quality of life of the patients and also has potentially deleterious repercussions on adherence and compliance to opioid therapy. With the current guidelines advocating liberal use of opioids by physicians even for non-cancer chronic pain, the situation is further complicated as these individuals are not undergoing palliative care and hence there cannot be any justification to subject these patients to the severe constipation brought on by opioid therapy which is no less debilitating than the chronic pain. The aim in these patients is to prevent the opioid-induced constipation but at the same time allow the analgesic activity of opioids. Many drugs have been used with limited success but the most specific among them were the peripherally acting mu opioid receptor antagonists (PAMORA). Methylnaltrexone and alvimopan were the early drugs in this group but were not approved for oral use in OIC. However naloxegol, the latest PAMORA has been very recently approved as the first oral drug for OIC. This article gives an overview of OIC, its current management and more specifically the development and approval of naloxegol, including pharmacokinetics, details of various clinical trials, adverse effects and its current status for the management of OIC. PMID:26312011

  3. Functional polymorphism of the mu-opioid receptor gene (OPRM1) influences reinforcement learning in humans.

    PubMed

    Lee, Mary R; Gallen, Courtney L; Zhang, Xiaochu; Hodgkinson, Colin A; Goldman, David; Stein, Elliot A; Barr, Christina S

    2011-01-01

    Previous reports on the functional effects (i.e., gain or loss of function), and phenotypic outcomes (e.g., changes in addiction vulnerability and stress response) of a commonly occurring functional single nucleotide polymorphism (SNP) of the mu-opioid receptor (OPRM1 A118G) have been inconsistent. Here we examine the effect of this polymorphism on implicit reward learning. We used a probabilistic signal detection task to determine whether this polymorphism impacts response bias to monetary reward in 63 healthy adult subjects: 51 AA homozygotes and 12 G allele carriers. OPRM1 AA homozygotes exhibited typical responding to the rewarded response--that is, their bias to the rewarded stimulus increased over time. However, OPRM1 G allele carriers exhibited a decline in response to the rewarded stimulus compared to the AA homozygotes. These results extend previous reports on the heritability of performance on this task by implicating a specific polymorphism. Through comparison with other studies using this task, we suggest a possible mechanism by which the OPRM1 polymorphism may confer reduced response to natural reward through a dopamine-mediated decrease during positive reinforcement learning.

  4. The Behavioral Effects of the Antidepressant Tianeptine Require the Mu Opioid Receptor.

    PubMed

    Samuels, Benjamin Adam; Nautiyal, Katherine M; Kruegel, Andrew C; Levinstein, Marjorie R; Magalong, Valerie M; Gassaway, Madalee M; Grinnell, Steven G; Han, Jaena; Ansonoff, Michael A; Pintar, John E; Javitch, Jonathan A; Sames, Dalibor; Hen, René

    2017-03-17

    Depression is a debilitating chronic illness that affects around 350 million people worldwide. Current treatments, such as selective serotonin reuptake inhibitors (SSRIs), are not ideal because only a fraction of patients achieve remission. Tianeptine is an effective antidepressant with a previously unknown mechanism of action. We recently reported that tianeptine is a full agonist at the mu-opioid receptor (MOR). Here we demonstrate that the acute and chronic antidepressant-like behavioral effects of tianeptine in mice require MOR. Interestingly, while tianeptine also produces many opiate-like behavioral effects such as analgesia and reward, it does not lead to tolerance or withdrawal. Furthermore, the primary metabolite of tianeptine (MC5), which has a longer half-life, mimics the behavioral effects of tianeptine in a MOR-dependent fashion. These results point to the possibility that MOR and its downstream signaling cascades may be novel targets for antidepressant drug development.Neuropsychopharmacology accepted article preview online, 17 March 2017. doi:10.1038/npp.2017.60.

  5. Genetic variation in mu-opioid-receptor-interacting proteins and smoking cessation in a nicotine replacement therapy trial.

    PubMed

    Ray, Riju; Jepson, Christopher; Wileyto, E Paul; Dahl, John P; Patterson, Freda; Rukstalis, Margaret; Pinto, Angela; Berrettini, Wade; Lerman, Caryn

    2007-11-01

    Extending a previous finding of an association between functional genetic variation in the mu-opioid receptor gene and response to nicotine replacement therapy, we explored the role of genetic variants in two genes encoding mu-opioid-receptor-interacting proteins, namely ARRB2 and HINT1. Participants were 374 smokers treated for nicotine dependence with either transdermal nicotine or nicotine nasal spray for 8 weeks in an open-label randomized trial. In a logistic regression model controlling for OPRM1 genotype, treatment type, and other covariates, we found no significant main effect of ARRB2 genotype on abstinence at either end of treatment or 6-month follow-up. Participants with the HINT1 TT genotype had significantly higher abstinence rates at 6-month follow-up, but this may not be a pharmacogenetic effect, given that the participants were drug free during this time. Haplotype analysis did not reveal any significant associations for either gene. We found an interaction of ARRB2 and OPRM1 genotype on abstinence at 6 months that approached significance; however, interpretation of this finding is limited by the small number of participants with the minor alleles for both genes. Although these data do not provide support for the role of genetic variation in these mu-opioid-receptor-interacting proteins and smoking cessation, further exploration of opioid pathway genes in larger prospective pharmacogenetic trials may be warranted.

  6. Variation in the mu-opioid receptor gene (OPRM1) is associated with dispositional and neural sensitivity to social rejection.

    PubMed

    Way, Baldwin M; Taylor, Shelley E; Eisenberger, Naomi I

    2009-09-01

    Scientific understanding of social pain--the hurt feelings resulting from social rejection, separation, or loss--has been facilitated by the hypothesis that such feelings arise, in part, from some of the same neural and neurochemical systems that generate the unpleasant feelings resulting from physical pain. Accordingly, in animals, the painkiller morphine not only alleviates the distress of physical pain, but also the distress of social separation. Because morphine acts on the mu-opioid receptor, we examined whether variation in the mu-opioid receptor gene (OPRM1), as measured by the functional A118G polymorphism, was associated with individual differences in rejection sensitivity. Participants (n = 122) completed a self-report inventory of dispositional sensitivity to social rejection and a subsample (n = 31) completed a functional MRI session in which they were rejected from an online ball-tossing game played with two supposed others. The A118G polymorphism was associated with dispositional sensitivity to rejection in the entire sample and in the fMRI subsample. Consistent with these results, G allele carriers showed greater reactivity to social rejection in neural regions previously shown to be involved in processing social pain as well as the unpleasantness of physical pain, particularly the dorsal anterior cingulate cortex (dACC) and anterior insula. Furthermore, dACC activity mediated the relationship between the A118G polymorphism and dispositional sensitivity to rejection, suggesting that this is a critical site for mu-opioid-related influence on social pain. Taken together, these data suggest that the A118G polymorphism specifically, and the mu-opioid receptor more generally, are involved in social pain in addition to physical pain.

  7. Expression of mu opioid receptor in dorsal diencephalic conduction system: new insights for the medial habenula.

    PubMed

    Gardon, O; Faget, L; Chu Sin Chung, P; Matifas, A; Massotte, D; Kieffer, B L

    2014-09-26

    The habenular complex, encompassing medial (MHb) and lateral (LHb) divisions, is a highly conserved epithalamic structure involved in the dorsal diencephalic conduction system (DDC). These brain nuclei regulate information flow between the limbic forebrain and the mid- and hindbrain, integrating cognitive with emotional and sensory processes. The MHb is also one of the strongest expression sites for mu opioid receptors (MORs), which mediate analgesic and rewarding properties of opiates. At present however, anatomical distribution and function of these receptors have been poorly studied in MHb pathways. Here we took advantage of a newly generated MOR-mcherry knock-in mouse line to characterize MOR expression sites in the DDC. MOR-mcherry fluorescent signal is weak in the LHb, but strong expression is visible in the MHb, fasciculus retroflexus (fr) and interpeduncular nucleus (IPN), indicating that MOR is mainly present in the MHb-IPN pathway. MOR-mcherry cell bodies are detected both in basolateral and apical parts of MHb, where the receptor co-localizes with cholinergic and substance P (SP) neurons, respectively, representing two main MHb neuronal populations. MOR-mcherry is expressed in most MHb-SP neurons, and is present in only a subpopulation of MHb-cholinergic neurons. Intense diffuse fluorescence detected in lateral and rostral parts of the IPN further suggests that MOR-mcherry is transported to terminals of these SP and cholinergic neurons. Finally, MOR-mcherry is present in septal regions projecting to the MHb, and in neurons of the central and intermediate IPN. Together, this study describes MOR expression in several compartments of the MHb-IPN circuitry. The remarkably high MOR density in the MHb-IPN pathway suggests that these receptors are in a unique position to mediate analgesic, autonomic and reward responses.

  8. Expression of mu opioid receptor in dorsal diencephalic conduction system: new insights for the medial habenula

    PubMed Central

    Gardon, O.; Faget, L.; Chu Sin Chung, P.; Matifas, A.; Massotte, D; Kieffer, B.L.

    2014-01-01

    The habenular complex, encompassing medial (MHb) and lateral (LHb) divisions, is a highly conserved epithalamic structure involved in the dorsal diencephalic conduction system (DDC). These brain nuclei regulate information flow between the limbic forebrain and the mid- and hindbrain, integrating cognitive with emotional and sensory processes. The MHb is also one of the strongest expression sites for mu opioid receptors (MORs), which mediate analgesic and rewarding properties of opiates. At present however, anatomical distribution and function of these receptors have been poorly studied in MHb pathways. Here we took advantage of a newly generated MOR-mcherry knock-in mouse line to characterize MOR expression sites in the DDC. MOR-mcherry fluorescent signal is weak in the lateral habenula, but strong expression is visible in the medial habenula, fasciculus retroflexus and interpeduncular nucleus (IPN), indicating that MOR is mainly present in the MHb-IPN pathway. MOR-mcherry cell bodies are detected both in basolateral and apical parts of MHb, where the receptor co-localizes with cholinergic and Substance P (SP) neurons, respectively, representing two main MHb neuronal populations. MOR-mcherry is expressed in most MHb-SP neurons, and is present in only a subpopulation of MHb-cholinergic neurons. Intense diffuse fluorescence detected in lateral and rostral parts of the IPN further suggests that MOR-mcherry is transported to terminals of these SP and cholinergic neurons. Finally, MOR-mcherry is present in septal regions projecting to the MHb, and in neurons of the central and intermediate IPN. Together, this study describes MOR expression in several compartments of the MHb-IPN circuitry. The remarkably high MOR density in the MHb-IPN pathway suggests that these receptors are in a unique position to mediate analgesic, autonomic and reward responses. PMID:25086313

  9. Studies of the Mu-Opioid Receptor/G-protein Complex Affinity Co-Purified and Membrane Preparations from 7315c Cells

    DTIC Science & Technology

    1989-06-09

    population of mu-opioid receptors. Morphine, an opioid agonist, inhibits prolactin secretion, in part, by inhibiting adenylyl cyclase activity via...interaction with multiple G-proteins in the 7315c cell membrane is that mu-opioid agonists inhibit prolactin secretion from this cell via the generation of...secretion. The receptor mediated activation of either Gil or Gi2 and subsequent inhibition of adenylyl cyclase activity would inhibit prolactin secretion

  10. Density of mu-opioid receptors in the hippocampus of adult male and female rats is altered by prenatal morphine exposure and gonadal hormone treatment.

    PubMed

    Slamberová, Romana; Rimanóczy, Agnes; Bar, Noffar; Schindler, Cheryl J; Vathy, Ilona

    2003-01-01

    The present in vitro autoradiography study demonstrates that prenatal exposure to morphine alters the density of mu-opioid receptors in the hippocampus of adult female but not adult male rats. Prenatal morphine exposure increased the mu-opioid receptor density in the CA1 of ovariectomized (OVX) females and in the CA3 of OVX, estradiol benzoate-plus progesterone (EB+P)-treated females, but decreased it in CA3 of OVX females. There were also hormonal effects on mu-opioid receptor density in adult female rats. In the CA1, only morphine-exposed but not saline-exposed, hormone-treated females (EB, P, or EB+P) had a decrease in mu-opioid receptor density relative to OVX females. Both saline-exposed and morphine-exposed, OVX females after gonadal hormone replacement had a lower density of mu-opioid receptors in the CA3 and in the dentate gyrus (DG) than OVX females. In male rats, there was a decrease in mu-opioid receptor density in the CA1 and CA3 of gonadectomized (GNX), testosterone 17beta-proprionate (TP)-treated males relative to GNX males regardless of prenatal morphine exposure. In the DG, the mu-opioid receptor density was reduced only in morphine-exposed but not in saline-exposed, TP-treated males compared with GNX males. Thus, our data demonstrate that mu-opioid receptor density in the hippocampus is affected by prenatal morphine exposure and by male and female gonadal hormones.

  11. Lobeline, a potential pharmacotherapy for drug addiction, binds to mu opioid receptors and diminishes the effects of opioid receptor agonists.

    PubMed

    Miller, Dennis K; Lever, John R; Rodvelt, Kelli R; Baskett, James A; Will, Matthew J; Kracke, George R

    2007-07-10

    Lobeline diminishes the behavioral and neurochemical effects of nicotine and amphetamines, and is considered a potential pharmacotherapy for drug abuse and addiction. Lobeline has high affinity for nicotinic acetylcholine receptors and inhibits the function of vesicular monoamine and dopamine transporters. The present study investigated the less-explored interaction of lobeline and the endogenous opioid system. In guinea pig brain homogenates, lobeline displaced (K(i)=0.74 microM) the binding of [(3)H]DAMGO [(D-Ala(2), N-ME-Phe(4), Gly(5)-ol)-enkephalin]. In a functional assay system comprised of MOR-1 mu opioid receptors and GIRK2 potassium channels expressed in Xenopus oocytes, lobeline had no effect on the resting current, but maximally inhibited (IC(50)=1.1 microM) morphine- and DAMGO-activated potassium current in a concentration-dependent manner. In a second functional assay, lobeline-evoked [(3)H]overflow from rat striatal slices preloaded with [(3)H]dopamine was not blocked by naltrexone. Importantly, concentrations of lobeline (0.1-0.3 microM) that did not have intrinsic activity attenuated ( approximately 50%) morphine-evoked [(3)H]overflow. Overall, the results suggest that lobeline functions as a mu opioid receptor antagonist. The ability of lobeline to block psychostimulant effects may be mediated by opioid receptor antagonism, and lobeline could be investigated as a treatment for opiate addiction.

  12. Allosteric modulation model of the mu opioid receptor by herkinorin, a potent not alkaloidal agonist.

    PubMed

    Marmolejo-Valencia, A F; Martínez-Mayorga, K

    2017-05-01

    Modulation of opioid receptors is the primary choice for pain management and structural information studies have gained new horizons with the recently available X-ray crystal structures. Herkinorin is one of the most remarkable salvinorin A derivative with high affinity for the mu opioid receptor, moderate selectivity and lack of nitrogen atoms on its structure. Surprisingly, binding models for herkinorin are lacking. In this work, we explore binding models of herkinorin using automated docking, molecular dynamics simulations, free energy calculations and available experimental information. Our herkinorin D-ICM-1 binding model predicted a binding free energy of -11.52 ± 1.14 kcal mol(-1) by alchemical free energy estimations, which is close to the experimental values -10.91 ± 0.2 and -10.80 ± 0.05 kcal mol(-1) and is in agreement with experimental structural information. Specifically, D-ICM-1 molecular dynamics simulations showed a water-mediated interaction between D-ICM-1 and the amino acid H297(6.52), this interaction coincides with the co-crystallized ligands. Another relevant interaction, with N127(2.63), allowed to rationalize herkinorin's selectivity to mu over delta opioid receptors. Our suggested binding model for herkinorin is in agreement with this and additional experimental data. The most remarkable observation derived from our D-ICM-1 model is that herkinorin reaches an allosteric sodium ion binding site near N150(3.35). Key interactions in that region appear relevant for the lack of β-arrestin recruitment by herkinorin. This interaction is key for downstream signaling pathways involved in the development of side effects, such as tolerance. Future SAR studies and medicinal chemistry efforts will benefit from the structural information presented in this work.

  13. Allosteric modulation model of the mu opioid receptor by herkinorin, a potent not alkaloidal agonist

    NASA Astrophysics Data System (ADS)

    Marmolejo-Valencia, A. F.; Martínez-Mayorga, K.

    2017-05-01

    Modulation of opioid receptors is the primary choice for pain management and structural information studies have gained new horizons with the recently available X-ray crystal structures. Herkinorin is one of the most remarkable salvinorin A derivative with high affinity for the mu opioid receptor, moderate selectivity and lack of nitrogen atoms on its structure. Surprisingly, binding models for herkinorin are lacking. In this work, we explore binding models of herkinorin using automated docking, molecular dynamics simulations, free energy calculations and available experimental information. Our herkinorin D-ICM-1 binding model predicted a binding free energy of -11.52 ± 1.14 kcal mol-1 by alchemical free energy estimations, which is close to the experimental values -10.91 ± 0.2 and -10.80 ± 0.05 kcal mol-1 and is in agreement with experimental structural information. Specifically, D-ICM-1 molecular dynamics simulations showed a water-mediated interaction between D-ICM-1 and the amino acid H2976.52, this interaction coincides with the co-crystallized ligands. Another relevant interaction, with N1272.63, allowed to rationalize herkinorin's selectivity to mu over delta opioid receptors. Our suggested binding model for herkinorin is in agreement with this and additional experimental data. The most remarkable observation derived from our D-ICM-1 model is that herkinorin reaches an allosteric sodium ion binding site near N1503.35. Key interactions in that region appear relevant for the lack of β-arrestin recruitment by herkinorin. This interaction is key for downstream signaling pathways involved in the development of side effects, such as tolerance. Future SAR studies and medicinal chemistry efforts will benefit from the structural information presented in this work.

  14. Vicia villosa agglutinin labels a subset of neurons coexpressing both the mu opioid receptor and parvalbumin in the developing rat subiculum.

    PubMed

    Bausch, S B; Chavkin, C

    1996-12-23

    Vicia villosa agglutinin (VVA), anti-parvalbumin antiserum and an affinity-purified anti-mu opioid receptor antibody were used to triple-label neurons in the postnatal rat subiculum. VVA labeled a subset of mu opioid receptor-positive neurons that were also immunoreactive for parvalbumin. The morphology of the triple-labeled neurons was heterogeneous, and included multipolar, ovoid and pyramidal-shaped neurons. Neurons single-labeled for the mu opioid receptor, VVA or parvalbumin were also morphologically heterogeneous. The postnatal development of mu opioid receptor immunoreactivity (IR), parvalbumin-IR and VVA binding was investigated using triple-labeling immunocytochemistry. Mu opioid receptor-IR appeared first and was present at postnatal day 1 (P1). Parvalbumin-IR was first observed in somata at P10, followed by proximal and distal dendrites at P15 and P20 respectively. Faint VVA labeling was seen first at P10 and surrounded a limited number of neurons. The intensity of labeling and the number of neurons labeled with VVA increased between P10 and P20; however, both measures remained below adult levels at P20. This study further illustrates the neurochemical heterogeneity of interneurons in the hippocampal formation and shows the developmentally early appearance of mu opioid receptor-IR compared to the late appearance of VVA binding and parvalbumin-IR.

  15. Synaptic mechanism for functional synergism between delta- and mu-opioid receptors

    PubMed Central

    Zhang, Zhi; Pan, Zhizhong Z.

    2010-01-01

    By sustained activation of mu-opioid receptors (MOR), chronic opioids cause analgesic tolerance, physical dependence and opioid addiction, common clinical problems for which an effective treatment is still lacking. Chronic opioids recruit delta-opioid receptors (DOR) to plasma membrane through exocytotic trafficking, but the role of this new DOR and its interaction with existing MOR in brain functions and in the clinical problems remains largely unknown. In this study, we investigated the mechanisms underlying synaptic and behavioral actions of chronic morphine-induced DOR and its interaction with MOR in Nucleus Raphe Magnus (NRM) neurons important for opioid analgesia. We found that the emerged DOR inhibited GABAergic IPSCs through both the phospholipase A2 (PLA2) and cAMP/PKA signaling pathways. MOR inhibition of IPSCs, normally mediated predominantly by the PLA2 pathway, was additionally mediated by the cAMP/PKA pathway, with MOR potency significantly increased after chronic morphine treatment. Isobologram analysis revealed a synergistic DOR-MOR interaction in their IPSC inhibition, which was dependent on upregulated activities of both the PLA2 and cAMP/PKA pathways. Furthermore, DOR and MOR agonists microinjected into the NRM in vivo also produced a PLA2–dependent synergism in their antinociceptive effects. These findings suggest that the cAMP/PKA pathway, upregulated by chronic opioids, becomes more important in the mechanisms of both MOR and DOR inhibition of GABA synaptic transmission after chronic opioid exposure, and DOR and MOR are synergic both synaptically and behaviorally in producing analgesic effects in a PLA2-dependent fashion, supporting the potential therapeutic use of DOR agonists in pain management under chronic opioid conditions. PMID:20357124

  16. THE ROLE OF AMYGDALAR MU OPIOID RECEPTORS IN ANXIETY-RELATED RESPONSES IN TWO RAT MODELS

    PubMed Central

    Wilson, Marlene A.; Junor, Lorain

    2009-01-01

    Amygdala opioids such as enkephalin appear to play some role in the control of anxiety and the anxiolytic effects of benzodiazepines, although the opioid receptor subtypes mediating such effects are unclear. This study compared the influences of mu opioid receptor (MOR) activation in the central nucleus of the amygdala (CEA) on unconditioned fear or anxiety-like responses in two models, the elevated plus maze and the defensive burying test. The role of MOR in the anxiolytic actions of the benzodiazepine agonist diazepam was also examined using both models. Either the MOR agonist [D-Ala2, NMe-Phe4, Gly-ol5]-enkephalin (DAMGO) or the MOR antagonists Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) or β-funaltrexamine (FNA) were bilaterally infused into the CEA of rats prior to testing. The results show that microinjection of DAMGO in the CEA decreased open arm time in the plus maze, while CTAP increased open arm behaviors. In contrast, DAMGO injections in the CEA reduced burying behaviors and increased rearing following exposure to a predator odor, suggesting a shift in the behavioral response in this context. Amygdala injections of the MOR agonist DAMGO or the MOR antagonist CTAP failed to change the anxiolytic effects of diazepam in either test. Our results demonstrate that MOR activation in the central amygdala exerts distinctive effects in two different models of unconditioned fear or anxiety-like responses, and suggest that opioids may exert context-specific regulation of amygdala output circuits and behavioral responses during exposure to potential threats (open arms of the maze) versus discrete threats (predator odor). PMID:18216773

  17. Epigenetic Variation in the Mu-opioid Receptor Gene in Infants with Neonatal Abstinence Syndrome

    PubMed Central

    Wachman, Elisha M; Hayes, Marie J; Lester, Barry M; Terrin, Norma; Brown, Mark S; Nielsen, David A; Davis, Jonathan M

    2014-01-01

    Objective Neonatal abstinence syndrome (NAS) from in utero opioid exposure is highly variable with genetic factors appearing to play an important role. Epigenetic changes in cytosine:guanine (CpG) dinucleotide methylation can occur after drug exposure and may help to explain NAS variability. We correlated DNA methylation levels in the mu-opioid receptor (OPRM1) promoter in opioid-exposed infants and correlate them with NAS outcomes. Study design DNA samples from cord blood or saliva were analyzed for 86 infants being treated for NAS according to institutional protocol. Methylation levels at 16 OPRM1 CpG sites were determined and correlated with NAS outcome measures, including need for treatment, treatment with >2 medications, and length of hospital stay. We adjusted for co-variates and multiple genetic testing. Results Sixty-five percent of infants required treatment for NAS, and 24% required ≥2 medications. Hypermethylation of the OPRM1 promoter was measured at the −10 CpG in treated versus non-treated infants [adjusted difference δ=3.2% (95% CI 0.3–6.0%), p=0.03; NS after multiple testing correction]. There was hypermethylation at the −14 [δ=4.9% (95% CI 1.8–8.1%), p=0.003], −10 [δ=5.0% (95% CI 2.3–7.7%), p=0.0005)], and +84 [δ=3.5% (95% CI 0.6 – 6.4), p=0.02] CpG sites in infants requiring ≥2 medications which remained significant for −14 and −10 after multiple testing correction. Conclusions Increased methylation within the OPRM1 promoter is associated with worse NAS outcomes, consistent with gene silencing. PMID:24996986

  18. Epigenetic variation in the mu-opioid receptor gene in infants with neonatal abstinence syndrome.

    PubMed

    Wachman, Elisha M; Hayes, Marie J; Lester, Barry M; Terrin, Norma; Brown, Mark S; Nielsen, David A; Davis, Jonathan M

    2014-09-01

    Neonatal abstinence syndrome (NAS) from in utero opioid exposure is highly variable with genetic factors appearing to play an important role. Epigenetic changes in cytosine:guanine (CpG) dinucleotide methylation can occur after drug exposure and may help to explain NAS variability. We correlated DNA methylation levels in the mu-opioid receptor (OPRM1) promoter in opioid-exposed infants with NAS outcomes. DNA samples from cord blood or saliva were analyzed for 86 infants who were being treated for NAS according to institutional protocol. Methylation levels at 16 OPRM1 CpG sites were determined and correlated with NAS outcome measures, including need for treatment, treatment with ≥ 2 medications, and length of hospital stay. We adjusted for covariates and multiple genetic testing. Sixty-five percent of infants required treatment for NAS, and 24% required ≥ 2 medications. Hypermethylation of the OPRM1 promoter was measured at the -10 CpG in treated vs nontreated infants (adjusted difference δ = 3.2% [95% CI, 0.3-6.0%], P = .03; nonsignificant after multiple testing correction). There was hypermethylation at the -14 (δ = 4.9% [95% CI, 1.8%-8.1%], P = .003), -10 (δ = 5.0% [95% CI, 2.3-7.7%], P = .0005), and +84 (δ = 3.5% [95% CI, 0.6-6.4], P = .02) CpG sites in infants requiring ≥ 2 medications, which remained significant for -14 and -10 after multiple testing correction. Increased methylation within the OPRM1 promoter is associated with worse NAS outcomes, consistent with gene silencing. Copyright © 2014 Elsevier Inc. All rights reserved.

  19. Ovarian steroids alter mu opioid receptor trafficking in hippocampal parvalbumin GABAergic interneurons.

    PubMed

    Torres-Reveron, Annelyn; Williams, Tanya J; Chapleau, Jeanette D; Waters, Elizabeth M; McEwen, Bruce S; Drake, Carrie T; Milner, Teresa A

    2009-09-01

    The endogenous hippocampal opioid systems are implicated in learning associated with drug use. Recently, we showed that ovarian hormones regulate enkephalin levels in the mossy fiber pathway. This pathway overlaps with parvalbumin (PARV)-basket interneurons that contain the enkephalin-activated mu opioid receptors (MORs) and are important for controlling the "temporal timing" of granule cells. Here, we evaluated the influence of ovarian steroids on the trafficking of MORs in PARV interneurons. Two groups of female rats were analyzed: cycling rats in proestrus (relatively high estrogens) or diestrus; and ovariectomized rats euthanized 6, 24 or 72 h after estradiol benzoate (10 microg, s.c.) administration. Dorsal hippocampal sections were dually immunolabeled for MOR and PARV and examined by light and electron microscopy. As in males, in females MOR-immunoreactivity (-ir) was in numerous PARV-labeled perikarya, dendrites and terminals in the dentate hilar region. Variation in ovarian steroid levels altered the subcellular distribution of MORs in PARV-labeled dendrites but not terminals. In normal cycling rats, MOR-gold particles on the plasma membrane of small PARV-labeled dendrites (area <1 microm2) had higher density in proestrus rats than in diestrus rats. Likewise, in ovariectomized rats MORs showed higher density on the plasma membrane of small PARV-labeled dendrites 72 h after estradiol exposure. The number of PARV-labeled cells was not affected by estrous cycle phase or estrogen levels. These results demonstrate that estrogen levels positively regulate the availability of MORs on GABAergic interneurons in the dentate gyrus, suggesting cooperative interaction between opioids and estrogens in modulating principal cell excitability.

  20. Brain Mu-Opioid Receptor Binding: Relationship to Relapse to Cocaine Use after Monitored Abstinence

    PubMed Central

    Gorelick, David A.; Kim, Yu Kyeong; Bencherif, Badreddine; Boyd, Susan J.; Nelson, Richard; Copersino, Marc L.; Dannals, Robert F.; Frost, J. James

    2008-01-01

    Rationale Cocaine users have increased regional brain mu-opioid receptor (mOR) binding which correlates with cocaine craving. The relationship of mOR binding to relapse is unknown. Objectives To evaluate regional brain mOR binding as a predictor of relapse to cocaine use. Methods Fifteen non-treatment-seeking, adult cocaine users were housed on a closed research ward for 12 weeks of monitored abstinence, then followed for up to one year after discharge. Regional brain mOR binding was measured after one and 12 weeks using positron emission tomography (PET) with [11C]carfentanil (a selective mOR agonist). Time to first cocaine use (lapse) and to first two consecutive days of cocaine use (relapse) after discharge was based on self-report and urine toxicology. Results A shorter interval before relapse was associated with increased mOR binding in frontal and temporal cortical regions at one and 12 weeks of abstinence (P’s < 0.001) and with a lesser decrease in binding between one and 12 weeks (P’s < 0.0008). There were significant positive correlations between mOR binding at 12 weeks and % days of cocaine use during first month after relapse (P’s < 0.002). In multiple linear regression analysis, mOR binding contributed significantly to the prediction of time to relapse (R2 = 0.79, P < 0.001), even after accounting for clinical variables. Conclusions Increased brain mOR binding in frontal and temporal cortical regions is a significant independent predictor of time to relapse to cocaine use, suggesting an important role for the brain endogenous opioid system in cocaine addiction. PMID:18762918

  1. Brain mu-opioid receptor binding predicts treatment outcome in cocaine-abusing outpatients

    PubMed Central

    Ghitza, Udi E.; Preston, Kenzie L.; Epstein, David H.; Kuwabara, Hiroto; Endres, Christopher J.; Bencherif, Badreddine; Boyd, Susan J.; Copersino, Marc L.; Frost, J. James; Gorelick, David A.

    2010-01-01

    Background Cocaine users not seeking treatment have increased regional brain mu-opioid receptor (mOR) binding that correlates with cocaine craving and tendency to relapse. In cocaine-abusing outpatients in treatment, the relationship of mOR binding and treatment outcome is unknown. Methods We determined whether regional brain mOR binding before treatment correlates with outcome and compared it to standard clinical predictors of outcome. Twenty-five individuals seeking outpatient treatment for cocaine abuse or dependence (DSM-IV) received up to 12 weeks of cognitive-behavioral therapy and cocaine-abstinence reinforcement whereby each cocaine-free urine was reinforced with vouchers redeemable for goods. Regional brain mOR binding was measured before treatment using positron emission tomography (PET) with [11C] carfentanil (a selective mOR agonist). Main outcome measures were: 1) overall percentage of urines positive for cocaine during first month of treatment, 2) longest duration (weeks) of abstinence from cocaine during treatment, all verified by urine toxicology. Results Elevated mOR binding in the medial frontal and middle frontal gyri before treatment correlated with greater cocaine use during treatment. Elevated mOR binding in the anterior cingulate, medial frontal, middle frontal, middle temporal, and sub-lobar insular gyri correlated with shorter duration of cocaine abstinence during treatment. Regional mOR binding contributed significant predictive power for treatment outcome beyond that of standard clinical variables such as baseline drug and alcohol use. Conclusions Elevated mOR binding in brain regions associated with reward sensitivity is a significant independent predictor of treatment outcome in cocaine-abusing outpatients, suggesting a key role for the brain endogenous opioid system in cocaine addiction. PMID:20579973

  2. Ultrastructural relationship between the mu opioid receptor and its interacting protein, GPR177, in striatal neurons.

    PubMed

    Reyes, Arith-Ruth S; Levenson, Robert; Berrettini, Wade; Van Bockstaele, Elisabeth J

    2010-10-28

    GPR177, the mammalian ortholog of Drosophila Wntless/Evi/Sprinter, was recently identified as a novel mu-opioid receptor (MOR) interacting protein. GPR177 is a trans-membrane protein pivotal to mediating the secretion of Wnt signaling proteins. Wnt proteins, in turn, are essential in regulating neuronal development, a phenomenon inhibited upon chronic exposure to MOR agonists such as morphine and heroin. We previously showed that GPR177 and MOR are co-localized in the mouse dorsolateral striatum; however, the nature of this interaction was not fully elucidated. Therefore, in the present study, we examined cellular substrates for interactions between GPR177 and MOR using a combined immunogold-silver and peroxidase detection approach in coronal sections in the dorsolateral segment of the striatum. Semi-quantitative analysis of the ultrastructural distribution of GPR177 and MOR in striatal somata and in dendritic processes showed that, of the somata and dendritic processes exhibiting GPR177, 32% contained MOR immunolabeling while for profiles exhibiting MOR, 37% also contained GPR177 immunoreactivity. GPR177-labeled particles were localized predominantly along both the plasma membrane and within the cytoplasm of MOR-labeled dendrites. Somata and dendritic processes that contained both GPR177 and MOR more often received symmetric (inhibitory-type) synapses from unlabeled axon terminals. To further define the phenotype of GPR177 and MOR-containing cellular profiles, triple immunofluorescence detection showed that GPR177 and MOR are localized in neurons containing the opioid peptide, enkephalin, within the dorsolateral striatum. The results provide an anatomical substrate for interactions between MOR and its interacting protein, GPR177, in striatal opioid-containing neurons that may underlie the morphological alterations produced in neurons by chronic opiate use.

  3. Induced association of mu opioid (MOP) and type 2 cholecystokinin (CCK2) receptors by novel bivalent ligands

    PubMed Central

    Zheng, Yaguo; Akgün, Eyup; Harikumar, Kaleeckal G.; Hopson, Jessika; Powers, Michael D.; Lunzer, Mary M.; Miller, Laurence J.; Portoghese, Philip S.

    2009-01-01

    Both mu opioid (MOP)† and type 2 cholecystokinin (CCK2) receptors are present in areas of the central nervous system that are involved in modulation of pain processing. We conducted bioluminescence resonance energy transfer (BRET) studies on COS cells coexpressing MOP and CCK2 receptors to determine whether receptor heterodimerization is involved in such modulation. These studies revealed the absence of constitutive or monovalent ligand-induced heterodimerization. Heterodimerization of MOP and CCK2 receptors therefore is unlikely to be responsible for the opposing effects between morphine and CCK in the CNS. However, association was induced, as indicated by a positive BRET signal, on exposure of the cells to bivalent ligands containing mu-opioid agonist and CCK2 receptor antagonist pharmacophores linked through spacers containing 16 to 22 atoms, but not with a shorter (9-atom) spacer. These studies demonstrate for the first time that an appropriately designed bivalent ligand is capable of inducing association of G protein-coupled receptors. The finding that opioid tolerance studies with these ligands in mice showed no correlation with the BRET data is consistent with the absence of association of MOP and CCK2 receptors in vivo. PMID:19113864

  4. Mu opioid receptor modulation in the nucleus accumbens lowers voluntary wheel running in rats bred for high running motivation.

    PubMed

    Ruegsegger, Gregory N; Toedebusch, Ryan G; Will, Matthew J; Booth, Frank W

    2015-10-01

    The exact role of opioid receptor signaling in mediating voluntary wheel running is unclear. To provide additional understanding, female rats selectively bred for motivation of low (LVR) versus high voluntary running (HVR) behaviors were used. Aims of this study were 1) to identify intrinsic differences in nucleus accumbens (NAc) mRNA expression of opioid-related transcripts and 2) to determine if nightly wheel running is differently influenced by bilateral NAc injections of either the mu-opioid receptor agonist D-Ala2, NMe-Phe4, Glyo5-enkephalin (DAMGO) (0.25, 2.5 μg/side), or its antagonist, naltrexone (5, 10, 20 μg/side). In Experiment 1, intrinsic expression of Oprm1 and Pdyn mRNAs were higher in HVR compared to LVR. Thus, the data imply that line differences in opioidergic mRNA in the NAc could partially contribute to differences in wheel running behavior. In Experiment 2, a significant decrease in running distance was present in HVR rats treated with 2.5 μg DAMGO, or with 10 μg and 20 μg naltrexone between hours 0-1 of the dark cycle. Neither DAMGO nor naltrexone had a significant effect on running distance in LVR rats. Taken together, the data suggest that the high nightly voluntary running distance expressed by HVR rats is mediated by increased endogenous mu-opioid receptor signaling in the NAc, that is disturbed by either agonism or antagonism. In summary, our findings on NAc opioidergic mRNA expression and mu-opioid receptor modulations suggest HVR rats, compared to LVR rats, express higher running levels mediated by an increase in motivation driven, in part, by elevated NAc opioidergic signaling.

  5. The Relationship between naloxone-induced cortisol and mu opioid receptor availability in mesolimbic structures is disrupted in alcohol dependent subjects

    PubMed Central

    Wand, Gary S.; Weerts, Elise M.; Kuwabara, Hiroto; Wong, Dean F.; Xu, Xiaoqiang; McCaul, Mary E.

    2012-01-01

    The mu opioid receptor system is altered in alcohol dependent (AD) subjects. Cortisol responses to opioid receptor antagonists are assumed to impart information about opioid receptor activity. In the present study we examined naloxone-induced cortisol responses in 18 healthy control (HC) and 25 recently detoxified AD subjects and then correlated the cortisol response with mu opioid receptor availability across 15 brain regions using positron emission tomography (PET) and the mu opioid receptor selective ligand [11C] Carfentanil (CFN). On average the AD subjects required twice the dose of naloxone to induce a peak cortisol response compared to the HC subjects. Using the rising slope of the cortisol curve (placebo to peak) as a metric we then went on to examine the relationship between cortisol responses to naloxone and [11C]CFN BPND. There were significant negative relationships between cortisol and [11C]CFN binding potential (BPND) in multiple brain regions of HC subjects. However, cortisol responses did not correlate with [11C]CFN BPND across any brain region in AD subjects. In summary, naloxone imparts information about individual differences in mu opioid receptor availability throughout the mesolimbic system in healthy individuals. However pathways governing the relationship between naloxone-induced cortisol and mu opioid receptor availability are disrupted during early abstinence in AD subjects. PMID:22717196

  6. Characterizing exons 11 and 1 promoters of the mu opioid receptor (Oprm) gene in transgenic mice

    PubMed Central

    Xu, Jin; Xu, Mingming; Pan, Ying-Xian

    2006-01-01

    Background The complexity of the mouse mu opioid receptor (Oprm) gene was demonstrated by the identification of multiple alternatively spliced variants and promoters. Our previous studies have identified a novel promoter, exon 11 (E11) promoter, in the mouse Oprm gene. The E11 promoter is located ~10 kb upstream of the exon 1 (E1) promoter. The E11 promoter controls the expression of nine splice variants in the mouse Oprm gene. Distinguished from the TATA-less E1 promoter, the E11 promoter resembles a typical TATA-containing eukaryote class II promoter. The aim of this study is to further characterize the E11 and E1 promoters in vivo using a transgenic mouse model. Results We constructed a ~20 kb transgenic construct in which a 3.7 kb E11 promoter region and an 8.9 kb E1 promoter region controlled expression of tau/LacZ and tau/GFP reporters, respectively. The construct was used to establish a transgenic mouse line. The expression of the reporter mRNAs, determined by a RT-PCR approach, in the transgenic mice during embryonic development displayed a temporal pattern similar to that of the endogenous promoters. X-gal staining for tau/LacZ reporter and GFP imaging for tau/GFP reporter showed that the transgenic E11 and E1 promoters were widely expressed in various regions of the central nervous system (CNS). The distribution of tau/GFP reporter in the CNS was similar to that of MOR-1-like immunoreactivity using an exon 4-specific antibody. However, differential expression of both promoters was observed in some CNS regions such as the hippocampus and substantia nigra, suggesting that the E11 and E1 promoters were regulated differently in these regions. Conclusion We have generated a transgenic mouse line to study the E11 and E1 promoters in vivo using tau/LacZ and tau/GFP reporters. The reasonable relevance of the transgenic model was demonstrated by the temporal and spatial expression of the transgenes as compared to those of the endogenous transcripts. We believe

  7. Nucleus accumbens mu opioid receptors mediate immediate postictal decrease in locomotion after an amygdaloid kindled seizure in rats.

    PubMed

    Ma, Jingyi; Boyce, Richard; Leung, L Stan

    2010-02-01

    Postictal movement dysfunction is a common symptom in patients with epilepsy. We investigated the involvement of opioid receptors in the nucleus accumbens (NAC) in amygdaloid kindling-induced postictal decrease in locomotion (PDL) in rats. Seizures were induced by daily electrical stimulation of the basolateral amygdala until four consecutive stage 5 seizures were elicited. Locomotion was quantified before and after infusion of an opioid receptor antagonist or saline into the NAC. Whereas PDL was induced after a stage 5 seizure in saline-infused rats, pre-infusion of the mu opioid receptor antagonist H-D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH(2) (CTAP, 5 microg/1 microL/side) into the NAC prevented PDL. Pre-infusion of delta (naltrindole, 30 microg/1 microL/side), kappa (nor-binaltorphimine, 1.8 microg/1 microL/side), or nonselective (naloxone, 10 microg/1 microL/side) opioid receptor antagonists did not block PDL, but late postictal hyperactivity was blocked by naltrindole. None of the antagonists affected amygdaloid evoked afterdischarge duration. It is suggested that mu opioid receptors in the NAC participate in amygdaloid seizure-induced PDL without affecting seizure duration.

  8. Antinociceptive role of oxytocin in the nucleus raphe magnus of rats, an involvement of mu-opioid receptor.

    PubMed

    Wang, Jing-Wen; Lundeberg, Thomas; Yu, Long-Chuan

    2003-10-15

    Recent studies showed that oxytocin plays an important role in nociceptive modulation in the central nervous system. The present study was undertaken to investigate the role of oxytocin in antinociception in the nucleus raphe magnus (NRM) of rats and the possible interaction between oxytocin and the opioid systems. Intra-NRM injection of oxytocin induced dose-dependent increases in hindpaw withdrawal latencies (HWLs) to noxious thermal and mechanical stimulation in rats. The antinociceptive effect of oxytocin was significantly attenuated by subsequent intra-NRM injection of the oxytocin antagonist 1-deamino-2-D-Tyr-(Oet)-4-Thr-8-Orn-oxytocin. Intra-NRM injection of naloxone dose-dependently antagonized the increased HWLs induced by preceding intra-NRM injection of oxytocin, indicating an involvement of opioid receptors in oxytocin-induced antinociception in the NRM of rats. Furthermore, the antinociceptive effect of oxytocin was dose-dependently attenuated by subsequent intra-NRM injection of the mu-opioid antagonist beta-funaltrexamine (beta-FNA), but not by the kappa-opioid antagonist nor-binaltorphimine (nor-BNI) or the delta-opioid antagonist naltrindole. The results demonstrated that oxytocin plays an antinociceptive role in the NRM of rats through activating the oxytocin receptor. Moreover, mu-opioid receptors, not kappa and delta receptors, are involved in the oxytocin-induced antinociception in the NRM of rats.

  9. Characterization of 6α- and 6β-N-Heterocyclic Substituted Naltrexamine Derivatives as Novel Leads to Development of Mu Opioid Receptor Selective Antagonists

    PubMed Central

    2011-01-01

    As important pharmacological probes, highly selective opioid receptor antagonists are essential in opioid receptor structural characterization and opioid agonist functional studies. At present, a nonpeptidyl, highly selective, and reversible mu opioid receptor antagonist is still not available. Among a series of novel naltrexamine derivatives that have been designed and synthesized following molecular modeling studies, two compounds, NAP and NAQ, were identified as leads based on the results of in vitro and in vivo pharmacological assays. Both of them displayed high binding affinity and selectivity to the mu opioid receptor. Further pharmacokinetic and functional characterization revealed that NAP seems to be a peripheral nervous system agent while NAQ seems to be a central one. Such characteristics provide two distinguished potential application routes for these two agents and their derivatives. These results also supported our hypothesis that they may serve as leads to develop more potent and selective antagonists for the mu opioid receptor. PMID:22816021

  10. Deficit in attachment behavior in mice lacking the mu-opioid receptor gene.

    PubMed

    Moles, Anna; Kieffer, Brigitte L; D'Amato, Francesca R

    2004-06-25

    Endogenous opioid binding to micro receptors is hypothesized to mediate natural rewards and has been proposed to be the basis of infant attachment behavior. Here, we report that micro-opioid receptor knockout mouse pups emit fewer ultrasonic vocalizations when removed from their mothers but not when exposed to cold or male mice odors. Moreover these knockout pups do not show a preference toward their mothers' cues and do not show ultrasonic calls potentiation after brief maternal exposure. Results from this study may indicate a molecular mechanism for diseases characterized by deficits in attachment behavior, such as autism or reactive attachment disorder.

  11. Broad spectrum analgesic efficacy of IBNtxA is mediated by exon 11-associated splice variants of the mu-opioid receptor gene

    PubMed Central

    Wieskopf, Jeffrey S.; Pan, Ying-Xian; Marcovitz, Jaclyn; Tuttle, Alexander H.; Majumdar, Susruta; Pidakala, John

    2014-01-01

    Mu-opioids remain vastly important for the treatment of pain, and would represent ideal analgesics if their analgesic effects could be separated from their many side effects. A recently synthesized compound, iodobenzoylnaltrexamide (IBNtxA), acting at 6-transmembrane (6-TM) splice variants of the mu-opioid receptor gene, was shown to have potent analgesic actions against acute, thermal pain accompanied by a vastly improved side-effect profile compared to 7-TM-acting drugs such as morphine. Whether such analgesia can be seen in longer-lasting and non-thermal algesiometric assays is not known. The current study demonstrates potent and efficacious IBNtxA inhibition of a wide variety of assays, including inflammatory and neuropathic hypersensitivity and spontaneous pain. We further demonstrate the dependence of such analgesia on 6-TM mu-opioid receptor variants using isobolographic analysis and the testing of Oprm1 (the mu-opioid receptor gene) exon 11 null mutant mice. Finally, the effect of nerve damage (spared nerve injury) and inflammatory injury (complete Freund’s adjuvant) on expression of mu-opioid receptor variant genes in pain-relevant central nervous system loci was examined, revealing a downregulation of the mMOR-1D splice variant in the dorsal root ganglion after spared nerve injury. These findings are supportive of the potential value of 6-TM-acting drugs as novel analgesics. PMID:25093831

  12. Nanoconjugated NAP as a Potent and Periphery Selective Mu Opioid Receptor Modulator To Treat Opioid-Induced Constipation.

    PubMed

    Xu, Guoyan G; Zolotarskaya, Olga Yu; Williams, Dwight A; Yuan, Yunyun; Selley, Dana E; Dewey, William L; Akbarali, Hamid I; Yang, Hu; Zhang, Yan

    2017-01-12

    Opioids are the mainstay for cancer and noncancer pain management. However, their use is often associated with multiple adverse effects. Among them, the most common and persistent one is probably opioid-induced constipation (OIC). Periphery selective opioid antagonists may alleviate the symptoms of OIC without compromising the analgesic effects of opioids. Recently our laboratories have identified one novel lead compound, 17-cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-[(4'-pyridyl)acetamido]morphinan (NAP), as a peripherally selective mu opioid receptor ligand carrying subnanomolar affinity to the mu opioid receptor and over 100-folds of selectivity over both the delta and kappa opioid receptors, with reasonable oral availability and half-life, and potential to treat OIC. Nanoparticle-based drug delivery systems are now widely considered due to their technological advantages such as good stability, high carrier capacity, low therapeutic side effects, etc. Herein we report nanoparticle supported NAP as a potential candidate for OIC treatment with improved peripheral selectivity over the original lead compound NAP.

  13. In vivo binding of [125I]NH2-carfentanil to mu opioid receptors in mouse brain.

    PubMed

    Tafani, J A; Francés, B; Coulais, Y; Méjean-Galzi, A; Goeldner, M; Hirth, C; Guiraud, R; Zajac, J M

    1994-02-01

    A functionalized derivative of the mu opioid agonist carfentanil was synthesized (NH2-carfentanil) and showed high specific activity when radiolabeled with iodine. [127I]NH2-carfentanil displayed high affinity and pronounced mu-binding selectivity with a delta/mu selectivity ratio of over 1200. The ability of [125I]NH2-carfentanil to interact in vivo with opioid receptors was determined in mouse brain using ex vivo binding techniques. Twenty minutes after intraperitoneal injection, 0.1% of the [125I]NH2-carfentanil injected into the mouse was present in the brain. [125I]NH2-carfentanil specific binding was inhibited by co-injection of naloxone or morphine while naltrindole, a delta-selective antagonist, was unable to displace the bound radioligand. Autoradiographic experiments revealed a heterogeneous distribution of [125I]NH2-carfentanil specific binding sites, maximal binding occurred in areas with high densities of mu receptors. Peripherally administered iodo-NH2-carfentanil selectively labelled central mu opioid receptors in mouse indicating great potential for single photon emission computed tomography studies.

  14. Methylnaltrexone, a new peripherally acting mu-opioid receptor antagonist being evaluated for the treatment of postoperative ileus.

    PubMed

    Kraft, Michael D

    2008-09-01

    Postoperative ileus (POI), a transient impairment of bowel function, is considered an inevitable response after open abdominal surgery. It leads to significant patient morbidity and increased hospital costs and length of stay. The pathophysiology is multifactorial, involving neurogenic, hormonal, inflammatory and pharmacologic mediators. Several treatments have been shown to reduce the duration of POI, and a multimodal approach combining several of these interventions seems to be the most effective treatment option. Various drug therapies have been evaluated for the treatment of POI, although most have not shown any benefit. Peripherally active mu-opioid receptor antagonists are a new class of compounds that selectively block the peripheral (i.e., gastrointestinal [GI]) effects of opioids while preserving centrally mediated analgesia. Recently, alvimopan was approved in the US for the treatment of POI after abdominal surgery with bowel resection. Methylnaltrexone is a peripherally active mu-opioid receptor antagonist that has been shown to antagonize the inhibitory effects of opioids on GI transit without impairing analgesia. Phase II data indicated that methylnaltrexone was effective for improving GI recovery, reducing POI and shortening the time to discharge readiness in patients who underwent segmental colectomy. Two Phase III trials have been completed, and one is underway at present. Preliminary results from the two completed trials indicate that methylnaltrexone was not better than placebo for the primary or secondary outcomes. Further analyses of these data, clinical trial designs and the various dosage forms are necessary to determine the potential role of methylnaltrexone in the treatment of POI.

  15. Changes in mu-opioid receptor expression and function in the mesolimbic system after long-term access to a palatable diet.

    PubMed

    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.

  16. Characterization of the complex morphinan derivative BU72 as a high efficacy, long-lasting mu-opioid receptor agonist.

    PubMed

    Neilan, Claire L; Husbands, Stephen M; Breeden, Simon; Ko, M C Holden; Aceto, Mario D; Lewis, John W; Woods, James H; Traynor, John R

    2004-09-19

    The development of buprenorphine as a treatment for opiate abuse and dependence has drawn attention to opioid ligands that have agonist actions followed by long-lasting antagonist actions. In a search for alternatives to buprenorphine, we discovered a bridged pyrrolidinomorphinan (BU72). In vitro, BU72 displayed high affinity and efficacy for mu-opioid receptors, but was also a partial delta-opioid receptor agonist and a full kappa-opioid receptor agonist. BU72 was a highly potent and long-lasting antinociceptive agent against both thermal and chemical nociception in the mouse and against thermal nociception in the monkey. These effects were prevented by mu-, but not kappa- or delta-, opioid receptor antagonists. Once the agonist effects of BU72 had subsided, the compound acted to attenuate the antinociceptive action of morphine. BU72 is too efficacious for human use but manipulation to reduce efficacy could provide a lead to the development of a treatment for opioid dependence.

  17. The endogenous mu-opioid receptor agonists endomorphins 1 and 2 have novel hypotensive activity in the rabbit.

    PubMed

    Champion, H C; Zadina, J E; Kastin, A J; Hackler, L; Ge, L J; Kadowitz, P J

    1997-06-27

    The endogenous peptides endomorphins 1 and 2 are newly isolated, potent, and selective mu-opioid receptor agonists. In the present study, responses to the endomorphin peptides were investigated in the systemic vascular bed of the rabbit. Endomorphins 1 and 2 induced dose-related decreases in systemic arterial pressure when injected in doses of 1-30 nmol/kg i.v. In terms of relative vasodepressor activity, endomorphins 1 and 2 were similar to the ORL1 receptor ligand, nociceptin (Orphanin FQ), and met-enkephalin in decreasing systemic arterial pressure. Vasodepressor responses to endomorphins 1 and 2 were inhibited by the opioid receptor antagonist, naloxone, in a dose of 2 mg/kg i.v. These results demonstrate that endomorphins 1 and 2 have significant naloxone-sensitive, vasodepressor activity in the rabbit.

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

    PubMed Central

    2012-01-01

    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 (p<0.01). People with schizophrenia who had died as a result of suicide had lower binding than control subjects (p<0.001), whilst people with bipolar disorder (non- suicide) had higher levels of binding (p<0.05). [3 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<0.01). 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. PMID:22925223

  19. Structure Selectivity Relationship Studies of 17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-[(4'-pyridyl)carboxamido]morphinan Derivatives Toward the Development of the Mu Opioid Receptor Antagonists

    PubMed Central

    Yuan, Yunyun; Elbegdorj, Orgil; Chen, Jianyang; Akubathini, Shashidhar K.; Beletskaya, Irina O.; Selley, Dana E.; Zhang, Yan

    2011-01-01

    Mu opioid receptor antagonists have been applied to target a variety of diseases clinically. The current study is designed to explore the structure selectivity relationship (SSR) of 17-cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-[(4'-pyridyl)carboxamido]morphinan (NAP), a lead compound identified as a selective mu opioid receptor antagonist based on the previous study. Among a series of NAP derivatives synthesized, compounds 6 (NMP) and 9 (NGP) maintained comparable binding affinity, selectivity and efficacy to the lead compound. Particularly, the mu opioid receptor selectivity over kappa opioid receptor of NGP was considerably enhanced compared to that of NAP. Overall, the preliminary SSR supported our original hypothesis that an alternate “address” domain may exist in the mu opioid receptor, which favours the ligands carrying a hydrogen bond acceptor and an aromatic system to selectively recognize the mu opioid receptor. PMID:21788135

  20. Structure selectivity relationship studies of 17-cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-[(4'-pyridyl)carboxamido]morphinan derivatives toward the development of the mu opioid receptor antagonists.

    PubMed

    Yuan, Yunyun; Elbegdorj, Orgil; Chen, Jianyang; Akubathini, Shashidhar K; Beletskaya, Irina O; Selley, Dana E; Zhang, Yan

    2011-09-15

    Mu opioid receptor antagonists have been applied to target a variety of diseases clinically. The current study is designed to explore the structure selectivity relationship (SSR) of 17-cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-[(4'-pyridyl)carboxamido]morphinan (NAP), a lead compound identified as a selective mu opioid receptor antagonist based on the previous study. Among a series of NAP derivatives synthesized, compounds 6 (NMP) and 9 (NGP) maintained comparable binding affinity, selectivity and efficacy to the lead compound. Particularly, the mu opioid receptor selectivity over kappa opioid receptor of NGP was considerably enhanced compared to that of NAP. Overall, the preliminary SSR supported our original hypothesis that an alternate 'address' domain may exist in the mu opioid receptor, which favors the ligands carrying a hydrogen bond acceptor and an aromatic system to selectively recognize the mu opioid receptor.

  1. Oxycodone plus ultra-low-dose naltrexone attenuates neuropathic pain and associated mu-opioid receptor-Gs coupling.

    PubMed

    Largent-Milnes, Tally M; Guo, Wenhong; Wang, Hoau-Yan; Burns, Lindsay H; Vanderah, Todd W

    2008-08-01

    Both peripheral nerve injury and chronic opioid treatment can result in hyperalgesia associated with enhanced excitatory neurotransmission at the level of the spinal cord. Chronic opioid administration leads to a shift in mu-opioid receptor (MOR)-G protein coupling from G(i/o) to G(s) that can be prevented by cotreatment with an ultra-low-dose opioid antagonist. In this study, using lumbar spinal cord tissue from rats with L(5)/L(6) spinal nerve ligation (SNL), we demonstrated that SNL injury induces MOR linkage to G(s) in the damaged (ipsilateral) spinal dorsal horn. This MOR-G(s) coupling occurred without changing G(i/o) coupling levels and without changing the expression of MOR or Galpha proteins. Repeated administration of oxycodone alone or in combination with ultra-low-dose naltrexone (NTX) was assessed on the SNL-induced MOR-G(s) coupling as well as on neuropathic pain behavior. Repeated spinal oxycodone exacerbated the SNL-induced MOR-G(s) coupling, whereas ultra-low-dose NTX cotreatment slightly but significantly attenuated this G(s) coupling. Either spinal or oral administration of oxycodone plus ultra-low-dose NTX markedly enhanced the reductions in allodynia and thermal hyperalgesia produced by oxycodone alone and minimized tolerance to these effects. The MOR-G(s) coupling observed in response to SNL may in part contribute to the excitatory neurotransmission in spinal dorsal horn in neuropathic pain states. The antihyperalgesic and antiallodynic effects of oxycodone plus ultra-low-dose NTX (Oxytrex, Pain Therapeutics, Inc., San Mateo, CA) suggest a promising new treatment for neuropathic pain. The current study investigates whether Oxytrex (oxycodone with an ultra-low dose of naltrexone) alleviates mechanical and thermal hypersensitivities in an animal model of neuropathic pain over a period of 7 days, given locally or systemically. In this report, we first describe an injury-induced shift in mu-opioid receptor coupling from G(i/o) to G(s), suggesting

  2. Heterologous Regulation of Mu-Opioid (MOP) Receptor Mobility in the Membrane of SH-SY5Y Cells*

    PubMed Central

    Carayon, Kévin; Moulédous, Lionel; Combedazou, Anne; Mazères, Serge; Haanappel, Evert; Salomé, Laurence; Mollereau, Catherine

    2014-01-01

    The dynamic organization of G protein-coupled receptors in the plasma membrane is suspected of playing a role in their function. The regulation of the diffusion mode of the mu-opioid (MOP) receptor was previously shown to be agonist-specific. Here we investigate the regulation of MOP receptor diffusion by heterologous activation of other G protein-coupled receptors and characterize the dynamic properties of the MOP receptor within the heterodimer MOP/neuropeptide FF (NPFF2) receptor. The data show that the dynamics and signaling of the MOP receptor in SH-SY5Y cells are modified by the activation of α2-adrenergic and NPFF2 receptors, but not by the activation of receptors not described to interact with the opioid receptor. By combining, for the first time, fluorescence recovery after photobleaching at variable radius experiments with bimolecular fluorescence complementation, we show that the MOP/NPFF2 heterodimer adopts a specific diffusion behavior that corresponds to a mix of the dynamic properties of both MOP and NPFF2 receptors. Altogether, the data suggest that heterologous regulation is accompanied by a specific organization of receptors in the membrane. PMID:25183007

  3. Effect of agmatine on spinal nociceptive reflexes: lack of interaction with alpha2-adrenoceptor or mu-opioid receptor mechanisms.

    PubMed

    Bradley, K J; Headley, P M

    1997-07-23

    Agmatine has been tested i.v. in alpha-chloralose anaesthetised rats for its effects on spinal nociceptive reflexes evoked by mechanical and electrical stimuli. Agmatine did not affect reflexes until very high doses (200 mg/kg, i.v.) which also caused complex cardiovascular disturbances. In spinally intact rats agmatine reduced reflexes; it was slightly less potent when there was carrageenan-induced hind paw inflammation. The alpha2-adrenoceptor antagonist atipamezole (80 microg/kg) did not significantly affect these reductions. In spinalised animals, agmatine caused a generalised increase in background firing which in animals with a non-inflamed paw was significantly reduced after atipamezole. There was no significant change in evoked responses once corrected for background activity. In all groups of animals agmatine, when administered at various doses and times prior to the mu-opioid receptor agonist fentanyl, had no effect on the ID50 of fentanyl.

  4. The stereoisomer (+)-naloxone potentiates G-protein coupling and feeding associated with stimulation of mu opioid receptors in the parabrachial nucleus.

    PubMed

    Chaijale, Nayla N; Aloyo, Vincent J; Simansky, Kenny J

    2013-03-01

    Classically, opioids produce their effects by activating Gi-proteins that inhibit adenylate cyclase activity. Previous studies proposed that mu-opioid receptors can also stimulate adenylate cyclase due to an initial transient coupling to Gs-proteins. Treatment with ultra-low doses of the nonselective opioid antagonist (-)-naloxone or its inactive enantiomer (+)-naloxone blocks this excitatory effect and enhances Gi-coupling. Previously we reported that infusion of the mu-opioid receptor agonist [D-Ala2, N-Me-Phe4, Glycinol5]-Enkephalin (DAMGO) into the mu-opioid receptor expressing lateral parabrachial nucleus increases feeding. Pretreatment with (-)-naloxone blocks this effect. We used this parabrachial circuit as a model to assess cellular actions of ultra-low doses of (-)-naloxone and (+)-naloxone in modifying the effects of DAMGO. Our results showed that an ultra-low concentration of (-)-naloxone (0.001 nM) and several concentrations of (+)-naloxone (0.01-10 nM) enhanced DAMGO-stimulated guanosine-5'-0-(γ-thio)-triphosphate incorporation in parabrachial sections in vitro. Further, we analyzed the relevance of these effects in vivo. In the present study, we show that (+)-naloxone can potentiate DAMGO-induced feeding at doses at which (-)-naloxone was an antagonist. These results implicated (+)-naloxone as a novel tool for studying mu-opioid receptor functions and suggest that (+)-naloxone may have therapeutic value to enhance clinical actions of opiate drugs.

  5. Effects of chronic opioid exposure on guinea pig mu opioid receptor in Chinese hamster ovary cells: Comparison with human and rat receptor

    PubMed Central

    Wallisch, Michael; Nelson, Cole S.; Mulvaney, Julia M.; Hernandez, Heather S.; Smith, Sue Ann; Olsen, George D.

    2007-01-01

    Chronic opioid treatment leads to agonist-specific effects at the mu opioid receptor. The molecular mechanisms resulting from chronic opioid exposure include desensitization, internalization and down-regulation of membrane-bound mu opioid receptors (MOP). The purpose of this study was to compare the cellular regulation of guinea pig, human and rat MOP expressed in Chinese hamster ovary (CHO) cells, following exposure to two clinically important opioids, morphine and methadone. MOP expressing CHO cells were treated in culture with methadone or morphine for up to 48 hours. Radioligand diprenorphine and [D-AIa2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO)-stimulated GTPγS binding assays were carried out using paired control and opioid-exposed CHO cells. Methadone induced downregulation of the mu opioid receptor, while morphine induced desensitization of the receptor for all three species. Furthermore, morphine predominantly decreased the potency of DAMGO to stimulate GTPγS binding, whereas methadone primarily reduced its efficacy. Changes in DAMGO potency and efficacy differed among species and depended on the opioid used to treat the cells. Our results showed similarities between guinea pig and human MOP for morphine-induced desensitization, but identified differences between the two for methadone-induced desensitization. In contrast, human and rat MOP differed in response to morphine treatment, but were not distinct in their response to methadone treatment. The guinea pig is an excellent and established animal model to study opioid effects, but its molecular opioid pharmacology has not been investigated thus far. These results can assist in understanding species differences in the effects of opioid ligands activating the mu opioid receptor. PMID:17343833

  6. Neural and Behavioral Effects of a Novel Mu Opioid Receptor Antagonist in Binge-Eating Obese People

    PubMed Central

    Cambridge, Victoria C.; Ziauddeen, Hisham; Nathan, Pradeep J.; Subramaniam, Naresh; Dodds, Chris; Chamberlain, Samuel R.; Koch, Annelize; Maltby, Kay; Skeggs, Andrew L.; Napolitano, Antonella; Farooqi, I. Sadaf; Bullmore, Edward T.; Fletcher, Paul C.

    2013-01-01

    Background Binge eating is associated with obesity and has been conceptualized as “food addiction.” However, this view has received only inconsistent support in humans, and limited evidence relates key neurocircuitry to the disorder. Moreover, relatively few studies have used pharmacologic functional magnetic resonance imaging to probe the underlying basis of altered eating behaviors. Methods In a double-blind, placebo-controlled, parallel group study, we explored the effects of a potent mu-opioid receptor antagonist, GSK1521498, in obese individuals with moderate binge eating. Subjects were tested during a baseline placebo run-in period and retested after 28-days of drug (n = 21) or placebo (n = 21) treatment. Using functional magnetic resonance imaging and behavioral measures, we determined the drug’s effects on brain responses to food images and, separately, on motivation to expend energy to view comparable images. Results Compared with placebo, GSK1521498 was associated with a significant reduction in pallidum/putamen responses to pictures of high-calorie food and a reduction in motivation to view images of high-calorie food. Intriguingly, although motivational responding was reduced, subjective liking for the same images actually increased following drug treatment. Conclusions Stimulus-specific putamen/pallidal responses in obese people with binge eating are sensitive to altered mu-opioid function. This neuromodulation was accompanied by reductions in motivational responding, as measured by grip force, although subjective liking responses to the same stimuli actually increased. As well as providing evidence for a link between the opioid system and food-related behavior in binge-eating obese individuals, these results support a dissociation across measures of motivation and liking associated with food-related stimuli in these individuals. PMID:23245760

  7. Melatonin attenuates the development of antinociceptive tolerance to delta-, but not to mu-opioid receptor agonist in mice.

    PubMed

    Dai, Xu; Cui, Shi-gang; Li, Shi-rong; Chen, Qiang; Wang, Rui

    2007-08-22

    The effects of melatonin (Mel) on the development of tolerance to antinociceptive actions induced by mu- and delta-opioid receptor agonists were determined in male Kunming mice. In the mouse tail-flick tests, selective mu and delta receptor agonists were repeatedly administered to mice supraspinally (intracerebroventricularly, i.c.v.) in the absence or presence of melatonin. Administration of endomorphin-1 (EM-1, a mu-opioid receptor agonist) or deltorphin I (del I, a delta-opioid receptor agonist) twice daily for 4 days produced antinociceptive tolerance compared with vehicle controls. Co-administration with melatonin prevented the development of tolerance to deltorphin I analgesia, and this effect was dose dependent. However, melatonin did not affect the development of antinociceptive tolerance to endomorphin-1. Additionally, the attenuation of deltorphin I tolerance by melatonin was reduced by chronic treatment with luzindole (luz), a selective antagonist on the MT(2) receptor subtype. Taken together, these data suggest that melatonin interferes with the neural mechanisms involved in the development of tolerance to delta-opioid agonist analgesia via its receptor.

  8. delta- and mu-opioid receptor mobilization of intracellular calcium in SH-SY5Y human neuroblastoma cells.

    PubMed Central

    Connor, M.; Henderson, G.

    1996-01-01

    1. In this study we have investigated delta and mu opioid receptor-mediated elevation of intracellular Ca2+ concentration ([Ca2+]i) in the human neuroblastoma cell line, SH-SY5Y. 2. The Ca(2+)-sensitive dye, fura-2, was used to measure [Ca2+]i in confluent monolayers of SH-SY5Y cells. Neither the delta-opioid agonist, DPDPE ([D-Pen2,5]-enkephalin) nor the mu-opioid agonist, DAMGO (Tyr-D-Ala-Gly-N-Me-Phe-Gly-ol enkephalin) elevated [Ca2+]i when applied alone. However, when either DPDPE or DAMGO was applied in the presence of the cholinoceptor agonist, carbachol (100 nM-1 mM) they evoked an elevation of [Ca2+]i above that caused by carbachol alone. 3. In the presence of 1 microM or 100 microM carbachol, DPDPE elevated [Ca2+]i with an EC50 of 10 nM. The elevation of [Ca2+]i was independent of the concentration of carbachol. The EC50 for DAMGO elevating [Ca2+]i in the presence of 1 microM and 100 microM carbachol was 270 nM and 145 nM respectively. 4. The delta-receptor antagonist, naltrindole (30 nM), blocked the elevations of [Ca2+]i by DPDPE (100 nM) without affecting those caused by DAMGO while the mu-receptor antagonist, CTAP (D-Phe-Cys-Tyr-D-Trp-Arg-Pen-Thr-NH2) (100 nM-1 microM) blocked the elevations of [Ca2+]i caused by DAMGO (1 microM) without affecting those caused by DPDPE. 5. Block of carbachol activation of muscarinic receptors with atropine (10 microM) abolished the elevation of [Ca2+]i by the opioids. The nicotinic receptor antagonist, mecamylamine (10 microM), did not affect the elevations of [Ca2+]i caused by opioids in the presence of carbachol. 6. Muscarinic receptor activation, not a rise in [Ca2+]i, was required to reveal the opioid response. The Ca2+ channel activator, maitotoxin (3 ng ml-1), also elevated [Ca2+]i but subsequent application of opioid in the presence of maitotoxin caused no further changes in [Ca2+]i. 7. The elevations of [Ca2+]i by DPDPE and DAMGO were abolished by pretreatment of the cells with pertussis toxin (200 ng ml-1, 16 h

  9. Co-administration of delta- and mu-opioid receptor agonists promotes peripheral opioid receptor function

    PubMed Central

    Schramm, Cicely L.; Honda, Christopher N.

    2010-01-01

    Enhancement of peripheral opioid analgesia following tissue injury or inflammation in animal models is well-documented, but clinical results of peripheral opioid therapy remain inconsistent. Previous studies in the central nervous system have shown that co-administration of μ- and δ-opioid receptor agonists can enhance analgesic outcomes; however, less is known about the functional consequences of opioid receptor interactions in the periphery. The present study examines the effects of intraplantar injection of the μ- and δ-opioid receptor agonists, morphine and deltorphin, alone and in combination on behavioral tests of nociception in naïve rats and on potassium-evoked release of CGRP from sciatic nerves of naïve rats. Neither drug alone affected nociceptive behaviors or CGRP release. Two separate measures of mechanical nociceptive sensitivity remained unchanged after co-administration of the two drugs. In contrast, when deltorphin was co-injected with morphine, dose-dependent and peripherally-restricted increases in paw withdrawal latencies to radiant heat were observed. Similarly, concentration-dependent inhibition of CGRP release was observed when deltorphin and morphine were administered in sequence prior to potassium stimulation. However, no inhibition was observed when morphine was administered prior to deltorphin. All combined opioid effects were blocked by co-application of antagonists. Deltorphin exposure also enhanced the in vivo and in vitro effects of another μ-opioid receptor agonist, DAMGO. Together, these results suggest that under normal conditions, δ-opioid receptor agonists enhance the effect of μ-opioid receptor agonists in the periphery, and local co-administration of δ- and μ-opioid receptor agonists may improve results of peripheral opioid therapy for the treatment of pain. PMID:20970925

  10. Mu-opioid receptor activation in the medial shell of nucleus accumbens promotes alcohol consumption, self-administration and cue-induced reinstatement

    PubMed Central

    Richard, Jocelyn M; Fields, Howard L

    2016-01-01

    Endogenous opioid signaling in ventral cortico-striatal-pallidal circuitry is implicated in elevated alcohol consumption and relapse to alcohol seeking. Mu-opioid receptor activation in the medial shell of the nucleus accumbens (NAc), a region implicated in multiple aspects of reward processing, elevates alcohol consumption while NAc opioid antagonists reduce it. However, the precise nature of the increases in alcohol consumption, and the effects of mu-opioid agonists on alcohol seeking and relapse are not clear. Here, we tested the effects of the mu-opioid agonist [D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO) in rat NAc shell on lick microstructure in a free-drinking test, alcohol seeking during operant self-administration, extinction learning and expression, and cue-reinforced reinstatement of alcohol seeking. DAMGO enhanced the number, but not the size of drinking bouts. DAMGO also enhanced operant alcohol self-administration and cue-induced reinstatement, but did not affect extinction learning or elicit reinstatement in the absence of cues. Our results suggest that mu-opioid agonism in NAc shell elevates alcohol consumption, seeking and conditioned reinforcement primarily by enhancing the incentive motivational properties of alcohol and alcohol-paired cues, rather than by modulating palatability, satiety, or reinforcement. PMID:27089981

  11. mu-opioid receptor-stimulated synthesis of reactive oxygen species is mediated via phospholipase D2.

    PubMed

    Koch, Thomas; Seifert, Anja; Wu, Dai-Fei; Rankovic, Marija; Kraus, Jürgen; Börner, Christine; Brandenburg, Lars-Ove; Schröder, Helmut; Höllt, Volker

    2009-08-01

    We have recently shown that the activation of the rat mu-opioid receptor (MOPr, also termed MOR1) by the mu-agonist [D-Ala(2), Me Phe(4), Glyol(5)]enkephalin (DAMGO) leads to an increase in phospholipase D2 (PLD2) activity and an induction of receptor endocytosis, whereas the agonist morphine which does not induce opioid receptor endocytosis fails to activate PLD2. We report here that MOPr-mediated activation of PLD2 stimulates production of reactive oxygen molecules via NADH/NADPH oxidase. Oxidative stress was measured with the fluorescent probe dichlorodihydrofluorescein diacetate and the role of PLD2 was assessed by the PLD inhibitor D-erythro-sphingosine (sphinganine) and by PLD2-small interfering RNA transfection. To determine whether NADH/NADPH oxidase contributes to opioid-induced production of reactive oxygen species, mu-agonist-stimulated cells were pre-treated with the flavoprotein inhibitor, diphenylene iodonium, or the specific NADPH oxidase inhibitor, apocynin. Our results demonstrate that receptor-internalizing agonists (like DAMGO, beta-endorphin, methadone, piritramide, fentanyl, sufentanil, and etonitazene) strongly induce NADH/NADPH-mediated ROS synthesis via PLD-dependent signaling pathways, whereas agonists that do not induce MOPr endocytosis and PLD2 activation (like morphine, buprenorphine, hydromorphone, and oxycodone) failed to activate ROS synthesis in transfected human embryonic kidney 293 cells. These findings indicate that the agonist-selective PLD2 activation plays a key role in the regulation of NADH/NADPH-mediated ROS formation by opioids.

  12. Association of mu-opioid receptor gene polymorphism A118G with alcohol dependence in a Japanese population.

    PubMed

    Nishizawa, Daisuke; Han, Wenhua; Hasegawa, Junko; Ishida, Takafumi; Numata, Yukio; Sato, Tadahiro; Kawai, Atsuko; Ikeda, Kazutaka

    2006-01-01

    Ethanol is considered to activate the brain reward system by increasing the release of an endogenous opioid receptor ligand, beta-endorphin. The polymorphism A118G in the mu-opioid receptor gene (OPRM1) causes the amino acid change Asn40Asp and has been reported to affect the affinity of the ligand for the receptor. The association of this polymorphism with the vulnerability to alcohol dependence has been studied in many populations, but not yet in Japanese people. In the present study, we compared the frequencies of the polymorphism OPRM1 A118G between patients with alcohol dependence and healthy control subjects living in a Japanese provincial prefecture. We also genotyped a polymorphism, G1510A, in the acetaldehyde dehydrogenase 2 gene (ALDH2), in which the A allele causes poor metabolism of acetaldehyde, a major metabolite of alcohol. Both OPRM1 118G and ALDH2 1510G were significantly associated with alcohol dependence. These results suggest that OPRM1 118G in addition to ALDH2 1510G might be one of the risk factors for alcohol dependence in Japanese people.

  13. Reconstitution of rate brain /mu/ opioid receptors with purified guanine nucleotide-binding regulatory proteins, G/sub i/ and G/sub o/

    SciTech Connect

    Ueda, Hiroshi; Harada, Hitoshi; Nozaki, Masakatsu; Katada, Toshiaki; Ui, Michio; Satoh, Masamichi; Takagi, Hiroshi

    1988-09-01

    Reconstitution of purified /mu/ opioid receptors with purified guanine nucleotide-binding regulatory proteins (G proteins) was investigated. The purified /mu/ opioid receptor (pI 5.6) migrated as a single M/sub r/ 58,000 polypeptide by NaDodSO/sub 4//PAGE, a value identical to that obtained by affinity cross-linking purified /mu/ receptors. When purified /mu/ receptors were reconstituted with purified G/sub i/, the G protein that mediates the inhibition of adenylate cyclase, the displacement of (/sup 3/H)naloxone binding by (D-Ala/sup 2/,MePhe/sup 4/,Gly-ol/sup 5/)enkephalin was increased 215-fold; this increase was abolished by adding 100 /mu/M guanosine 5'-(/gamma/-thio)triphosphate. Similar increases in agonist displacement of (/sup 3/H)naloxone binding (33-fold) and its abolition by guanosine 5'-(/gamma/-thio)triphosphate were observed with G/sub o/, the G protein of unknown function, but not with the v-Ki-ras protein p.21. The stoichiometry was such that the stimulation of 1 mol of /mu/ receptor led to the binding of (/sup 3/H)guanosine 5'-(/beta/,/gamma/-imido)triphosphate to 2.5 mol of G/sub i/ or to 1.37 mol of G/sub o/. These results suggest that the purified /mu/ opioid receptor is functionally coupled to G/sub i/ and G/sub o/ in the reconstituted phospholipid vesicles.

  14. Argon prevents the development of locomotor sensitization to amphetamine and amphetamine-induced changes in mu opioid receptor in the nucleus accumbens.

    PubMed

    David, Hélène N; Dhilly, Martine; Poisnel, Géraldine; Degoulet, Mickael; Meckler, Cédric; Vallée, Nicolas; Blatteau, Jean-Éric; Risso, Jean-Jacques; Lemaire, Marc; Debruyne, Danièle; Abraini, Jacques H

    2014-01-01

    Systemic administration of γ-amino-butyric acid type A (GABA-A) and benzodiazepine receptor agonists has been reported to block the development of locomotor sensitization to amphetamine. Here, we investigated whether the non-anesthetic noble gas argon, shown to possess agonistic properties at these receptors, may block the acquisition of amphetamine-induced locomotor sensitization and mu opioid receptor activation in the nucleus accumbens. Rats were pretreated with saline solution or amphetamine (1 mg/kg) from day 1 to day 3 and then exposed, immediately after injection of amphetamine, to medicinal air or argon at 75 vol% (with the remainder being oxygen). After a 3-day period of withdrawal, rats were challenged with amphetamine on day 7. Rats pretreated with amphetamine and argon had lower locomotor activity (U = 5, P < 0.005) and mu opioid receptor activity in the nucleus accumbens (U = 0, P < 0.001) than rats pretreated with amphetamine and air. In contrast, argon had effect on locomotor and mu receptor activity neither in rats pretreated with saline and challenged with amphetamine (acute amphetamine) nor in rats pretreated and challenged with saline solution (controls). These results indicate that argon inhibits the development of both locomotor sensitization and mu opioid receptor activation induced by repeated administration of amphetamine.

  15. Ligand-Directed Functional Selectivity at the Mu Opioid Receptor Revealed by Label-Free Integrative Pharmacology On-Target

    PubMed Central

    Morse, Megan; Tran, Elizabeth; Sun, Haiyan; Levenson, Robert; Fang, Ye

    2011-01-01

    Development of new opioid drugs that provide analgesia without producing dependence is important for pain treatment. Opioid agonist drugs exert their analgesia effects primarily by acting at the mu opioid receptor (MOR) sites. High-resolution differentiation of opioid ligands is crucial for the development of new lead drug candidates with better tolerance profiles. Here, we use a label-free integrative pharmacology on-target (iPOT) approach to characterize the functional selectivity of a library of known opioid ligands for the MOR. This approach is based on the ability to detect dynamic mass redistribution (DMR) arising from the activation of the MOR in living cells. DMR assays were performed in HEK-MOR cells with and without preconditioning with probe molecules using label-free resonant waveguide grating biosensors, wherein the probe molecules were used to modify the activity of specific signaling proteins downstream the MOR. DMR signals obtained were then translated into high resolution heat maps using similarity analysis based on a numerical matrix of DMR parameters. Our data indicate that the iPOT approach clearly differentiates functional selectivity for distinct MOR signaling pathways among different opioid ligands, thus opening new avenues to discover and quantify the functional selectivity of currently used and novel opioid receptor drugs. PMID:22003401

  16. Morphine-6β-glucuronide has a higher efficacy than morphine as a mu-opioid receptor agonist in the rat locus coeruleus

    PubMed Central

    Osborne, Peregrine B; Chieng, Billy; Christie, MacDonald J

    2000-01-01

    The pharmacological properties of the active morphine metabolite, morphine-6β-D-glucuronide (M6G), and the parent compound were compared in rat locus coeruleus neurons by electrophysiological recording in brain slices.M6G and morphine activated potassium currents in voltage clamped neurons, which were blocked by the opioid receptor antagonist naloxone.Both M6G and morphine behaved as partial agonists that produced maximal responses smaller than the system maximum, which was measured using [Met5]-enkephalin. M6G produced a larger maximal response (78%) than morphine (62%), which we estimated was due to a 2–4 fold difference in the relative efficacy of the agonists.3-O-methoxynaltrexone, which has been reported to behave as a selective antagonist of a M6G preferring receptor, was equally effective at blocking currents produced by M6G and the selective mu-opioid receptor agonist DAMGO.M6G currents were occluded by a prior application of morphine, and were reduced when mu-opioid receptors were desensitized by using [Met5]-enkephalin.Morphine-3β-D-glucuronide did not affect action potential firing or membrane currents in locus coeruleus neurons and had no effect on currents produced by M6G.These results show that the relative efficacy of M6G is higher than morphine in locus coeruleus neurons, contrary to what has been shown using mu-opioid receptors expressed in cell clones. PMID:11090116

  17. Morphine withdrawal syndrome and its prevention with baclofen: Autoradiographic study of mu-opioid receptors in prepubertal male and female mice.

    PubMed

    Diaz, Silvina L; Barros, Virginia G; Antonelli, Marta C; Rubio, Modesto C; Balerio, Graciela N

    2006-08-01

    Although the expression of the morphine (MOR) withdrawal syndrome is more marked in male mice than in females, we have demonstrated that the GABAB agonist baclofen (BAC) is able to attenuate MOR withdrawal signs in either sex. In order to extend these previous observations, the aim of the present study was to evaluate the mu-opioid receptor labeling in various brain areas in mice of either sex, during MOR withdrawal and its prevention with BAC. Prepubertal Swiss-Webster mice were rendered dependent by intraperitonial (i.p.) injection of MOR (2 mg/kg) twice daily for 9 days. On the 10th day, dependent animals received naloxone (NAL; 6 mg/kg, i.p.) 60 min after MOR, and another pool of dependent mice received BAC (2 mg/kg, i.p.) previous to NAL. Thirty minutes after NAL, mice were sacrificed and autoradiography with [3H]-[D-Ala2, N-Me-Phe4, -glycol5] enkephalin (DAMGO) was carried out on mice brains at five different anatomical levels. Autoradiographic mapping showed a significant increase of mu-opioid receptor labeling during MOR withdrawal in nucleus accumbens core (NAcC), caudate putamen (CPu), mediodorsal thalamic nucleus (MDTh), basolateral and basomedial amygdala, and ventral tegmental area vs. respective control groups in male mice. In contrast, opiate receptor labeling was not significantly modified in any of the brain areas studied in withdrawn females. BAC reestablished mu-opioid receptor binding sites during MOR withdrawal only in NAcC of males, and a similar tendency was observed in CPu and MDTh, even when it was not statistically significant. The sexual dimorphism observed in the present study confirms previous reports indicating a greater sensitivity of males in response to MOR pharmacological properties. The present results suggest that the effect of BAC in preventing the expression of MOR withdrawal signs could be related with the ability of BAC to reestablish the mu-opioid receptor labeling in certain brain areas.

  18. Mu-opioid receptor A118G polymorphism in healthy volunteers affects hypothalamic–pituitary–adrenal axis adrenocorticotropic hormone stress response to metyrapone

    PubMed Central

    Ducat, Elizabeth; Ray, Brenda; Bart, Gavin; Umemura, Yoshie; Varon, Jack; Ho, Ann; Kreek, Mary Jeanne

    2013-01-01

    The mu-opioid receptor encoded by the gene OPRM1 plays a primary role in opiate, alcohol, cocaine and nicotine addiction. Studies using opioid antagonists demonstrate that the mu-opioid receptor (MOP-r) also mediates the hypothalamic–pituitary–adrenal (HPA) axis stress response. A common polymorphism in exon one of the MOP-r gene, A118G, has been shown to significantly alter receptor function and MOP-r gene expression; therefore, this variant likely affects HPA-axis responsivity. In the current study, we have investigated whether the presence of the 118AG variant genotype affects HPA axis responsivity to the stressor metyrapone, which transiently blocks glucocorticoid production in the adrenal cortex. Forty-eight normal and healthy volunteers (32 men, 16 women) were studied, among whom nine men and seven women had the 118AG genotype. The 118G allele blunted the adrenocorticotropic hormone (ACTH) response to metyrapone. Although there was no difference in basal levels of ACTH, subjects with the 118AG genotype had a more modest rise and resultant significantly lower ACTH levels than those with the prototype 118AA at the 8-hour time point (P < 0.02). We found no significant difference between genders. These findings suggest a relatively greater tonic inhibition at hypothalamic–pituitary sites through the mu-opioid receptor and relatively less cyclical glucocorticoid inhibition in subjects with the 118G allele. PMID:21507151

  19. Mu-opioid receptor A118G polymorphism in healthy volunteers affects hypothalamic-pituitary-adrenal axis adrenocorticotropic hormone stress response to metyrapone.

    PubMed

    Ducat, Elizabeth; Ray, Brenda; Bart, Gavin; Umemura, Yoshie; Varon, Jack; Ho, Ann; Kreek, Mary Jeanne

    2013-03-01

    The mu-opioid receptor encoded by the gene OPRM1 plays a primary role in opiate, alcohol, cocaine and nicotine addiction. Studies using opioid antagonists demonstrate that the mu-opioid receptor (MOP-r) also mediates the hypothalamic-pituitary-adrenal (HPA) axis stress response. A common polymorphism in exon one of the MOP-r gene, A118G, has been shown to significantly alter receptor function and MOP-r gene expression; therefore, this variant likely affects HPA-axis responsivity. In the current study, we have investigated whether the presence of the 118AG variant genotype affects HPA axis responsivity to the stressor metyrapone, which transiently blocks glucocorticoid production in the adrenal cortex. Forty-eight normal and healthy volunteers (32 men, 16 women) were studied, among whom nine men and seven women had the 118AG genotype. The 118G allele blunted the adrenocorticotropic hormone (ACTH) response to metyrapone. Although there was no difference in basal levels of ACTH, subjects with the 118AG genotype had a more modest rise and resultant significantly lower ACTH levels than those with the prototype 118AA at the 8-hour time point (P < 0.02). We found no significant difference between genders. These findings suggest a relatively greater tonic inhibition at hypothalamic-pituitary sites through the mu-opioid receptor and relatively less cyclical glucocorticoid inhibition in subjects with the 118G allele.

  20. Neurons in the rat arcuate nucleus are hyperpolarized by GABAB and mu-opioid receptor agonists: evidence for convergence at a ligand-gated potassium conductance.

    PubMed

    Loose, M D; Ronnekleiv, O K; Kelly, M J

    1991-12-01

    Both gamma-aminobutyric acid (GABA) and the endogenous opioid peptides have pervasive effects on neuroendocrine function. This study examined the effects of selective activation of GABAB and/or mu-opioid receptors on neurons of the arcuate nucelus (ARC) of the rat hypothalamus using intracellular recording of cells in a hypothalamic slice. Some recorded neurons were filled with biocytin allowing subsequent identification and immunocytochemical evaluation for the presence of beta-endorphin. ARC neurons exhibited a broad array of active and passive conductances. Tyr-D-Ala-Gly-MePhe-Gly-ol (DAGOL), a mu-opioid receptor agonist, inhibited spontaneous firing, hyperpolarized 68% of ARC cells in a dose-dependent manner and increased cell conductance. Baclofen, a GABAB receptor agonist, hyperpolarized all cells tested. The reversal potentials for both the DAGOL- and baclofen-induced currents were near that of a potassium conductance. Maximal activation by either of the agonists blocked the effects of the other agonist. Identified beta-endorphin cells were inhibited by both DAGOL and baclofen. The results of these in vitro studies suggest that GABAB and mu-opioid receptors are coupled to the same set of potassium channels and that these channels directly and powerfully inhibit most ARC cells, including beta-endorphin neurons. We propose that convergence of inhibitory influences at the ligand-gated potassium conductance described here may be an important site of interaction for opioidergic, GABAergic and other putative neurotransmitter systems in the control of neuroendocrine circuits by the ARC.

  1. Exposure to ethanol on prenatal days 19-20 increases ethanol intake and palatability in the infant rat: involvement of kappa and mu opioid receptors.

    PubMed

    Díaz-Cenzano, Elena; Gaztañaga, Mirari; Gabriela Chotro, M

    2014-09-01

    Prenatal exposure to ethanol on gestation Days 19-20, but not 17-18, increases ethanol acceptance in infant rats. This effect seems to be a conditioned response acquired prenatally, mediated by the opioid system, which could be stimulated by ethanol's pharmacological properties (mu-opioid receptors) or by a component of the amniotic fluid from gestation-day 20 (kappa-inducing factor). The latter option was evaluated administering non-ethanol chemosensory stimuli on gestation Days 19-20 and testing postnatal intake and palatability. However, prenatal exposure to anise or vanilla increased neither intake nor palatability of these tastants on postnatal Day 14. In experiment 2, the role of ethanol's pharmacological effect was tested by administering ethanol and selective antagonists of mu and kappa opioid receptors prenatally. Blocking the mu-opioid receptor system completely reversed the effects on intake and palatability, while antagonizing kappa receptors only partially reduced the effects on palatability. This suggests that the pharmacological effect of ethanol on the fetal mu opioid system is the appetitive reinforcer, which induces the prenatally conditioned preference detected in the preweanling period.

  2. Regional differences in mu-opioid receptor-dependent modulation of basal dopamine transmission in rat striatum.

    PubMed

    Campos-Jurado, Y; Martí-Prats, L; Zornoza, T; Polache, A; Granero, L; Cano-Cebrián, M J

    2017-01-18

    The nigrostriatal dopamine system is implicated in the regulation of reward and motor activity. Dopamine (DA) release in dorsal striatum (DS) is controlled by the firing rate of DA neurons in substantia nigra pars compacta. However, influences at terminal level, such as those involving activation of mu opioid receptors (MORs), can play a key role in determining DA levels in striatum. Nonetheless, published data also suggest that the effect of opioid drugs on DA levels may differ depending on the DS subregion analyzed. In this study, in vivo microdialysis in rats was used to explore this regional dependence. Changes in basal DA levels induced by local retrodialysis application of DAMGO (selective MORs agonist) in three different subregions of DS along the rostro-caudal axis were studied. Our results indicate that whereas administration of 10μM DAMGO into the rostral and caudal DS significantly reduced DA levels, in medial DS an increase in DA levels was observed. These data reveal a regional-dependent MOR modulation of DA release in DS, similar to that described in the ventral striatum. Our findings may lead to a better understanding of the nigrostriatal DA system regulation. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  3. Mu opioid receptor in the human endometrium: dynamics of its expression and localization during the menstrual cycle.

    PubMed

    Totorikaguena, Lide; Olabarrieta, Estibaliz; Matorras, Roberto; Alonso, Edurne; Agirregoitia, Ekaitz; Agirregoitia, Naiara

    2017-04-01

    To study the dynamics of the expression and localization of the mu opioid receptor (MOR) in human endometrium throughout the menstrual cycle. Analysis of human endometrial samples from different menstrual cycle phases (menstrual, early/midproliferative, late proliferative/early secretory, midsecretory, and late secretory) by reverse transcription-polymerase chain reaction, Western blot, and immunohistochemistry. Academic research laboratory. Women from the Human Reproduction Unit of the Cruces University Hospital, fulfilling the following criteria: normal uterine vaginal ultrasound; absence of endometriosis, polycystic ovary syndrome, implantation failure, or recurrent miscarriage; and no history of opioid drug use. Endometrial samples of 86 women categorized into groups for the menstrual cycle phases: 12 menstrual, 21 early/midproliferative, 16 late proliferative/early secretory, 17 midsecretory, and 20 late secretory. MOR gene and protein expression and localization in the different compartments of the human endometrium at different stages of the menstrual cycle. The expression of MOR mRNA and protein changed throughout the cycle in human endometrium. MOR expression increased during the proliferative phase and decreased during the secretory one. Lower values were found at menstruation, and maximum values around the time of ovulation. Small variations for each endometrial compartment were found. The presence of MOR in human endometrium and the dynamic changes during the menstrual cycle suggest a possible role for opioids in reproduction events related to the human endometrium or endometriosis. Copyright © 2017 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  4. Naloxone-induced cortisol predicts mu opioid receptor binding potential in specific brain regions of healthy subjects

    PubMed Central

    Wand, Gary S.; Weerts, Elise M.; Kuwabara, Hiroto; Frost, J. James; Xu, Xiaoqiang; McCaul, Mary E.

    2011-01-01

    Investigators have administered the opioid receptor antagonist, naloxone, to interrogate the hypothalamic-pituitary-adrenal (HPA) axis response under the assumption that this technique provides a measure of endogenous opioid activity. However it has never been tested whether provocation of the HPA axis with naloxone provides a surrogate marker for direct measurement of endogenous opioid activity using PET imaging as the gold standard. To test this hypothesis, eighteen healthy subjects underwent a PET scan with the mu-opioid receptor (MOR) selective ligand [11C]carfentanil (CFN). The following day ACTH and cortisol responses were assessed using a technique which allows administration of 5 incremental doses of naloxone (0, 25, 50, 100 and 250 µg/kg) in a single session. Relationships between ACTH and cortisol responses and [11C]CFN binding potential (BPND) were examined in 5 brain regions involved in the regulation of the HPA axis and/or regions with high concentrations of MOR. All subjects mounted graded ACTH and cortisol responses to naloxone administrations. There were significant negative relationships between cortisol response to naloxone and [11C]CFN BPND in ventral striatum, putamen and caudate. When sex and smoking were added as covariates to the model, these correlations were strengthened and there was a significant correlation with the hypothalamus. There were no significant correlations between ACTH and any volumes of interest. The opioid receptor antagonist naloxone is not merely a non-specific pharmacologic activator of the HPA axis; it provides information about individual differences in opioid receptor availability. PMID:21549509

  5. Differential effects of gestational buprenorphine, naloxone, and methadone on mesolimbic mu opioid and ORL1 receptor G protein coupling.

    PubMed

    Hou, Yanning; Tan, Yun; Belcheva, Mariana M; Clark, Amy L; Zahm, Daniel S; Coscia, Carmine J

    2004-07-19

    In addition to its use for heroin addiction pharmacotherapy in general, buprenorphine has advantages in treating maternal heroin abuse. To examine the gestational effects of buprenorphine on opioid receptor signaling, the [(35)S]-GTP gamma S in situ binding induced by the mu agonist [D-Ala(2),MePhe(4),Gly(5)-ol] enkephalin (DAMGO) or the nociceptin/orphanin FQ (N/OFQ) agonist was measured in mesolimbic structures of pup brains from pregnant rats administered with buprenorphine +/- naloxone, naloxone, or methadone by osmotic minipump. Drug- and gender-based changes in DAMGO- and N/OFQ-induced GTP gamma S binding were discovered in mesolimbic regions of dam, P2, and P7 brains. Buprenorphine and/or methadone gestational treatment attenuated DAMGO-induced GTP gamma S binding in some dam and male P2 mesolimbic regions. Methadone diminished DAMGO-induced GTP gamma S binding in almost all monitored brain regions of the dam but had few effects on their N/OFQ-induced GTP gamma S binding. Naloxone used in combination with buprenorphine blocked the inhibition by buprenorphine alone on DAMGO-induced GTP gamma S binding. In contrast to its inhibitory effects on DAMGO-induced GTP gamma S binding, buprenorphine stimulated N/OFQ-induced GTP gamma S binding in male P2 nucleus accumbens and lateral septum. Brain region-dependent gender differences in DAMGO-induced GTP gamma S binding were seen in P2 pups, and males showed greater sensitivity to buprenorphine and methadone than females. Our findings on mu opioid receptor (MOR) GTP-binding regulatory protein (G protein) coupling and its gender dependency are consistent with our earlier studies on mu receptor binding adaptation induced by buprenorphine in dams and neonatal rats after in utero treatment regimens, and they extend the gestational effects of this opiate to mu and N/OFQ receptor functionality.

  6. Preparation and biodistribution in mice of [11C]carfentanil: a radiopharmaceutical for studying brain mu-opioid receptors by positron emission tomography.

    PubMed

    Saji, H; Tsutsumi, D; Magata, Y; Iida, Y; Konishi, J; Yokoyama, A

    1992-02-01

    A potent mu-opioid agonist, [11C]carfentanil, was prepared by the methylation of carfentanil carboxylic acid with [11C]methyl iodide in order to study brain mu-opioid receptors by positron emission tomography. Synthesis (including purification) was completed within 25 min and the radiochemical yield was approximately 40%. The radiochemical purity of the product was more than 99% and its specific activity was 3.7-7.4 GBq/mumol. Biodistribution studies performed in mice after intravenous injection showed a high brain uptake and rapid blood clearance, so a high brain/blood ratio of 1.5-1.8 was found from 5 to 30 min. Regional cerebral distribution studies in the mouse showed a significantly higher uptake of [11C]carfentanil by the thalamus and striatum than by the cerebellum, with the radioactivity in the striatum disappearing more rapidly than that in the thalamus. Treatment with naloxone significantly reduced the uptake of [11C]carfentanil by the thalamus and striatum. These results indicate that [11C]carfentanil binds specifically to brain mu-opioid receptors.

  7. Direct association of Mu-opioid and NMDA glutamate receptors supports their cross-regulation: molecular implications for opioid tolerance.

    PubMed

    Garzón, Javier; Rodríguez-Muñoz, María; Sánchez-Blázquez, Pilar

    2012-09-01

    In the nervous system, the interaction of opioids like morphine and its derivatives, with the G protein-coupled Mu-opioid receptor (MOR) provokes the development of analgesic tolerance, as well as physical dependence. Tolerance implies that increasing doses of the drug are required to achieve the same effect, a phenomenon that contributes significantly to the social problems surrounding recreational opioid abuse. In recent years, our understanding of the mechanisms that control MOR function in the nervous system, and that eventually produce opioid tolerance, has increased greatly. Pharmacological studies have identified a number of signaling proteins involved in morphine-induced tolerance, including the N-methyl-D-aspartate acid glutamate receptor (NMDAR), nitric oxide synthase (NOS), protein kinase C (PKC), protein kinase A (PKA), calcium (Ca²⁺)/calmodulin (CaM)-dependent kinase II (CaMKII), delta-opioid receptor (DOR) and the regulators of G-protein signaling (RGS) proteins. There is general agreement on the critical role of the NMDAR/nNOS/CaMKII pathway in this process, which is supported by the recent demonstration of a physical association between MORs and NMDARs in post-synaptic structures. Indeed, it is feasible that treatments that diminish morphine tolerance may target distinct elements within the same regulatory MOR-NMDAR pathway. Accordingly, we propose a model that incorporates the most relevant signaling components implicated in opioid tolerance in which, certain signals originating from the activated MOR are perceived by the associated NMDAR, which in turn exerts a negative feedback effect on MOR signaling. MOR- and NMDAR-mediated signals work together in a sequential and interconnected manner to ultimately induce MOR desensitization. Future studies of these phenomena should focus on adding further components to this signaling pathway in order to better define the mechanism underlying MOR desensitization in neural cells.

  8. Effect of electromagnetic field on cyclic adenosine monophosphate (cAMP) in a human mu-opioid receptor cell model.

    PubMed

    Ross, Christina L; Teli, Thaleia; Harrison, Benjamin S

    2016-01-01

    During the cell communication process, endogenous and exogenous signaling affect normal as well as pathological developmental conditions. Exogenous influences such as extra-low-frequency electromagnetic field (EMF) have been shown to effect pain and inflammation by modulating G-protein receptors, down-regulating cyclooxygenase-2 activity, and affecting the calcium/calmodulin/nitric oxide pathway. Investigators have reported changes in opioid receptors and second messengers, such as cyclic adenosine monophosphate (cAMP), in opiate tolerance and dependence by showing how repeated exposure to morphine decreases adenylate cyclase activity causing cAMP to return to control levels in the tolerant state, and increase above control levels during withdrawal. Resonance responses to biological systems using exogenous EMF signals suggest that frequency response characteristics of the target can determine the EMF biological response. In our past research we found significant down regulation of inflammatory markers tumor necrosis factor alpha (TNF-α) and nuclear factor kappa B (NFκB) using 5 Hz EMF frequency. In this study cAMP was stimulated in Chinese Hamster Ovary (CHO) cells transfected with human mu-opioid receptors, then exposed to 5 Hz EMF, and outcomes were compared with morphine treatment. Results showed a 23% greater inhibition of cAMP-treating cells with EMF than with morphine. In order to test our results for frequency specific effects, we ran identical experiments using 13 Hz EMF, which produced results similar to controls. This study suggests the use of EMF as a complementary or alternative treatment to morphine that could both reduce pain and enhance patient quality of life without the side-effects of opiates.

  9. G9a inhibits CREB-triggered expression of mu opioid receptor in primary sensory neurons following peripheral nerve injury

    PubMed Central

    Liang, Lingli; Zhao, Jian-Yuan; Gu, Xiyao; Wu, Shaogen; Mo, Kai; Xiong, Ming; Marie Lutz, Brianna; Bekker, Alex

    2016-01-01

    Neuropathic pain, a distressing and debilitating disorder, is still poorly managed in clinic. Opioids, like morphine, remain the mainstay of prescribed medications in the treatment of this disorder, but their analgesic effects are highly unsatisfactory in part due to nerve injury-induced reduction of opioid receptors in the first-order sensory neurons of dorsal root ganglia. G9a is a repressor of gene expression. We found that nerve injury-induced increases in G9a and its catalyzed repressive marker H3K9m2 are responsible for epigenetic silencing of Oprm1, Oprk1, and Oprd1 genes in the injured dorsal root ganglia. Blocking these increases rescued dorsal root ganglia Oprm1, Oprk1, and Oprd1 gene expression and morphine or loperamide analgesia and prevented the development of morphine or loperamide-induced analgesic tolerance under neuropathic pain conditions. Conversely, mimicking these increases reduced the expression of three opioid receptors and promoted the mu opioid receptor-gated release of primary afferent neurotransmitters. Mechanistically, nerve injury-induced increases in the binding activity of G9a and H3K9me2 to the Oprm1 gene were associated with the reduced binding of cyclic AMP response element binding protein to the Oprm1 gene. These findings suggest that G9a participates in the nerve injury-induced reduction of the Oprm1 gene likely through G9a-triggered blockage in the access of cyclic AMP response element binding protein to this gene. PMID:27927796

  10. N-methyl-D-aspartate receptors and large conductance calcium-sensitive potassium channels inhibit the release of opioid peptides that induce mu-opioid receptor internalization in the rat spinal cord.

    PubMed

    Song, B; Marvizón, J C G

    2005-01-01

    Endogenous opioids in the spinal cord play an important role in nociception, but the mechanisms that control their release are poorly understood. To simultaneously detect all opioids able to activate the mu-opioid receptor, we measured mu-opioid receptor internalization in rat spinal cord slices stimulated electrically or chemically to evoke opioid release. Electrical stimulation of the dorsal horn in the presence of peptidase inhibitors produced mu-opioid receptor internalization in half of the mu-opioid receptor neurons. This internalization was rapidly abolished by N-methyl-D-aspartate (IC50=2 microM), and N-methyl-D-aspartate antagonists prevented this effect. mu-Opioid receptor internalization evoked by high K+ or veratridine was also inhibited by N-methyl-D-aspartate receptor activation. N-methyl-D-aspartate did not affect mu-opioid receptor internalization induced by exogenous endomorphins, confirming that the effect of N-methyl-D-aspartate was on opioid release. We hypothesized that this inhibition was mediated by large conductance Ca2+-sensitive K+ channels BK(Ca2+). Indeed, inhibition by N-methyl-D-aspartate was prevented by tetraethylammonium and by the selective BK(Ca2+) blockers paxilline, penitrem A and verruculogen. Paxilline did not increase mu-opioid receptor internalization in the absence of N-methyl-D-aspartate, indicating that it does not produce an increase in opioid release unrelated to the inhibition by N-methyl-d-aspartate. The BK(Ca2+) involved appears to be a subtype with slow association kinetics for iberiotoxin, which was effective only with long incubations. The BK(Ca2+) opener NS-1619 also inhibited the evoked mu-opioid receptor internalization, and iberiotoxin prevented this effect. We concluded that Ca2+ influx through N-methyl-D-aspartate receptors causes the opening of BK(Ca2+) and hyperpolarization in opioid-containing dorsal horn neurons, resulting in the inhibition of opioid release. Since mu-opioid receptors in the dorsal horn

  11. Naloxone's pentapeptide binding site on filamin A blocks Mu opioid receptor-Gs coupling and CREB activation of acute morphine.

    PubMed

    Wang, Hoau-Yan; Burns, Lindsay H

    2009-01-01

    Chronic morphine causes the mu opioid receptor (MOR) to switch its coupling from Gi/o to Gs, resulting in excitatory signaling via both Galphas and its Gbetagamma dimer. Ultra-low-dose naloxone (NLX) prevents this switch and attenuates opioid tolerance and dependence. This protective effect is mediated via a high-affinity interaction of NLX to a pentapeptide region in c-terminal filamin A (FLNA), a scaffolding protein interacting with MOR. In organotypic striatal slice cultures, we now show that acute morphine induces a dose-dependent Go-to-Gs coupling switch at 5 and 15 min that resolves by 1 hr. The acute Gs coupling induced by 100 microM morphine was completely prevented by co-treatment with 100 pM NLX, (+)NLX, or naltrexone (NTX), or their pentapeptide binding site (FLNA(2561-2565)), which we show can act as a decoy for MOR or bind to FLNA itself. All of these co-treatments presumably prevent the MOR-FLNA interaction. Since ultra-low-dose NTX also attenuates the addictive properties of opioids, we assessed striatal cAMP production and CREB phosphorylation at S(133). Correlating with the Gs coupling, acute morphine induced elevated cAMP levels and a several-fold increase in pS(133)CREB that were also completely blocked by NLX, NTX or the FLNA pentapeptide. We propose that acute, robust stimulation of MOR causes an interaction with FLNA that allows an initially transient MOR-Gs coupling, which recovers with receptor recycling but persists when MOR stimulation is repeated or prolonged. The complete prevention of this acute, morphine-induced MOR-Gs coupling by 100 pM NLX/NTX or 10 microM pentapeptide segment of FLNA further elucidates both MOR signaling and the mechanism of action of ultra-low-dose NLX or NTX in attenuating opioid tolerance, dependence and addictive potential.

  12. It's MORe exciting than mu: crosstalk between mu opioid receptors and glutamatergic transmission in the mesolimbic dopamine system.

    PubMed

    Chartoff, Elena H; Connery, Hilary S

    2014-01-01

    Opioids selective for the G protein-coupled mu opioid receptor (MOR) produce potent analgesia and euphoria. Heroin, a synthetic opioid, is considered one of the most addictive substances, and the recent exponential rise in opioid addiction and overdose deaths has made treatment development a national public health priority. Existing medications (methadone, buprenorphine, and naltrexone), when combined with psychosocial therapies, have proven efficacy in reducing aspects of opioid addiction. Unfortunately, these medications have critical limitations including those associated with opioid agonist therapies (e.g., sustained physiological dependence and opioid withdrawal leading to high relapse rates upon discontinuation), non-adherence to daily dosing, and non-renewal of monthly injection with extended-release naltrexone. Furthermore, current medications fail to ameliorate key aspects of addiction such as powerful conditioned associations that trigger relapse (e.g., cues, stress, the drug itself). Thus, there is a need for developing novel treatments that target neural processes corrupted with chronic opioid use. This requires a basic understanding of molecular and cellular mechanisms underlying effects of opioids on synaptic transmission and plasticity within reward-related neural circuits. The focus of this review is to discuss how crosstalk between MOR-associated G protein signaling and glutamatergic neurotransmission leads to immediate and long-term effects on emotional states (e.g., euphoria, depression) and motivated behavior (e.g., drug-seeking, relapse). Our goal is to integrate findings on how opioids modulate synaptic release of glutamate and postsynaptic transmission via α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and N-methyl-D-aspartate receptors in the nucleus accumbens and ventral tegmental area with the clinical (neurobehavioral) progression of opioid dependence, as well as to identify gaps in knowledge that can be addressed in future studies.

  13. Opiate agonist-induced re-distribution of Wntless, a mu-opioid receptor interacting protein, in rat striatal neurons.

    PubMed

    Reyes, B A S; Vakharia, K; Ferraro, T N; Levenson, R; Berrettini, W H; Van Bockstaele, E J

    2012-01-01

    Wntless (WLS), a mu-opioid receptor (MOR) interacting protein, mediates Wnt protein secretion that is critical for neuronal development. We investigated whether MOR agonists induce re-distribution of WLS within rat striatal neurons. Adult male rats received either saline, morphine or [d-Ala2, N-Me-Phe4, Gly-ol5]-enkephalin (DAMGO) directly into the lateral ventricles. Following thirty minutes, brains were extracted and tissue sections were processed for immunogold silver detection of WLS. In saline-treated rats, WLS was distributed along the plasma membrane and within the cytoplasmic compartment of striatal dendrites as previously described. The ratio of cytoplasmic to total dendritic WLS labeling was 0.70±0.03 in saline-treated striatal tissue. Morphine treatment decreased this ratio to 0.48±0.03 indicating a shift of WLS from the intracellular compartment to the plasma membrane. However, following DAMGO treatment, the ratio was 0.85±0.05 indicating a greater distribution of WLS intracellularly. The difference in the re-distribution of the WLS following different agonist exposure may be related to DAMGO's well known ability to induce internalization of MOR in contrast to morphine, which is less effective in producing receptor internalization. Furthermore, these data are consistent with our hypothesis that MOR agonists promote dimerization of WLS and MOR, thereby preventing WLS from mediating Wnt secretion. In summary, our findings indicate differential agonist-induced trafficking of WLS in striatal neurons following distinct agonist exposure. Adaptations in WLS trafficking may represent a novel pharmacological target in the treatment of opiate addiction and/or pain.

  14. Mu opioid receptor up-regulation and participation in excitability of hippocampal pyramidal cell electrophysiology

    SciTech Connect

    Moudy, A.M.

    1988-01-01

    Chronic administration of opiate antagonists to rats results in up-regulation of their brain opioid receptors. Using subcellular fractionation techniques, brain opioid receptors were resolved into two membrane populations, one associated with synaptic plasma membranes (SPM) and the other enriched in smooth endoplasmic reticulum and Golgi (microsomes). This study addressed in part the question of whether an antagonist induces up-regulation uniformly in these two populations. Rats were administered naltrexone by subcutaneously implanted osmotic minipumps. Forebrain mu receptor levels were determined by homologous displacement of ({sup 3}H)D-ala{sup 2}-mePhe{sup 4}-gly-ol{sup 5}-enkephalin (DAGO) followed by computer estimation of binding parameters. Receptor levels in crude membranes rose 77% after treatment. Microsomes displayed a 92% increase, a two-fold greater change than in SPMs (51%). These results establish that naltrexone induces up-regulation of both membrane populations; and that microsomal and SPM receptors represent discrete populations of intracellular and cell surface sites, respectively. Binding experiments on isolated hippocampi also demonstrated up-regulation (71%) of mu receptors. To demonstrate up-regulation of opioid receptors electrophysiologically, hippocampal slices were prepared from rats which had been chronically treated with naltrexone. After superfusion with DAGO, these slices showed a 42% greater population spike output than controls in response to the same EPSP input. Hippocampi from animals treated for two weeks showed an additional increase in sensitivity. The results support a disinhibitory role for opioids in pyramidal cell hyper-excitability. More importantly, they demonstrate a significant physiological correlate to opioid receptor up-regulation.

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

    PubMed

    Lam, Hoa; Maga, Matthew; Pradhan, Amynah; Evans, Christopher J; Maidment, Nigel T; Hales, Tim G; Walwyn, Wendy

    2011-04-12

    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.

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

    PubMed Central

    2011-01-01

    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. PMID:21486473

  17. A structural feature of the non-peptide ligand interactions with mice mu-opioid receptors.

    PubMed

    Noori, Hamid R; Mucksch, Christian; Urbassek, Herbert M

    2014-01-01

    By binding to and activating the G-protein coupled μ-, κ- and δ-opioid receptors in the central nervous system, opiates are known to induce analgesic and sedative effects. In particular, non-peptide opioid ligands are often used in clinical applications to induce these therapeutically beneficial effects, due to their superior pharmacokinetics and bioavailability in comparison to endogenous neuropeptides. However, since opioid alkaloids are highly addictive substances, it is necessary to understand the exact mechanisms of their actions, specifically the ligand-binding properties of the target receptors, in order to safely apply opiates for therapeutic purposes. Using an in silico molecular docking approach (AutoDock Vina) combined with two-step cluster analysis, we have computationally obtained the docking scores and the ligand-binding pockets of twelve representative non-peptide nonendogenous agonists and antagonists at the crystallographically identified μ-opioid receptor. Our study predicts the existence of two main binding sites that are congruently present in all opioid receptor types. Interestingly, in terms of the agonist or antagonist properties of the substances on the receptors, the clustering analysis suggests a relationship with the position of the ligand-binding pockets, particularly its depth within the receptor structure. Furthermore, the binding affinity of the substances is directly correlated to the proximity of the binding pockets to the extracellular space. In conclusion, the results provide further insights into the structural features of the functional pharmacology of opioid receptors, suggesting the importance of the binding position of non-peptide agonists and antagonists- specifically the distance and the level of exposure to the extracellular space- to their dissociation kinetics and subsequent potency.

  18. The mu opioid receptor A118G gene polymorphism moderates effects of trait anger-out on acute pain sensitivity.

    PubMed

    Bruehl, Stephen; Chung, Ok Y; Burns, John W

    2008-10-15

    Both trait anger-in (managing anger through suppression) and anger-out (managing anger through direct expression) are related to pain responsiveness, but only anger-out effects involve opioid mechanisms. Preliminary work suggested that the effects of anger-out on postoperative analgesic requirements were moderated by the A118G single nucleotide polymorphism of the mu opioid receptor gene. This study further explored these potential genotypexphenotype interactions as they impact acute pain sensitivity. Genetic samples and measures of anger-in and anger-out were obtained in 87 subjects (from three studies) who participated in controlled laboratory acute pain tasks (ischemic, finger pressure, thermal). McGill Pain Questionnaire (MPQ) Sensory and Affective ratings for each pain task were standardized within studies, aggregated across pain tasks, and combined for analyses. Significant anger-outxA118G interactions were observed (p's<.05). Simple effects tests for both pain measures revealed that whereas anger-out was nonsignificantly hyperalgesic in subjects homozygous for the wild-type allele, anger-out was significantly hypoalgesic in those with the variant G allele (p's<.05). For the MPQ-Affective measure, this interaction arose both from low pain sensitivity in high anger-out subjects with the G allele and heightened pain sensitivity in low anger-out subjects with the G allele relative to responses in homozygous wild-type subjects. No genetic moderation was observed for anger-in, although significant main effects on MPQ-Affective ratings were noted (p<.005). Anger-in main effects were due to overlap with negative affect, but anger-outxA118G interactions were not, suggesting unique effects of expressive anger regulation. Results support opioid-related genotypexphenotype interactions involving trait anger-out.

  19. The Mu Opioid Receptor A118G Gene Polymorphism Moderates Effects of Trait Anger-Out on Acute Pain Sensitivity

    PubMed Central

    Bruehl, Stephen; Chung, Ok Y.; Burns, John W.

    2008-01-01

    Both trait anger-in (managing anger through suppression) and anger-out (managing anger through direct expression) are related to pain responsiveness, but only anger-out effects involve opioid mechanisms. Preliminary work suggested the effects of anger-out on post-operative analgesic requirements were moderated by the A118G single nucleotide polymorphism of the mu opioid receptor gene. This study further explored these potential genotype X phenotype interactions as they impact acute pain sensitivity. Genetic samples and measures of anger-in and anger-out were obtained in 87 subjects (from three studies) who participated in controlled laboratory acute pain tasks (ischemic, finger pressure, thermal). McGill Pain Questionnaire (MPQ) Sensory and Affective ratings for each pain task were standardized within studies, aggregated across pain tasks, and combined for analyses. Significant anger-out X A118G interactions were observed (p’s<.05). Simple effects tests for both pain measures revealed that whereas anger-out was nonsignificantly hyperalgesic in subjects homozygous for the wild-type allele, anger-out was significantly hypoalgesic in those with the variant G allele (p’s<.05). For the MPQ-Affective measure, this interaction arose both from low pain sensitivity in high anger-out subjects with the G allele and heightened pain sensitivity in low anger-out subjects with the G allele relative to responses in homozygous wild-type subjects. No genetic moderation was observed for anger-in, although significant main effects on MPQ-Affective ratings were noted (p<.005). Anger-in main effects were due to overlap with negative affect, but anger-out X A118G interactions were not, suggesting unique effects of expressive anger regulation. Results support opioid-related genotype X phenotype interactions involving trait anger-out. PMID:18579306

  20. Functional mu opioid receptors are expressed in cholinergic interneurons of the rat dorsal striatum: territorial specificity and diurnal variation.

    PubMed

    Jabourian, Maritza; Venance, Laurent; Bourgoin, Sylvie; Ozon, Sylvie; Pérez, Sylvie; Godeheu, Gérard; Glowinski, Jacques; Kemel, Marie-Louise

    2005-06-01

    Striatal cholinergic interneurons play a crucial role in the control of movement as well as in motivational and learning aspects of behaviour. Neuropeptides regulate striatal cholinergic transmission and particularly activation of mu opioid receptor (MOR) inhibits acetylcholine (ACh) release in the dorsal striatum. In the present study we investigated whether this cholinergic transmission could be modulated by an enkephalin/MOR direct process. We show that mRNA and protein of MORs are expressed by cholinergic interneurons in the limbic/prefrontal territory but not by those in the sensorimotor territory of the dorsal striatum. These MORs are functional because potassium-evoked release of ACh from striatal synaptosomes was dose-dependently reduced by a selective MOR agonist, this effect being suppressed by a MOR antagonist. The MOR regulation of cholinergic interneurons presented a diurnal variation. (i) The percentage of cholinergic interneurons containing MORs that was 32% at the beginning of the light period (morning) increased to 80% in the afternoon. (ii) The MOR-mediated inhibition of synaptosomal ACh release was higher in the afternoon than in the morning. (iii) While preproenkephalin mRNA levels remained stable, enkephalin tissue content was the lowest (-32%) in the afternoon when the spontaneous (+35%) and the N-methyl-d-aspartate-evoked (+140%) releases of enkephalin (from microsuperfused slices) were the highest. Therefore, by acting on MORs present on cholinergic interneurons, endogenously released enkephalin reduces ACh release. This direct enkephalin/MOR regulation of cholinergic transmission that operates only in the limbic/prefrontal territory of the dorsal striatum might contribute to information processing in fronto-cortico-basal ganglia circuits.

  1. Ethanol/Naltrexone Interactions at the mu-Opioid Receptor. CLSM/FCS Study in Live Cells

    PubMed Central

    D'Addario, Claudio; Rigler, Rudolf; Johansson, Björn; Terenius, Lars

    2008-01-01

    Background Alcoholism is a widespread chronic disorder of complex aetiology with a significant negative impact on the individual and the society. Mechanisms of ethanol action are not sufficiently well understood at the molecular level and the pharmacotherapy of alcoholism is still in its infancy. Our study focuses at the cellular and molecular level on ethanol-induced effects that are mediated through the mu-opioid receptor (MOP) and on the effects of naltrexone, a well-known antagonist at MOP that is used clinically to prevent relapse in alcoholism. Methodology/Principal Findings Advanced fluorescence imaging by Confocal Laser Scanning Microscopy (CLSM) and Fluorescence Correlation Spectroscopy (FCS) are used to study ethanol effects on MOP and plasma membrane lipid dynamics in live PC12 cells. We observed that relevant concentrations of ethanol (10–40 mM) alter MOP mobility and surface density, and affect the dynamics of plasma membrane lipids. Compared to the action of specific ligands at MOP, ethanol-induced effects show complex kinetics and point to a biphasic underlying mechanism. Pretreatment with naloxone or naltrexone considerably mitigates the effects of ethanol. Conclusions/Significance We suggest that ethanol acts by affecting the sorting of MOP at the plasma membrane of PC12 cells. Naltrexone exerts opposite effects on MOP sorting at the plasma membrane, thereby countering the effects of ethanol. Our experimental findings give new insight on MOP-mediated ethanol action at the cellular and molecular level. We suggest a new hypothesis to explain the well established ethanol-induced increase in the activity of the endogenous opioid system. PMID:19104662

  2. The influences of reproductive status and acute stress on the levels of phosphorylated mu opioid receptor immunoreactivity in rat hippocampus.

    PubMed

    Gonzales, Keith L; Chapleau, Jeanette D; Pierce, Joseph P; Kelter, David T; Williams, Tanya J; Torres-Reveron, Annelyn; McEwen, Bruce S; Waters, Elizabeth M; Milner, Teresa A

    2011-08-19

    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.

  3. Mu Opioid Receptors in Gamma-Aminobutyric Acidergic Forebrain Neurons Moderate Motivation for Heroin and Palatable Food.

    PubMed

    Charbogne, Pauline; Gardon, Olivier; Martín-García, Elena; Keyworth, Helen L; Matsui, Aya; Mechling, Anna E; Bienert, Thomas; Nasseef, Taufiq; Robé, Anne; Moquin, Luc; Darcq, Emmanuel; Ben Hamida, Sami; Robledo, Patricia; Matifas, Audrey; Befort, Katia; Gavériaux-Ruff, Claire; Harsan, Laura-Adela; von Elverfeldt, Dominik; Hennig, Jurgen; Gratton, Alain; Kitchen, Ian; Bailey, Alexis; Alvarez, Veronica A; Maldonado, Rafael; Kieffer, Brigitte L

    2017-05-01

    Mu opioid receptors (MORs) are central to pain control, drug reward, and addictive behaviors, but underlying circuit mechanisms have been poorly explored by genetic approaches. Here we investigate the contribution of MORs expressed in gamma-aminobutyric acidergic forebrain neurons to major biological effects of opiates, and also challenge the canonical disinhibition model of opiate reward. We used Dlx5/6-mediated recombination to create conditional Oprm1 mice in gamma-aminobutyric acidergic forebrain neurons. We characterized the genetic deletion by histology, electrophysiology, and microdialysis; probed neuronal activation by c-Fos immunohistochemistry and resting-state functional magnetic resonance imaging; and investigated main behavioral responses to opiates, including motivation to obtain heroin and palatable food. Mutant mice showed MOR transcript deletion mainly in the striatum. In the ventral tegmental area, local MOR activity was intact, and reduced activity was only observed at the level of striatonigral afferents. Heroin-induced neuronal activation was modified at both sites, and whole-brain functional networks were altered in live animals. Morphine analgesia was not altered, and neither was physical dependence to chronic morphine. In contrast, locomotor effects of heroin were abolished, and heroin-induced catalepsy was increased. Place preference to heroin was not modified, but remarkably, motivation to obtain heroin and palatable food was enhanced in operant self-administration procedures. Our study reveals dissociable MOR functions across mesocorticolimbic networks. Thus, beyond a well-established role in reward processing, operating at the level of local ventral tegmental area neurons, MORs also moderate motivation for appetitive stimuli within forebrain circuits that drive motivated behaviors. Copyright © 2017 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

  4. Effects of defeat stress on behavioral flexibility in males and females: modulation by the mu-opioid receptor.

    PubMed

    Laredo, Sarah A; Steinman, Michael Q; Robles, Cindee F; Ferrer, Emilio; Ragen, Benjamin J; Trainor, Brian C

    2015-02-01

    Behavioral flexibility is a component of executive functioning that allows individuals to adapt to changing environmental conditions. Independent lines of research indicate that the mu opioid receptor (MOR) is an important mediator of behavioral flexibility and responses to psychosocial stress. The current study bridges these two lines of research and tests the extent to which social defeat and MOR affect behavioral flexibility and whether sex moderates these effects in California mice (Peromyscus californicus). Males and females assigned to social defeat or control conditions were tested in a Barnes maze. In males, defeat impaired behavioral flexibility but not acquisition. Female performance was unaffected by defeat. MOR binding in defeated and control mice in the orbitofrontal cortex (OFC), striatum and hippocampus was examined via autoradiography. Stressed males had reduced MOR binding in the OFC whereas females were unaffected. The MOR antagonist beta-funaltrexamine (1 mg/kg) impaired performance in males naïve to defeat during the reversal phase but had no effect on females. Finally, we examined the effects of the MOR agonist morphine (2.5 and 5 mg/kg) on stressed mice. As expected, morphine improved behavioral flexibility in stressed males. The stress-induced deficits in behavioral flexibility in males are consistent with a proactive coping strategy, including previous observations that stressed male California mice exhibit strong social approach and aggression. Our pharmacological data suggest that a down-regulation of MOR signaling in males may contribute to sex differences in behavioral flexibility following stress. This is discussed in the framework of coping strategies for individuals with mood disorders.

  5. Mu-opioid receptor (OPRM1) variation, oxytocin levels and maternal attachment in free-ranging rhesus macaques

    PubMed Central

    Higham, James P.; Barr, Christina S.; Hoffman, Christy L.; Mandalaywala, Tara M.; Parker, Karen J.; Maestripieri, Dario

    2014-01-01

    Understanding the genetic and neuroendocrine basis of the mother-infant bond is critical to understanding mammalian affiliation and attachment. Functionally similar non-synonymous mu-opioid receptor (OPRM1) SNPs have arisen and been maintained in humans (A118G) and rhesus macaques (C77G). In rhesus macaques, variation in OPRM1 predicts individual differences in infant affiliation for mothers. Specifically, infants carrying the G allele show increased distress on separation from their mothers, and spend more time with them upon reunion, than individuals homozygous for the C allele. In humans, individuals possessing the G allele report higher perceptions of emotional pain on receiving rejection by social partners. We studied maternal behavior over the course of a year among free-ranging female rhesus macaques on Cayo Santiago, Puerto Rico. We then trapped females and collected blood samples, from which we assessed OPRM1 genotype; we also collected CSF samples from which we measured oxytocin (OT) levels. We show that females possessing the G allele restrain their infants more (i.e. prevent infants from separating from them by pulling them back) than females homozygous for the C allele. Females possessing the G allele also show higher OT levels when lactating, and lower OT levels when neither lactating nor pregnant, than females homozygous for the C allele. This is the first study to demonstrate an association between OPRM1 genotype and maternal attachment for infants, and is one of the first studies of any free-ranging primate population to link functional genetic variation to behavior via potentially related neuroendocrine mechanisms. PMID:21463018

  6. Internalization and down-regulation of mu opioid receptors by endomorphins and morphine in SH-SY5Y human neuroblastoma cells.

    PubMed

    Horner, Kristen A; Zadina, James E

    2004-12-03

    The human neuroblastoma cell line, SH-SY5Y, was used to examine the effects of morphine and the endogenous opioid peptides, endomorphin-1 (EM-1) and endomorphin-2 (EM-2), on mu opioid receptor (MOR) internalization and down-regulation. Treatment for 24 h with EM-1, EM-2 or morphine at 100 nM, 1 microM and 10 microM resulted in a dose-dependent down-regulation of mu receptors. Exposure of cells to 10 microM EM-1 for 2.5, 5 and 24 h resulted in a time-dependent down-regulation of mu receptors. Down-regulation of mu receptors by morphine and EM-1 was blocked by treatment with hypertonic sucrose, consistent with an endocytosis-dependent mechanism. Sensitive cell-surface binding studies with a radiolabeled mu antagonist revealed that morphine was able to induce internalization of mu receptors naturally expressed in SH-SY5Y cells. EM-1 produced a more rapid internalization of mu receptors than morphine, but hypertonic sucrose blocked the internalization induced by each of these agonists. This study demonstrates that, like morphine, the endomorphins down-regulate mu opioid receptors in a dose- and time-dependent manner. This study also demonstrates that morphine, as well as EM-1, can induce rapid, endocytosis-dependent internalization of mu opioid receptors in SH-SY5Y cells. These results may help elucidate the ability of mu agonists to regulate the number and responsiveness of their receptors.

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

    PubMed Central

    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

  8. Loss of the mu opioid receptor on different genetic backgrounds leads to increased bromodeoxyuridine labeling in the dentate gyrus only after repeated injection.

    PubMed

    Cominski, T P; Turchin, C E; Hsu, M S; Ansonoff, M A; Pintar, J E

    2012-03-29

    The endogenous opioid system is involved in various physiological processes, including neurogenesis in the dentate gyrus (DG) of the hippocampus. In the current study, we investigated the role of the mu opioid receptor (MOR-1) on DG neurogenesis and measured glucocorticoid levels following several injection paradigms to supplement the neurogenesis experiments. MOR-1 knockout (KO) mice on C57BL/6 and 129S6 backgrounds were injected with bromodeoxyuridine (BrdU) using either a single injection or two different repeated injection protocols and then sacrificed at different time points. The total number of BrdU and proliferating cell nuclear antigen (PCNA) positive cells in the DG is significantly increased in MOR-1 KO mice compared with wild type (WT) on both strains after repeated injection, but not after a single injection. Plasma corticosterone (CORT) levels increased similarly in MOR-1 KO and WT mice following both single and repeated injection, indicating that the stress response is activated following any injection protocol, but that the mechanism responsible for the increase in BrdU labeling in MOR-1 KO mice is CORT-level independent. Finally, WT 129S6 mice, independent of genotype, showed higher levels of plasma CORT compared with WT C57BL/6 mice in both noninjected controls and following injection at two separate time points; these levels were inversely correlated with low numbers of BrdU cells in the DG in 129S6 mice compared with C57BL/6 mice. In summary, these data demonstrate that loss of MOR-1 increases BrdU labeling in the DG independent of CORT levels, but only following a repeated injection, illustrating the capability of injection paradigms to influence cell-proliferative responses in a genotype-dependent manner.

  9. Role of the mu opioid receptor in opioid modulation of immune function

    PubMed Central

    Ninković, Jana; Roy, Sabita

    2014-01-01

    SUMMARY Endogenous opioids are synthesized in vivo in order to modulate pain mechanisms and inflammatory pathways. Endogenous and exogenous opioids mediate analgesia in response to painful stimuli by binding to opioid receptors on neuronal cells. However, wide distribution of opioid receptors on tissues and organ systems outside the CNS, such as the cells of the immune system, indicate that opioids are capable of exerting additional effects in the periphery, such as immunomodulation. The increased prevalence of infections in opioid abusers based epidemiological studies further highlights the immunosuppressive effects of opioids. In spite of their many debilitating side effects, prescription opioids remain a gold standard for treatment of chronic pain. Therefore, given the prevalence of opioid use and abuse, opioid mediated immune suppression presents a serious concern in our society today. It is imperative to understand the mechanisms by which exogenous opioids modulate immune processes. In this review we will discuss the role of opioid receptors and their ligands in mediating immune suppressive functions. We will summarize recent studies on direct and indirect opioid modulation of the cells of the immune system as well as the role of opioids in exacerbation of certain disease states. PMID:22170499

  10. "Top-Down" Mu-Opioid System Function in Humans: Mu-Opioid Receptors in Ventrolateral Prefrontal Cortex Mediate the Relationship Between Hedonic Tone and Executive Function in Major Depressive Disorder.

    PubMed

    Light, Sharee N; Bieliauskas, Linas A; Zubieta, Jon-Kar

    2017-01-01

    Cognitive dysfunction and anhedonia, the reduced ability to experience pleasure, are commonly comorbid symptoms that are persistent following successful resolution of negative affect in major depressive disorder (MDD). Little is known about whether they share common etiology. In the present study, the relationship between ventrolateral prefrontal cortex (VLPFC) activity, cognitive dysfunction (i.e., executive dysfunction), and positive emotionality was investigated in conjunction with mu-opioid neurotransmission in a sample of 39 MDD patients. Results suggest that increased endogenous mu-opioid tone in the VLPFC mediates the relationship between increased trait positive emotionality and more efficient executive functioning.

  11. Neonatal administration of thimerosal causes persistent changes in mu opioid receptors in the rat brain.

    PubMed

    Olczak, Mieszko; Duszczyk, Michalina; Mierzejewski, Pawel; Bobrowicz, Teresa; Majewska, Maria Dorota

    2010-11-01

    Thimerosal added to some pediatric vaccines is suspected in pathogenesis of several neurodevelopmental disorders. Our previous study showed that thimerosal administered to suckling rats causes persistent, endogenous opioid-mediated hypoalgesia. Here we examined, using immunohistochemical staining technique, the density of μ-opioid receptors (MORs) in the brains of rats, which in the second postnatal week received four i.m. injections of thimerosal at doses 12, 240, 1,440 or 3,000 μg Hg/kg. The periaqueductal gray, caudate putamen and hippocampus were examined. Thimerosal administration caused dose-dependent statistically significant increase in MOR densities in the periaqueductal gray and caudate putamen, but decrease in the dentate gyrus, where it was accompanied by the presence of degenerating neurons and loss of synaptic vesicle marker (synaptophysin). These data document that exposure to thimerosal during early postnatal life produces lasting alterations in the densities of brain opioid receptors along with other neuropathological changes, which may disturb brain development.

  12. Mu opioid receptor agonist DAMGO-induced suppression of saccharin intake in Lewis and Fischer rats.

    PubMed

    Liu, Chuang; Sue Grigson, Patricia

    2005-12-07

    Rats suppress intake of a saccharin cue when paired with a drug of abuse such as morphine or cocaine. Relative to Lewis rats, Fischer rats exhibit greater avoidance of a saccharin cue following saccharin-morphine pairings. The present study used the mu agonist, [D-Ala2,N-MePhe4,Gly-ol5]enkephalin (DAMGO), to test whether strain differences in sensitivity of the mu receptor contribute to this effect. Water-deprived Lewis and Fischer rats were given 5 min access to 0.15% saccharin followed by an icv injection of either DAMGO (0.5 microg/1 microl/rat) or an equal volume of saline. There were six taste-drug pairings occurring at 48 h intervals. The results showed that, relative to the saline treated controls, all rats reduced intake of the saccharin cue following saccharin-DAMGO pairings. No differences occurred between strains. These data suggest that greater morphine-induced suppression of saccharin intake by the Fischer rats is not likely mediated by differences in sensitivity of the mu receptor. Other mechanisms are implicated.

  13. A role for the mu opioid receptor in the antidepressant effects of buprenorphine.

    PubMed

    Robinson, Shivon A; Erickson, Rebecca L; Browne, Caroline A; Lucki, Irwin

    2017-02-15

    Buprenorphine (BPN), a mixed opioid drug with high affinity for mu (MOR) and kappa (KOR) opioid receptors, has been shown to produce behavioral responses in rodents that are similar to those of antidepressant and anxiolytic drugs. Although recent studies have identified KORs as a primary mediator of BPN's effects in rodent models of depressive-like behavior, the role of MORs in BPN's behavioral effects has not been as well explored. The current studies investigated the role of MORs in mediating conditioned approach behavior in the novelty-induced hypophagia (NIH) test, a behavioral measure previously shown to be sensitive to chronic treatment with antidepressant drugs. The effects of BPN were evaluated in the NIH test 24h post-administration in mice with genetic deletion of the MOR (Oprm1(-/-)) or KOR (Oprk1(-/-)), or after pharmacological blockade with the non-selective opioid receptor antagonist naltrexone and selective MOR antagonist cyprodime. We found that behavioral responses to BPN in the NIH test were blocked in Oprm1(-/-) mice, but not in Oprk1(-/-) mice. Both cyprodime and naltrexone significantly reduced approach latency at doses experimentally proven to antagonize the MOR. In contrast the selective MOR agonist morphine and the selective KOR antagonist nor-BNI were both ineffective. Moreover, antinociceptive studies revealed persistence of the MOR antagonist properties of BPN at 24h post-administration, the period of behavioral reactivity. These data support modulation of MOR activity as a key component of BPN's antidepressant-like effects in the NIH paradigm.

  14. A novel alternatively spliced isoform of the mu-opioid receptor: functional antagonism

    PubMed Central

    2010-01-01

    Background Opioids are the most widely used analgesics for the treatment of clinical pain. They produce their therapeutic effects by binding to μ-opioid receptors (MORs), which are 7 transmembrane domain (7TM) G-protein-coupled receptors (GPCRs), and inhibiting cellular activity. However, the analgesic efficacy of opioids is compromised by side-effects such as analgesic tolerance, dependence and opioid-induced hyperalgesia (OIH). In contrast to opioid analgesia these side effects are associated with cellular excitation. Several hypotheses have been advanced to explain these phenomena, yet the molecular mechanisms underlying tolerance and OIH remain poorly understood. Results We recently discovered a new human alternatively spliced isoform of MOR (MOR1K) that is missing the N-terminal extracellular and first transmembrane domains, resulting in a 6TM GPCR variant. To characterize the pattern of cellular transduction pathways activated by this human MOR1K isoform, we conducted a series of pharmacological and molecular experiments. Results show that stimulation of MOR1K with morphine leads to excitatory cellular effects. In contrast to stimulation of MOR1, stimulation of MOR1K leads to increased Ca2+ levels as well as increased nitric oxide (NO) release. Immunoprecipitation experiments further reveal that unlike MOR1, which couples to the inhibitory Gαi/o complex, MOR1K couples to the stimulatory Gαs complex. Conclusion The major MOR1 and the alternative MOR1K isoforms mediate opposite cellular effects in response to morphine, with MOR1K driving excitatory processes. These findings warrant further investigations that examine animal and human MORK1 expression and function following chronic exposure to opioids, which may identify MOR1K as a novel target for the development of new clinically effective classes of opioids that have high analgesic efficacy with diminished ability to produce tolerance, OIH, and other unwanted side-effects. PMID:20525224

  15. Decreased motivation to eat in mu-opioid receptor-deficient mice.

    PubMed

    Papaleo, Francesco; Kieffer, Brigitte L; Tabarin, Antoine; Contarino, Angelo

    2007-06-01

    Altered motivational processes might participate to the physiopathology of eating-related disorders. The endogenous opioid system is thought to mediate the hedonic properties of food intake. To assess the role for the micro-opioid receptor (MOR) pathway in the motivational properties of food intake, in the present study we tested wild-type and MOR-deficient mice (MOR-/-) in a nose-poke operant paradigm for chow or sucrose pellets. To avoid confounding factors linked to food restriction/deprivation experience, mice were always provided with food ad libitum. Although less MOR-/- than wild-type mice initiated operant behaviour, under a fixed ratio-1 (FR-1) reinforcement schedule the two genotypes showed similar patterns of food-driven nose-poking, indicating preserved cognitive abilities in MOR-deficient mice. However, during FR-3 and progressive ratio (PR) reinforcement experiments, MOR-/- mice showed lower levels of nose-poking for either chow or sucrose pellets than wild-type mice, indicating a crucial role for the MOR pathway in the motivational properties of food intake. Moreover, under the PR reinforcement schedule mice nose-poking for sucrose pellets showed higher genotype-independent breakpoint levels than mice working for chow pellets, indicating that the MOR pathway is not essential for hedonic processing of palatable food intake. Finally, MOR-/- mice did not differ from wild-type mice in the rate of operant responding extinction, further supporting the notion of unaltered cognitive abilities in the MOR-deficient mice. The present findings strongly indicate that the MOR pathway mediates the motivational properties of food intake, but it is not essential for hedonic processing of ingestive behaviour.

  16. Cortisol Stress Response in Men and Women Modulated Differentially by the Mu-Opioid Receptor Gene Polymorphism OPRM1 A118G

    PubMed Central

    Lovallo, William R; Enoch, Mary-Anne; Acheson, Ashley; Cohoon, Andrew J; Sorocco, Kristen H; Hodgkinson, Colin A; Vincent, Andrea S; Glahn, David C; Goldman, David

    2015-01-01

    Differences in stress reactivity may affect long-term health outcomes, but there is little information on how these differences arise. The stress axis is regulated by, in part, the endogenous opioid, beta-endorphin, acting on mu-opioid receptors. Persons carrying one or two copies of the G allele of the mu-opioid receptor gene (OPRM1 A118G) may have higher receptor binding for beta-endorphin compared with AA homozygotes that may contribute to individual differences in cortisol reactivity to stress, leading to a relative blunting of cortisol stress reactivity in G allele genotypes. We measured cortisol in 251 young adults (69 GA/GG vs 182 AA genotypes) exposed to mental arithmetic plus public speaking stress relative to a resting control day. Women had smaller cortisol responses than men (F=10.2, p=0.002), and women with GA or GG genotypes (N=39) had an absence of cortisol response relative to AA carriers (N=110) (F=18.4, p<0.0001). Male genotypes had no such difference in response (F=0.29). Cortisol response following mu-opioid receptor blockade using naltrexone in 119 of these subjects unmasked a greater tonic opioid inhibition of cortisol secretion in women (N=64), consistent with their blunted stress reactivity. Compared with men, women may have cortisol stress responses that are more heavily regulated by endogenous opioid mechanisms, and the OPRM1 GA/GG genotypes may affect females differentially relative to males. Diminished cortisol responses to stress may have consequences for health behaviors in women with GA/GG genotypes. PMID:25881118

  17. Cortisol Stress Response in Men and Women Modulated Differentially by the Mu-Opioid Receptor Gene Polymorphism OPRM1 A118G.

    PubMed

    Lovallo, William R; Enoch, Mary-Anne; Acheson, Ashley; Cohoon, Andrew J; Sorocco, Kristen H; Hodgkinson, Colin A; Vincent, Andrea S; Glahn, David C; Goldman, David

    2015-10-01

    Differences in stress reactivity may affect long-term health outcomes, but there is little information on how these differences arise. The stress axis is regulated by, in part, the endogenous opioid, beta-endorphin, acting on mu-opioid receptors. Persons carrying one or two copies of the G allele of the mu-opioid receptor gene (OPRM1 A118G) may have higher receptor binding for beta-endorphin compared with AA homozygotes that may contribute to individual differences in cortisol reactivity to stress, leading to a relative blunting of cortisol stress reactivity in G allele genotypes. We measured cortisol in 251 young adults (69 GA/GG vs 182 AA genotypes) exposed to mental arithmetic plus public speaking stress relative to a resting control day. Women had smaller cortisol responses than men (F=10.2, p=0.002), and women with GA or GG genotypes (N=39) had an absence of cortisol response relative to AA carriers (N=110) (F=18.4, p<0.0001). Male genotypes had no such difference in response (F=0.29). Cortisol response following mu-opioid receptor blockade using naltrexone in 119 of these subjects unmasked a greater tonic opioid inhibition of cortisol secretion in women (N=64), consistent with their blunted stress reactivity. Compared with men, women may have cortisol stress responses that are more heavily regulated by endogenous opioid mechanisms, and the OPRM1 GA/GG genotypes may affect females differentially relative to males. Diminished cortisol responses to stress may have consequences for health behaviors in women with GA/GG genotypes.

  18. Expression of antinociception in response to a signal for shock is blocked after selective downregulation of mu-opioid receptors in the rostral ventromedial medulla.

    PubMed

    Foo, H; Helmstetter, F J

    2000-03-29

    Prior work has shown that release of endogenous ligands for mu-opioid receptors in the rostral ventromedial medulla (RVM) is critical for the modulation of spinal nociceptive reflexes observed during stress. In the present study, we used antisense oligodeoxynucleotides (AS ODN) to suppress synthesis of mu-opioid receptors in the RVM prior to activating descending antinociceptive systems with a signal for foot shock. Five groups of rats with RVM cannulae were trained with paired or unpaired exposures to white noise (WN) and foot shock. Over several days, they received RVM infusions of an AS ODN probe targeting exon 1 of the cloned MOR-1 receptor, an inactive missense (MS) ODN with the same base composition in which the sequence for four bases was changed, an AS ODN probe targeting exon 4, or saline. Tail-flick latencies (TFLs) were measured before, during, and after presentation of the auditory signal for shock. Rats given paired training and saline injections displayed longer TFLs than saline control rats given unpaired exposures to WN and shock, confirming the ability of the conditional stimuli (CS) to elicit antinociception. Expression of this conditional hypoalgesia (CHA) was attenuated by pretreatment with the AS ODN probe targeting exon 1, but was unaffected by pretreatment with AS ODN probe targeting exon 4 or MS ODN sequence for exon 1. However, pretreatment with the AS ODN probe targeting exon 1 did not affect expression of conditional freezing to other shock-associated cues. Testing of the same animals several days after the ODN injections showed that the attenuating effect on expression of CHA were reversible. These results support the idea that mu-opioid receptors in the RVM are critically involved in mediating expression of hypoalgesia following stress. They also provide further evidence for dissociation in the mechanisms mediating expression of aversive conditional responses.

  19. Neuropeptide FF-sensitive confinement of mu opioid receptor does not involve lipid rafts in SH-SY5Y cells

    SciTech Connect

    Mouledous, Lionel

    2008-08-15

    *: Mu opioid (MOP) receptor activation can be functionally modulated by stimulation of Neuropeptide FF 2 (NPFF{sub 2}) G protein-coupled receptors. Fluorescence recovery after photobleaching experiments have shown that activation of the NPFF{sub 2} receptor dramatically reduces the fraction of MOP receptors confined in microdomains of the plasma membrane of SH-SY5Y neuroblastoma cells. The aim of the present work was to assess if the direct observation of receptor compartmentation by fluorescence techniques in living cells could be related to indirect estimation of receptor partitioning in lipid rafts after biochemical fractionation of the cell. Our results show that MOP receptor distribution in lipid rafts is highly dependent upon the method of purification, questioning the interpretation of previous data regarding MOP receptor compartmentation. Moreover, the NPFF analogue 1DMe does not modify the distribution profile of MOP receptors, clearly demonstrating that membrane fractionation data do not correlate with direct measurement of receptor compartmentation in living cells.

  20. Examining the role of mu opioid receptor endocytosis in the beneficial and side-effects of prolonged opioid use: From a symposium on new concepts in mu-opioid pharmacology

    PubMed Central

    Whistler, Jennifer L.

    2014-01-01

    Opioid drugs remain the gold standard for the treatment of severe pain, both acute/post-surgical and chronic. However, the utility of opioid drugs for the treatment of chronic pain is compromised by the development of analgesic tolerance which, in turn, leads to dose-escalation and increased likelihood of dangerous side effects, including dependence. Consequently, there remains resistance among clinicians and the general population to using opiates for pain management because of risk of “addiction.” These fears are not unwarranted. More than 2.5 million people begin abusing opioid painkillers each year, and prescription opioid abuse is now the second most common type of illegal drug use after marijuana. Some abusers become dependent due to recreational use of prescription painkillers. However, many abusers are among the 40 million people suffering from chronic pain, and developed dependence while using the drugs for legitimate purposes. Both of these trends highlight the need to develop opioid therapeutics with a reduced liability to cause tolerance, dependence and addiction. Identifying the ideal properties of opioid drugs that would retain analgesia but reduce these side-effects has been a goal of my laboratory for more than a decade. During this time, we have proposed the novel hypothesis that opioid drugs that promote desensitization, endocytosis and recycling of the mu-opioid-receptor (MOR) will retain analgesic efficacy, but will have a reduced liability to cause tolerance, dependence and addiction. We have generated substantial data, both pharmacological and genetic to suggest that our hypothesis is a valid one. These data are summarized in this review. PMID:22226706

  1. Activation of Peripheral Mu-Opioid Receptors by Dermorphin [D-Arg2, Lys4] (1-4) amide Leads to Modality-preferred Inhibition of Neuropathic Pain

    PubMed Central

    Tiwari, Vinod; Yang, Fei; He, Shao-Qiu; Shechter, Ronen; Zhang, Chen; Shu, Bin; Zhang, Tong; Tiwari, Vineeta; Wang, Yun; Dong, Xinzhong; Guan, Yun; Raja, Srinivasa N.

    2015-01-01

    Background Opioids have long been regarded as the most effective drugs for treatment of severe acute and chronic pain. Unfortunately, their therapeutic efficacy and clinical utility has been limited because of central and peripheral side effects. Methods To determine the therapeutic value of peripheral mu-opioid receptors as a target for neuropathic pain treatment, we examined the effects of DALDA, a hydrophilic, peripherally acting mu-opioid receptor agonist, in male and female rats with spinal nerve ligation-induced neuropathic pain. We also utilized behavioral, pharmacologic, electrophysiologic, and molecular biologic tools to characterize DALDA's possible mechanisms of action in male rats. Results DALDA, administered subcutaneously, had 70 times greater efficacy for inhibiting thermal (n=8–11/group) than mechanical hypersensitivity (n=6–8/group) in male rats. The pain inhibitory effects of DALDA on mechanical and heat hypersensitivity were abolished in animals pretreated with systemic methylnaltrexone (n=7–9/group), a peripheral mu-opioid receptor antagonist. In spinal wide-dynamic range neurons, systemic DALDA inhibited C-fiber–mediated, but not A-fiber–mediated, response in neuropathic male rats (n=13). In primary sensory neurons, DALDA inhibited the capsaicin-induced [Ca2+] increase more than the β-alanine–induced [Ca2+] increase (n=300); capsaicin and β-alanine activate subpopulations of neurons involved in the signaling of heat and mechanical pain, respectively. DALDA-treated rats (n=5–8/group) did not exhibit motor deficits and locomotor impairment suggesting that it does not induce central side effects. Conclusion These findings suggest that DALDA may represent a potential alternative to current opioid therapy for the treatment of neuropathic pain and is likely to be associated with minimal adverse effects. PMID:26756519

  2. Morphine-induced trafficking of a mu-opioid receptor interacting protein in rat locus coeruleus neurons.

    PubMed

    Jaremko, Kellie M; Thompson, Nicholas L; Reyes, Beverly A S; Jin, Jay; Ebersole, Brittany; Jenney, Christopher B; Grigson, Patricia S; Levenson, Robert; Berrettini, Wade H; Van Bockstaele, Elisabeth J

    2014-04-03

    Opiate addiction is a devastating health problem, with approximately 2million people currently addicted to heroin or non-medical prescription opiates in the United States alone. In neurons, adaptations in cell signaling cascades develop following opioid actions at the mu opioid receptor (MOR). A novel putative target for intervention involves interacting proteins that may regulate trafficking of MOR. Morphine has been shown to induce a re-distribution of a MOR-interacting protein Wntless (WLS, a transport molecule necessary for secretion of neurotrophic Wnt proteins), from cytoplasmic to membrane compartments in rat striatal neurons. Given its opiate-sensitivity and its well-characterized molecular and cellular adaptations to morphine exposure, we investigated the anatomical distribution of WLS and MOR in the rat locus coeruleus (LC)-norepinephrine (NE) system. Dual immunofluorescence microscopy was used to test the hypothesis that WLS is localized to noradrenergic neurons of the LC and that WLS and MOR co-exist in common LC somatodendritic processes, providing an anatomical substrate for their putative interactions. We also hypothesized that morphine would influence WLS distribution in the LC. Rats received saline, morphine or the opiate agonist [d-Ala2, N-Me-Phe4, Gly-ol5]-enkephalin (DAMGO), and tissue sections through the LC were processed for immunogold-silver detection of WLS and MOR. Statistical analysis showed a significant re-distribution of WLS to the plasma membrane following morphine treatment in addition to an increase in the proximity of gold-silver labels for MOR and WLS. Following DAMGO treatment, MOR and WLS were predominantly localized within the cytoplasmic compartment when compared to morphine and control. In a separate cohort of rats, brains were obtained from saline-treated or heroin self-administering male rats for pulldown co-immunoprecipitation studies. Results showed an increased association of WLS and MOR following heroin exposure. As the

  3. Morphine-induced trafficking of a mu-opioid receptor interacting protein in rat locus coeruleus neurons

    PubMed Central

    Jaremko, Kellie M.; Thompson, Nicholas L.; Reyes, Beverly A. S.; Jin, Jay; Ebersole, Brittany; Jenney, Christopher B.; Grigson, Patricia S.; Levenson, Robert; Berrettini, Wade H.; Van Bockstaele, Elisabeth J.

    2014-01-01

    Opiate addiction is a devastating health problem, with approximately 2 million people currently addicted to heroin or non-medical prescription opiates in the United States alone. In neurons, adaptations in cell signaling cascades develop following opioid actions at the mu opioid receptor (MOR). A novel putative target for intervention involves interacting proteins that may regulate trafficking of MOR. Morphine has been shown to induce a re-distribution of a MOR-interacting protein Wntless (WLS, a transport molecule necessary for secretion of neurotrophic Wnt proteins), from cytoplasmic to membrane compartments in rat striatal neurons. Given its opiate-sensitivity and its well-characterized molecular and cellular adaptations to morphine exposure, we investigated the anatomical distribution of WLS and MOR in the rat locus coeruleus (LC)-norepinephrine (NE) system. Dual immunofluorescence microscopy was used to test the hypothesis that WLS is localized to noradrenergic neurons of the LC and that WLS and MOR co-exist in common LC somatodendritic processes, providing an anatomical substrate for their putative interactions. We also hypothesized that morphine would influence WLS distribution in the LC. Rats received saline, morphine or the opiate agonist [D-Ala2, N-Me-Phe4, Gly-ol5]-enkephalin (DAMGO), and tissue sections through the LC were processed for immunogold-silver detection of WLS and MOR. Statistical analysis showed a significant re-distribution of WLS to the plasma membrane following morphine treatment in addition to an increase in the proximity of gold-silver labels for MOR and WLS. Following DAMGO treatment, MOR and WLS were predominantly localized within the cytoplasmic compartment when compared to morphine and control. In a separate cohort of rats, brains were obtained from saline-treated or heroin self-administering male rats for pulldown co-immunoprecipitation studies. Results showed an increased association of WLS and MOR following heroin exposure. As

  4. Opioid Peptidomimetics: Leads for the Design of Bioavailable Mixed Efficacy Mu Opioid Receptor (MOR) Agonist/Delta Opioid Receptor (DOR) Antagonist Ligands

    PubMed Central

    Mosberg, Henry I.; Yeomans, Larisa; Harland, Aubrie A.; Bender, Aaron M.; Sobczyk-Kojiro, Katarzyna; Anand, Jessica P.; Clark, Mary J.; Jutkiewicz, Emily M.; Traynor, John R.

    2013-01-01

    We have previously described opioid peptidomimetic, 1, employing a tetrahydroquinoline scaffold and modeled on a series of cyclic tetrapeptide opioid agonists. We have recently described modifications to these peptides that confer a mu opioid receptor (MOR) agonist, delta opioid receptor (DOR) antagonist profile, which has been shown to reduce the development of tolerance to the analgesic actions of MOR agonists. Several such bifunctional ligands have been reported, but none has been demonstrated to cross the blood brain barrier. Here we describe the transfer of structural features that evoked MOR agonist/DOR antagonist behavior in the cyclic peptides to the tetrahydroquinoline scaffold and show that the resulting peptidomimetics maintain the desired pharmacological profile. Further, the 4R diastereomer of 1 was fully efficacious and approximately equipotent to morphine in the mouse warm water tail withdrawal assay following intraperitoneal administration and thus a promising lead for the development of opioid analgesics with reduced tolerance. PMID:23419026

  5. /sup 125/I-FK 33-824: a selective probe for radioautographic labeling of mu opioid receptors in the brain

    SciTech Connect

    Moyse, E.; Pasquini, F.; Quirion, R.; Beaudet, A.

    1986-03-01

    The selectivity of the Met-enkephalin analog FK 33-824 (FK) for mu opioid receptors has been, over the years, a matter of controversy. We report here pharmacological and radioautographic data demonstrating that at nanomolar concentrations. /sup 125/I-FK interacts exclusively with mu sites. (1) Specific binding of /sup 125/I-FK to rat striatal membranes is totally inhibited by mu- and/or delta-preferring ligands according to monovalent, Michaelian kinetics, with a potency proportional to the affinity of competing drugs for mu receptors. (2) Unlabeled FK competes only at high concentration with the delta-selective ligand 3H-DPLPE and according to the same kinetics as the mu-selective agonist DAGO. (3) /sup 125/I-FK generates the same regional radioautographic labeling pattern as 3H-DAGO. We conclude that when used at nanomolar concentrations /sup 125/I-FK constitutes a selective probe for the radioautographic detection of mu opioid receptors at both light and electron microscopic levels.

  6. Argon blocks the expression of locomotor sensitization to amphetamine through antagonism at the vesicular monoamine transporter-2 and mu-opioid receptor in the nucleus accumbens

    PubMed Central

    David, H N; Dhilly, M; Degoulet, M; Poisnel, G; Meckler, C; Vallée, N; Blatteau, J-É; Risso, J-J; Lemaire, M; Debruyne, D; Abraini, J H

    2015-01-01

    We investigated the effects of the noble gas argon on the expression of locomotor sensitization to amphetamine and amphetamine-induced changes in dopamine release and mu-opioid neurotransmission in the nucleus accumbens. We found (1) argon blocked the increase in carrier-mediated dopamine release induced by amphetamine in brain slices, but, in contrast, potentiated the decrease in KCl-evoked dopamine release induced by amphetamine, thereby suggesting that argon inhibited the vesicular monoamine transporter-2; (2) argon blocked the expression of locomotor and mu-opioid neurotransmission sensitization induced by repeated amphetamine administration in a short-term model of sensitization in rats; (3) argon decreased the maximal number of binding sites and increased the dissociation constant of mu-receptors in membrane preparations, thereby indicating that argon is a mu-receptor antagonist; (4) argon blocked the expression of locomotor sensitization and context-dependent locomotor activity induced by repeated administration of amphetamine in a long-term model of sensitization. Taken together, these data indicate that argon could be of potential interest for treating drug addiction and dependence. PMID:26151922

  7. Argon blocks the expression of locomotor sensitization to amphetamine through antagonism at the vesicular monoamine transporter-2 and mu-opioid receptor in the nucleus accumbens.

    PubMed

    David, H N; Dhilly, M; Degoulet, M; Poisnel, G; Meckler, C; Vallée, N; Blatteau, J-É; Risso, J-J; Lemaire, M; Debruyne, D; Abraini, J H

    2015-07-07

    We investigated the effects of the noble gas argon on the expression of locomotor sensitization to amphetamine and amphetamine-induced changes in dopamine release and mu-opioid neurotransmission in the nucleus accumbens. We found (1) argon blocked the increase in carrier-mediated dopamine release induced by amphetamine in brain slices, but, in contrast, potentiated the decrease in KCl-evoked dopamine release induced by amphetamine, thereby suggesting that argon inhibited the vesicular monoamine transporter-2; (2) argon blocked the expression of locomotor and mu-opioid neurotransmission sensitization induced by repeated amphetamine administration in a short-term model of sensitization in rats; (3) argon decreased the maximal number of binding sites and increased the dissociation constant of mu-receptors in membrane preparations, thereby indicating that argon is a mu-receptor antagonist; (4) argon blocked the expression of locomotor sensitization and context-dependent locomotor activity induced by repeated administration of amphetamine in a long-term model of sensitization. Taken together, these data indicate that argon could be of potential interest for treating drug addiction and dependence.

  8. Design, synthesis, and biological evaluation of 6alpha- and 6beta-N-heterocyclic substituted naltrexamine derivatives as mu opioid receptor selective antagonists.

    PubMed

    Li, Guo; Aschenbach, Lindsey C; Chen, Jianyang; Cassidy, Michael P; Stevens, David L; Gabra, Bichoy H; Selley, Dana E; Dewey, William L; Westkaemper, Richard B; Zhang, Yan

    2009-03-12

    Opioid receptor selective antagonists are important pharmacological probes in opioid receptor structural characterization and opioid agonist functional study. Thus far, a nonpeptidyl, highly selective and reversible mu opioid receptor (MOR) antagonist is unavailable. On the basis of our modeling studies, a series of novel naltrexamine derivatives have been designed and synthesized. Among them, two compounds were identified as leads based on the results of in vitro and in vivo assays. Both of them displayed high binding affinity for the MOR (K(i) = 0.37 and 0.55 nM). Compound 6 (NAP) showed over 700-fold selectivity for the MOR over the delta receptor (DOR) and more than 150-fold selectivity over the kappa receptor (KOR). Compound 9 (NAQ) showed over 200-fold selectivity for the MOR over the DOR and approximately 50-fold selectivity over the KOR. Thus these two novel ligands will serve as leads to further develop more potent and selective antagonists for the MOR.

  9. Involvement of exon 11-associated variants of the mu opioid receptor MOR-1 in heroin, but not morphine, actions.

    PubMed

    Pan, Ying-Xian; Xu, Jin; Xu, Mingming; Rossi, Grace C; Matulonis, Joshua E; Pasternak, Gavril W

    2009-03-24

    Heroin remains a major drug of abuse and is preferred by addicts over morphine. Like morphine, heroin has high affinity and selectivity for mu-receptors, but its residual analgesia in exon 1 MOR-1 knockout mice that do not respond to morphine suggests a different mechanism of action. MOR-1 splice variants lacking exon 1 have been observed in mice, humans, and rats, raising the possibility that they might be responsible for the residual heroin and morphine-6beta-glucuronide (M6G) analgesia in the exon 1 knockout mice. To test this possibility, we disrupted exon 11 of MOR-1, which eliminates all of the variants that do not contain exon 1. Morphine and methadone analgesia in the exon 11 knockout mouse was normal, but the analgesic actions of heroin, M6G, and fentanyl were markedly diminished in the radiant heat tail-flick and hot-plate assays. Similarly, the ability of M6G to inhibit gastrointestinal transit was greatly diminished in these exon 11 knockout mice, whereas the ability of morphine was unchanged. These findings identify receptors selectively involved with heroin and M6G actions and confirm the relevance of the exon 11-associated variants and raise important issues regarding the importance of atypical truncated G-protein-coupled receptors.

  10. Nucleus accumbens dopamine and mu-opioid receptors modulate the reinstatement of food-seeking behavior by food-associated cues.

    PubMed

    Guy, Elizabeth G; Choi, Eugene; Pratt, Wayne E

    2011-06-01

    The high attrition rates for dietary interventions aimed at promoting a healthier body mass may be caused, at least in part, by constant exposure to environmental stimuli that are associated with palatable foods. In both humans and animals, conditioned stimuli (CSs) that signal reward availability reliably reinstate food- and drug-seeking behaviors. The nucleus accumbens (NAcc) is critically involved in the cue-evoked reinstatement of food-seeking, but the role of individual neurotransmitter systems within the NAcc remains to be determined. These experiments tested the effects of intra-accumbal pharmacological manipulations of dopamine (DA) D(1) and D(2) receptors, mu-opioid receptors, or serotonin (5-HT) receptors on cue-evoked relapse to food-seeking. Rats were trained to lever press for sucrose pellets and the concurrent presentation of a light-tone CS. Once training was complete, lever-pressing was extinguished in the absence of either sucrose or CS presentation. Once each rat had reached extinction criterion, they received two reinstatement sessions in which lever pressing was renewed by response-contingent presentation of the CS. Prior to each reinstatement test, rats received NAcc microinfusions of saline or the selective D(1) receptor antagonist SCH 23390, the D(2) receptor antagonist raclopride, the mu-opioid receptor agonist [D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO), or 5-HT hydrogen maleate. Compared to saline test days, intra-accumbens infusions of SCH 23390 (1 μg/0.5 μL), raclopride (1 μg/0.5 μL), or DAMGO (0.25 μg/0.5 μL) effectively blocked the cue-evoked reinstatement of food-seeking. In contrast, stimulation of serotonin (5-HT) receptors by 5-HT hydrogen maleate (5 μg/0.5 μL) had no effect on cue-induced reinstatement. These novel data support roles for NAcc DA D(1), D(2), and mu-opioid receptors in the cue-evoked reinstatement of food seeking.

  11. Mu opioid receptor antagonism in the nucleus accumbens shell blocks consumption of a preferred sucrose solution in an anticipatory contrast paradigm.

    PubMed

    Katsuura, Y; Taha, S A

    2014-03-07

    Binge eating, a central feature of multiple eating disorders, is characterized by excessive consumption occurring during discrete, often brief, intervals. Highly palatable foods play an important role in these binge episodes - foods chosen during bingeing are typically higher in fat or sugar than those normally consumed. Multiple lines of evidence suggest a central role for signaling by endogenous opioids in promoting palatability-driven eating. This role extends to binge-like feeding studied in animal models, which is reduced by administration of opioid antagonists. However, the neural circuits and specific opioid receptors mediating these effects are not fully understood. In the present experiments, we tested the hypothesis that endogenous opioid signaling in the nucleus accumbens promotes consumption in a model of binge eating. We used an anticipatory contrast paradigm in which separate groups of rats were presented sequentially with 4% sucrose and then either 20% or 0% sucrose solutions. In rats presented with 4% and then 20% sucrose, daily training in this paradigm produced robust intake of 20% sucrose, preceded by learned hypophagia during access to 4% sucrose. We tested the effects of site-specific infusions of naltrexone (a nonspecific opioid receptor antagonist: 0, 1, 10, and 50μg/side in the nucleus accumbens core and shell), naltrindole (a delta opioid receptor antagonist: 0, 0.5, 5, and 10μg/side in the nucleus accumbens shell) and beta-funaltrexamine (a mu opioid receptor antagonist: 0 and 2.5μg/side in the nucleus accumbens shell) on consumption in this contrast paradigm. Our results show that signaling through the mu opioid receptor in the nucleus accumbens shell is dynamically modulated during formation of learned food preferences, and promotes binge-like consumption of palatable foods based on these learned preferences.

  12. Mu-opioid receptor densities are depleted in regions implicated in agonistic and sexual behavior in male European starlings (Sturnus vulgaris) defending nest sites and courting females.

    PubMed

    Kelm, Cynthia A; Forbes-Lorman, Robin M; Auger, Catherine J; Riters, Lauren V

    2011-05-16

    Social status and resource availability can strongly influence individual behavioral responses to conspecifics. In European starlings, males that acquire nest sites sing in response to females and dominate other males. Males without nest sites sing, but not to females, and they do not interact agonistically with other males. Little is known about the neural regulation of status- or resource-appropriate behavioral responses to conspecifics. Opioid neuropeptides are implicated in birdsong and agonistic behavior, suggesting that opioids may underlie differences in the production of these behaviors in males with and without nest sites. Here, we examined densities of immunolabeled mu-opioid receptors in groups of male starlings. Males that defended nest boxes dominated other males and sang at higher rates when presented with a female than males without nest boxes, independent of testosterone concentrations. Multiple regression analyses showed nest box ownership (not agonistic behavior or singing) predicted the optical density of receptor labeling in the medial bed nucleus of stria terminalis, paraventricular nucleus, ventral tegmental area and the medial preoptic nucleus. Compared to males without nest boxes, males with nest boxes had lower densities of immunolabeled mu-opioid receptors in these regions. Singing additionally predicted the area covered by labeling in the ventral tegmental area. The results suggest that elevated opioid activity in these regions suppresses courtship and agonistic behavioral responses to conspecifics in males without nest boxes. The findings are consistent with a dynamic role for opioid receptors in adjusting social behavior so that it is appropriate given the resources available to an individual. Copyright © 2010 Elsevier B.V. All rights reserved.

  13. Persistent inflammatory pain decreases the antinociceptive effects of the mu opioid receptor agonist DAMGO in the locus coeruleus of male rats.

    PubMed

    Jongeling, Amy C; Johns, Malcolm E; Murphy, Anne Z; Hammond, Donna L

    2009-01-01

    Persistent inflammatory nociception increases levels of endogenous opioids with affinity for delta opioid receptors in the ventromedial medulla and enhances the antinociceptive effects of the mu opioid receptor (MOPr) agonist [D-Ala2, N-Me-Phe4, Gly5-ol]enkephalin (DAMGO) [Hurley, R.W., Hammond, D.L., 2001. Contribution of endogenous enkephalins to the enhanced analgesic effects of supraspinal mu opioid receptor agonists after inflammatory injury. J. Neurosci. 21, 2536-2545]. It also increases levels of endogenous opioids that act at MOPr elsewhere in the CNS [Zangen, A., Herzberg, U., Vogel, Z., Yadid, G., 1998. Nociceptive stimulus induces release of endogenous beta-endorphin in the rat brain. Neuroscience 85, 659-662]. This study tested the hypothesis that a sustained release of endogenous opioids leads to a downregulation of MOPr in the locus coeruleus (LC) and induces a state of endogenous opioid tolerance. Four days after injection of complete Freund's adjuvant (CFA) in the left hindpaw of the rat, both the magnitude and duration of the antinociception produced by microinjection of DAMGO in the right LC were reduced. Saturation isotherms demonstrated a 50% decrease in MOPr B(max) in homogenates of the LC from CFA-treated rats; K(d) was unchanged. Receptor autoradiography revealed that this decrease was bilateral. The decreased efficacy of DAMGO in CFA-treated rats most likely results from a decreased number of MOPr in the LC. Microinjection of the MOPr antagonist D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) in the LC did not exacerbate hyperalgesia in the ipsilateral hindpaw or produce hyperalgesia in the contralateral hindpaw of CFA-treated rats. The downregulation in MOPr is therefore unlikely to result from the induction of endogenous opioid tolerance in the LC. These results indicate that persistent inflammatory nociception alters the antinociceptive actions of MOPr agonists in the CNS by diverse mechanisms that are nucleus specific and likely to have

  14. Binding and structure-activity-relation of benzo[f]isoquinoline- and norcodeinone-derivatives at mu-opioid receptors in the rat cerebral cortex.

    PubMed Central

    Freissmuth, M.; Beindl, W.; Kratzel, M.

    1993-01-01

    1. We have probed the ligand binding site of the mu-opioid receptor using a series of isoquinoline- and norcodeinone-derivatives; in these morphine- and codeine-analogues, the position of the piperidine-nitrogen as well as its mobility is altered relative to that found in morphine. 2. The mu-receptor in rat cortical membranes was labelled with [3H]-naloxone and competition experiments were carried out in the absence and presence of Gpp(NH)p and NaCl: conditions, which are associated with affinity shifts for agonists whilst antagonist affinity remains unaffected. Moving the piperidine-nitrogen closer to the phenolic ring or reducing its mobility by incorporation into an additional ring drastically decreases the affinity. 3. In contrast, we find that the piperidine-nitrogen in a distal position is well tolerated provided that additional structural criteria, in particular a phenolic hydroxyl-group and a 6 carbon ring corresponding to ring C in morphine, are met. This assumption was verified by the synthesis of WB4/PH (4aR, 10bS, 11R)-10, 11-epoxy-1, 2, 3, 4, 5, 6-hexahydro-9-hydroxy-3-methyl-4a,10b-butano- benzo[f]isochinolin-12-on(10). This compound is an agonist with an affinity comparable to that of morphine. 4. We therefore conclude that both the mobility of the piperidine nitrogen of the ligand and of its counterpart anionic site in the ligand binding pocket of the mu-opioid receptor (presumably aspartic acid) are important determinants for fruitful interaction. The mobility of the anionic site is restricted in one direction but is sufficient to bridge the 2A distance that exists between the position of the nitrogen in morphine and WB4/PH. PMID:8306082

  15. Binding and structure-activity-relation of benzo[f]isoquinoline- and norcodeinone-derivatives at mu-opioid receptors in the rat cerebral cortex.

    PubMed

    Freissmuth, M; Beindl, W; Kratzel, M

    1993-12-01

    1. We have probed the ligand binding site of the mu-opioid receptor using a series of isoquinoline- and norcodeinone-derivatives; in these morphine- and codeine-analogues, the position of the piperidine-nitrogen as well as its mobility is altered relative to that found in morphine. 2. The mu-receptor in rat cortical membranes was labelled with [3H]-naloxone and competition experiments were carried out in the absence and presence of Gpp(NH)p and NaCl: conditions, which are associated with affinity shifts for agonists whilst antagonist affinity remains unaffected. Moving the piperidine-nitrogen closer to the phenolic ring or reducing its mobility by incorporation into an additional ring drastically decreases the affinity. 3. In contrast, we find that the piperidine-nitrogen in a distal position is well tolerated provided that additional structural criteria, in particular a phenolic hydroxyl-group and a 6 carbon ring corresponding to ring C in morphine, are met. This assumption was verified by the synthesis of WB4/PH (4aR, 10bS, 11R)-10, 11-epoxy-1, 2, 3, 4, 5, 6-hexahydro-9-hydroxy-3-methyl-4a,10b-butano- benzo[f]isochinolin-12-on(10). This compound is an agonist with an affinity comparable to that of morphine. 4. We therefore conclude that both the mobility of the piperidine nitrogen of the ligand and of its counterpart anionic site in the ligand binding pocket of the mu-opioid receptor (presumably aspartic acid) are important determinants for fruitful interaction. The mobility of the anionic site is restricted in one direction but is sufficient to bridge the 2A distance that exists between the position of the nitrogen in morphine and WB4/PH.

  16. The role of the Asn40Asp polymorphism of the mu opioid receptor gene (OPRM1) on alcoholism etiology and treatment: a critical review.

    PubMed

    Ray, Lara A; Barr, Christina S; Blendy, Julie A; Oslin, David; Goldman, David; Anton, Raymond F

    2012-03-01

    The endogenous opioid system has been implicated in the pathophysiology of alcoholism as it modulates the neurobehavioral effects of alcohol. A variant in the mu opioid receptor gene (OPRM1), the Asn40Asp polymorphism, has received attention as a functional variant that may influence a host of behavioral phenotypes for alcoholism as well as clinical response to opioid antagonists. This paper will review converging lines of evidence on the effect of the Asn40Asp SNP on alcoholism phenotypes, including: (i) genetic association studies; (ii) behavioral studies of alcoholism; (iii) neuroimaging studies; (iv) pharmacogenetic studies and clinical trials; and (v) preclinical animal studies. Together, these lines of research seek to elucidate the effects of this functional polymorphism on alcoholism etiology and treatment response. Copyright © 2011 by the Research Society on Alcoholism.

  17. The Role of the Asn40Asp Polymorphism of the Mu Opioid Receptor Gene (OPRM1) on Alcoholism Etiology and Treatment: A Critical Review

    PubMed Central

    Ray, Lara A.; Barr, Christina S.; Blendy, Julie A.; Oslin, David; Goldman, David; Anton, Raymond F.

    2011-01-01

    The endogenous opioid system has been implicated in the pathophysiology of alcoholism as it modulates the neurobehavioral effects of alcohol. A variant in the mu opioid receptor gene (OPRM1), the Asn40Asp polymorphism, has received attention as a functional variant that may influence a host of behavioral phenotypes for alcoholism as well as clinical response to opioid antagonists. This paper will review converging lines of evidence on the effect of the Asn40Asp SNP on alcoholism phenotypes, including: (i) genetic association studies; (ii) behavioral studies of alcoholism; (iii) neuroimaging studies; (iv) pharmacogenetic studies and clinical trials; and (v) preclinical animal studies. Together, these lines of research seek to elucidate the effects of this functional polymorphism on alcoholism etiology and treatment response. PMID:21895723

  18. Individual Variation in Sleep Quality and Duration Is Related to Cerebral Mu Opioid Receptor Binding Potential during Tonic Laboratory Pain in Healthy Subjects

    PubMed Central

    Campbell, Claudia M.; Bounds, Sara C.; Kuwabara, Hiroto; Edwards, Robert R.; Campbell, James N.; Haythornthwaite, Jennifer A.; Smith, Michael T.

    2014-01-01

    Objective Although poor sleep is a consequence of pain, sleep disturbance reciprocally induces hyperalgesia and exacerbates clinical pain. Conceptual models of chronic pain implicate dysfunctional supraspinal pain processing mechanisms, mediated in part by endogenous opioid peptides. Our preliminary work indicates that sleep disruption impairs psychophysical measures of descending pain modulation, but few studies have investigated whether insufficient sleep may be associated with alterations in endogenous opioid systems. This preliminary, exploratory investigation sought to examine the relationship between sleep and functioning of the cerebral mu opioid system during the experience of pain in healthy participants. Subjects and Design Twelve healthy volunteers participated in a 90-minute positron emission tomography imaging scan using [11C]Carfentanil, a mu opioid receptors agonist. During the session, pain responses to a 10% topical capsaicin cream were continuously rated on a 0–100 scale. Participants also completed the Pittsburgh Sleep Quality Index (PSQI). Results Poor sleep quality (PSQI) was positively and significantly associated with greater binding potential (BP) in regions within the frontal lobes. In addition, sleep duration was negatively associated with BP in these areas as well as the temporal lobe and anterior cingulate. Conclusions These findings suggest that poor sleep quality and short sleep duration are associated with endogenous opioid activity in these brain regions during the application of a noxious stimulus. Elucidating the role of the endogenous opioid system in mediating some of the associations between sleep and pain could significantly improve our understanding of the pathophysiology of chronic pain and might advance clinical practice by suggesting interventions that could buffer the adverse effects of poor sleep on pain. PMID:24102962

  19. Inhibition of Inflammatory and Neuropathic Pain by Targeting a Mu Opioid Receptor/Chemokine Receptor5 Heteromer (MOR-CCR5).

    PubMed

    Akgün, Eyup; Javed, Muhammad I; Lunzer, Mary M; Powers, Michael D; Sham, Yuk Y; Watanabe, Yoshikazu; Portoghese, Philip S

    2015-11-12

    Chemokine release promotes cross-talk between opioid and chemokine receptors that in part leads to reduced efficacy of morphine in the treatment of chronic pain. On the basis of the possibility that a MOR-CCR5 heteromer is involved in such cross-talk, we have synthesized bivalent ligands (MCC series) that contain mu opioid agonist and CCR5 antagonist pharmacophores linked through homologous spacers (14-24 atoms). When tested on lipopolysaccharide-inflamed mice, a member of the series (MCC22; 3e) with a 22-atom spacer exhibited profound antinociception (i.t. ED50 = 0.0146 pmol/mouse) that was 2000× greater than morphine. Moreover, MCC22 was ~3500× more potent than a mixture of mu agonist and CCR5 antagonist monovalent ligands. These data strongly suggest that MCC22 acts by bridging the protomers of a MOR-CCR5 heteromer having a TM5,6 interface. Molecular simulation studies are consistent with such bridging. This study supports the MOR-CCR5 heteromer as a novel target for the treatment of chronic pain.

  20. Ultrastructural relationship between the AMPA-GluR2 receptor subunit and the mu-opioid receptor in the mouse central nucleus of the amygdala

    PubMed Central

    Beckerman, Marc A.; Glass, Michael J.

    2010-01-01

    Activation of GluR2 expressing non-calcium permeable AMPA-type glutamate receptors in the central nucleus of the amygdala (CeA) may play an important role in integrating emotion and memory with goal directed behaviors involved in opioid addiction. The location of non-calcium permeable AMPA receptors within distinct neuronal compartments (i.e. soma, dendrite, or axon) is an important functional feature of these proteins, however, their ultrastructural location and subcellular relationship with mu-opioid receptors (μOR) in the CeA are unknown. Immunocytochemical electron microscopy was used to characterize the ultrastructural distribution of GluR2 and its association with μOR in the mouse CeA. A single labeling analysis of GluR2 distribution employing immunoperoxidase or immunogold markers revealed that this protein was frequently affiliated with intracellular vesicular organelles, as well as the plasma membrane of CeA neuronal profiles. Among all GluR2 labeled neuronal structures, over 85% were dendrites or somata. Unlabeled axon terminals frequently formed asymmetric excitatory-type synaptic junctions with GluR2 labeled dendritic profiles. Dual labeling immunocytochemical analysis showed that GluR2 and μOR were co-localized in neuronal compartments. Among all dual labeled structures, approximately 80% were dendritic. Synaptic inputs to these dual labeled dendrites were frequently from unlabeled axon terminals forming asymmetric excitatory-type synapses. The presence of GluR2 in dendritic profiles receiving asymmetric synapses suggests that activation of the non-calcium permeable AMPA receptor plays a role in the postsynaptic modulation of excitatory signaling involving CeA neuronal circuits that coordinate sensory, affective, and behavioral processes involved in drug addiction. Given the critical role of non-calcium permeable AMPA receptor function in neural and behavioral adaptability, their dendritic association with μOR in CeA dendrites provides a neuronal

  1. Association between single nucleotide polymorphisms in the mu opioid receptor gene (OPRM1) and self-reported responses to alcohol in American Indians.

    PubMed

    Ehlers, Cindy L; Lind, Penelope A; Wilhelmsen, Kirk C

    2008-04-23

    Variation in response to the hedonic and adverse effects of a substance is in part an inherited factor that may influence its use, abuse and dependence. The mu opioid receptor is the primary site of action for opiates and individuals with polymorphisms in this receptor appear to have variation in the CNS effects of opiates. Several studies have suggested that this receptor may also mediate some of the effects of non-opioid drugs, such as alcohol. The purpose of this study was to investigate associations between 13 single nucleotide polymorphisms in the mu opioid receptor gene (OPRM1) with self-reported responses to alcohol, an endophenotype associated with the development of alcohol dependence, in American Indians living on eight contiguous reservations. Each participant gave a blood sample and completed a structured diagnostic interview. Additionally, response to alcohol was indexed using the expectation version of the subjective high assessment scale (SHAS-E). SNPs were genotyped in 251 participants and data analyses were conducted using SOLAR. The estimated heritability (h2) for the SHAS-E phenotypes ranged from 0.01 to 0.28. Endorsing the expectation of a more intense response on one or more of the following items from the SHAS-E: buzzed, clumsy, dizzy, drunk, effects, high, nausea, sleepy, talkative, terrible, and/or uncomfortable after imbibing 2-3 drinks was significantly associated with having at least one minor allele for at least one of 7 SNPs (p < 0.01) in the OPRM1 receptor gene. These studies provide data to suggest that the minor allele, for most of the polymorphisms in the OPRM1 receptor gene investigated, was found to be associated with a more intense, and/or more adverse, response to alcohol, traits that are significantly correlated with lowered quantity of alcohol consumption and less susceptibility to dependence in this Indian population. These data further suggest that making conclusions on the role of the mu opiod receptor gene in the

  2. Molecular insights into mu opioid pharmacology: From the clinic to the bench.

    PubMed

    Pasternak, Gavril W

    2010-01-01

    Most of the opioids used in clinical practice exert their effects through mu opioid receptors. Yet, subtle but important pharmacological differences have been observed among the mu opioids. Their potency, effectiveness, and adverse effects can vary unpredictably among patients. These clinical differences among the mu opioids strongly argue against a single receptor mediating their actions. The cloning of the mu opioid receptor has greatly enhanced our understanding of the complexity of this system and has provided possible mechanisms to explain these observations. A single mu opioid receptor gene has been identified, but we now know that it generates a multitude of different mu opioid receptor subtypes through a mechanism commonly used to enhance protein diversity, alternative splicing. Early studies identified a number of splice variants involving the tip of the C-terminus. This region of the receptor is far away from the binding pocket, explaining why these variants still exhibit the same selectivity for mu opioids. However, the differences in structure at the C-terminus influence the activation patterns of the mu opioids. In addition, a second series of variants has been isolated that involves alternative splicing at the N-terminus. Together, these sets of mu opioid receptor splice variants may help explain the clinical variability of the mu drugs among patients and provide insights into why it is so important to individualize therapy for every patient in pain.

  3. Co-incident signalling between mu-opioid and M3 muscarinic receptors at the level of Ca2+ release from intracellular stores: lack of evidence for Ins(1,4,5)P3 receptor sensitization.

    PubMed Central

    Samways, Damien S K; Li, Wen-hong; Conway, Stuart J; Holmes, Andrew B; Bootman, Martin D; Henderson, Graeme

    2003-01-01

    Activation of G(i)/G(o)-coupled opioid receptors increases [Ca2+]i (intracellular free-Ca2+ concentration), but only if there is concomitant G(q)-coupled receptor activation. This G(i)/G(o)-coupled receptor-mediated [Ca2+]i increase does not appear to result from further production of Ins P3 [Ins(1,4,5) P3] in SH-SY5Y cells. In the present study, fast-scanning confocal microscopy revealed that activation of mu-opioid receptors alone by 1 muM DAMGO ([L-Ala, NMe-Phe, Gly-ol]-enkephalin) did not stimulate the Ins P3-dependent elementary Ca2+-signalling events (Ca2+ puffs), whereas DAMGO did evoke Ca2+ puffs when applied during concomitant activation of M3 muscarinic receptors with 1 muM carbachol. We next determined whether mu-opioid receptor activation might increase [Ca2+]i by sensitizing the Ins P3 receptor to Ins P3. DAMGO did not potentiate the amplitude of the [Ca2+]i increase evoked by flash photolysis of the caged Ins P3 receptor agonist, caged 2,3-isopropylidene-Ins P3, whereas the Ins P3 receptor sensitizing agent, thimerosal (10 muM), did potentiate this response. DAMGO also did not prolong the rate of decay of the increase in [Ca2+]i evoked by flash photolysis of caged 2,3-isopropylidene-Ins P3. Furthermore, DAMGO did not increase [Ca2+]i in the presence of the cell-membrane-permeable Ins P3 receptor agonist, Ins P3 hexakis(butyryloxymethyl) ester. Therefore it appears that mu-opioid receptors do not increase [Ca2+]i through either Ins P3 receptor sensitization, enhancing the releasable pool of Ca2+ or inhibition of Ca2+ removal from the cytoplasm. PMID:12880387

  4. Endomorphins inhibit high-threshold Ca2+ channel currents in rodent NG108-15 cells overexpressing mu-opioid receptors.

    PubMed

    Higashida, H; Hoshi, N; Knijnik, R; Zadina, J E; Kastin, A J

    1998-02-15

    1. Extracellular application of the novel brain peptides endomorphin 1 (EM1) and endomorphin 2 (EM2) inhibited high-threshold Ca2+ channel currents in NGMO-251 cells, a daughter clone of NG108-15 mouse neuroblastoma x rat glioma hybrid cells, in which mu-opioid receptors are overexpressed. 2. In contrast, EM1 and EM2 did not induce this inhibition in the parental NG108-15 cells that predominantly express endogenous delta-receptors. 3. The IC50 for EM1 and EM2 was 7.7 and 23.1 nM, respectively. 4. EM-induced Ca2+ channel current inhibition was blocked by treatment or pretreatment of the cells with 100 microM N-methylmaleimide or 100 ng ml-1 pertussis toxin. 5. These results show that a decrease in conductance of Ca2+ channels results following interaction of EMs with cloned mu-receptors, which couple via Gi/Go-type G proteins, and that EMs fulfill one of the necessary synaptic conditions for them to be identified as neurotransmitters.

  5. Morphine activation of mu opioid receptors causes disinhibition of neurons in the ventral tegmental area mediated by β-arrestin2 and c-Src.

    PubMed

    Bull, Fiona A; Baptista-Hon, Daniel T; Lambert, Jeremy J; Walwyn, Wendy; Hales, Tim G

    2017-08-30

    The tyrosine kinase, c-Src, participates in mu opioid receptor (MOP) mediated inhibition in sensory neurons in which β-arrestin2 (β-arr2) is implicated in its recruitment. Mice lacking β-arr2 exhibit increased sensitivity to morphine reinforcement; however, whether β-arr2 and/or c-Src participate in the actions of opioids in neurons within the reward pathway is unknown. It is also unclear whether morphine acts exclusively through MOPs, or involves delta opioid receptors (DOPs). We examined the involvement of MOPs, DOPs, β-arr2 and c-Src in the inhibition by morphine of GABAergic inhibitory postsynaptic currents (IPSCs) recorded from neurons in the mouse ventral tegmental area. Morphine inhibited spontaneous IPSC frequency, mainly through MOPs, with only a negligible effect remaining in MOP-/- neurons. However, a reduction in the inhibition by morphine for DOP-/- c.f. WT neurons and a DPDPE-induced decrease of IPSC frequency revealed a role for DOPs. The application of the c-Src inhibitor, PP2, to WT neurons also reduced inhibition by morphine, while the inactive PP3, and the MEK inhibitor, SL327, had no effect. Inhibition of IPSC frequency by morphine was also reduced in β-arr2-/- neurons in which PP2 caused no further reduction. These data suggest that inhibition of IPSCs by morphine involves a β-arr2/c-Src mediated mechanism.

  6. Binding of dynorphin A and related peptides to kappa- and mu-opioid receptors: sensitivity to Na+ ions and Gpp(NH)p.

    PubMed

    Gairin, J E; Botanch, C; Cros, J; Meunier, J C

    1989-10-17

    We have examined the effects of Na+ ions and 5'-guanylyl imidodiphosphate (Gpp(NH)p) on the equilibrium binding of dynorphin A and of a series of related agonist and antagonist peptides to kappa- and mu-opioid receptors in guinea pig (kappa) and rabbit (mu) cerebellum membrane preparations. The binding to kappa sites of dynorphin A and of the peptides displaying agonist properties was strongly inhibited in the presence of 120 mM NaCl and 50 microM Gpp(NH)p. In contrast, a somewhat lower sensitivity to the inhibitory effect of the two allosteric effectors was observed for the analogues of the series showing antagonist properties. The same general behavior, but more marked, was observed at mu sites, at both mu- and kappa-opioid receptors. The peptides had biochemical properties (binding sensitivity vs. insensitivity to sodium ions and guanine nucleotides) that correlated well with their biological activities (agonist vs. antagonist) previously determined in in vitro pharmacological bioassays.

  7. Mu-opioid receptor binding measured by [11C]carfentanil positron emission tomography is related to craving and mood in alcohol dependence.

    PubMed

    Bencherif, Badreddine; Wand, Gary S; McCaul, Mary E; Kim, Yu Kyeong; Ilgin, Nese; Dannals, Robert F; Frost, James J

    2004-02-01

    The endogenous opioid system has been linked to alcohol dependence through animal and human studies. We investigated the relationship between alcohol craving and brain mu opioid receptors (mu-OR) in alcohol-dependent subjects. Regional brain mu-OR binding potential (BP) was measured using [(11)C]carfentanil positron emission tomography in eight male alcohol-dependent subjects undergoing alcohol withdrawal and eight matched control subjects. Self-reported alcohol craving, withdrawal, and mood were measured. Lower mu-OR BP was associated with higher craving in the right dorsal lateral prefrontal cortex, the right anterior frontal cortex, and right parietal cortex. In these regions, alcoholics showed lower mean mu-OR BP compared with control subjects. Mu-OR BP in four other brain regions also correlated with craving, but there were no group differences in receptor binding potential. Mu-OR BP also correlated with depressive symptoms in five brain regions, three of which were identified in the craving analyses. Results show a strong functional relationship between alcohol craving, mood, and mu-OR binding in specific brain regions of recently abstinent, alcohol-dependent men.

  8. Colocalization and shared distribution of endomorphins with substance P, calcitonin gene-related peptide, gamma-aminobutyric acid, and the mu opioid receptor.

    PubMed

    Greenwell, Thomas N; Martin-Schild, Sheryl; Inglis, Fiona M; Zadina, James E

    2007-07-10

    The endomorphins are endogenous opioids with high affinity and selectivity for the mu opioid receptor (MOR, MOR-1, MOP). Endomorphin-1 (Tyr-Pro-Trp-Phe-NH(2); EM1) and endomorphin-2 (Tyr-Pro-Phe-Phe-NH(2); EM2) have been localized to many regions of the central nervous system (CNS), including those that regulate antinociception, autonomic function, and reward. Colocalization or shared distribution (overlap) of two neurotransmitters, or a transmitter and its cognate receptor, may imply an interaction of these elements in the regulation of functions mediated in that region. For example, previous evidence of colocalization of EM2 with substance P (SP), calcitonin gene-related peptide (CGRP), and MOR in primary afferent neurons suggested an interaction of these peptides in pain modulation. We therefore investigated the colocalization of EM1 and EM2 with SP, CGRP, and MOR in other areas of the CNS. EM2 was colocalized with SP and CGRP in the nucleus of the solitary tract (NTS) and with SP, CGRP and MOR in the parabrachial nucleus. Several areas in which EM1 and EM2 showed extensive shared distributions, but no detectable colocalization with other signaling molecules, are also described.

  9. Truncated G protein-coupled mu opioid receptor MOR-1 splice variants are targets for highly potent opioid analgesics lacking side effects.

    PubMed

    Majumdar, Susruta; Grinnell, Steven; Le Rouzic, Valerie; Burgman, Maxim; Polikar, Lisa; Ansonoff, Michael; Pintar, John; Pan, Ying-Xian; Pasternak, Gavril W

    2011-12-06

    Pain remains a pervasive problem throughout medicine, transcending all specialty boundaries. Despite the extraordinary insights into pain and its mechanisms over the past few decades, few advances have been made with analgesics. Most pain remains treated by opiates, which have significant side effects that limit their utility. We now describe a potent opiate analgesic lacking the traditional side effects associated with classical opiates, including respiratory depression, significant constipation, physical dependence, and, perhaps most important, reinforcing behavior, demonstrating that it is possible to dissociate side effects from analgesia. Evidence indicates that this agent acts through a truncated, six-transmembrane variant of the G protein-coupled mu opioid receptor MOR-1. Although truncated splice variants have been reported for a number of G protein-coupled receptors, their functional relevance has been unclear. Our evidence now suggests that truncated variants can be physiologically important through heterodimerization, even when inactive alone, and can comprise new therapeutic targets, as illustrated by our unique opioid analgesics with a vastly improved pharmacological profile.

  10. 6β-N-heterocyclic substituted naltrexamine derivative NAP as a potential lead to develop peripheral mu opioid receptor selective antagonists.

    PubMed

    Yuan, Yunyun; Stevens, David L; Braithwaite, Amanda; Scoggins, Krista L; Bilsky, Edward J; Akbarali, Hamid I; Dewey, William L; Zhang, Yan

    2012-07-15

    A 6β-N-heterocyclic substituted naltrexamine derivative, NAP, was proposed as a peripheral mu opioid receptor (MOR) selective antagonist based on the in vitro and in vivo pharmacological and pharmacokinetic studies. To further validate this notion, several functional assays were carried out to fully characterize this compound. In the charcoal gavage and intestinal motility assay in morphine-pelleted mice, when administered 0.3 mg/kg or higher doses up to 3 mg/kg subcutaneously, NAP significantly increased the intestinal motility compared to the saline treatment. The comparative opioid withdrawal precipitation study and the lower locomotor assay demonstrated that NAP showed only marginal intrinsic effect in the central nervous system either given subcutaneously or intravenously: no jumps were witnessed for the tested animals even given up to a dose of 50 mg/kg, while similar noticeable wet-dog shakes only occurred at the dose 50 times of those for naloxone or naltrexone, and significant reduction of the hyper-locomotion only happened at the dose as high as 32 mg/kg. Collectively, these results suggested that NAP may serve as a novel lead to develop peripheral MOR selective antagonist which might possess therapeutic potential for opioid-induced bowel dysfunction (OBD), such as opioid-induced constipation (OIC).

  11. Functional Mu Opioid Receptor Polymorphism (OPRM1 A118G) Associated With Heroin Use Outcomes in Caucasian Males: A Pilot Study

    PubMed Central

    Woodcock, Eric A.; Lundahl, Leslie H.; Burmeister, Margit; Greenwald, Mark K.

    2017-01-01

    Background Heroin’s analgesic, euphoric and dependence-producing effects are primarily mediated by the mu opioid receptor (MOR). A single gene, OPRM1, encodes the MOR. The functional polymorphism A118G, located in exon 1 of the OPRM1 gene, results in anatomically-specific reductions in MOR expression, which may alter an individual’s response to heroin. In prior studies 118G (rare allele) carriers demonstrated significantly greater opioid tolerance, overdose vulnerability, and pain sensitivity than 118AA homozygotes. Those findings suggest OPRM1 genotype may impact characteristics of heroin use. Methods The present pilot study characterized the impact of OPRM1 genotype (rs1799971, 118G allele carriers vs. 118AA homozygotes) on heroin-use phenotypes associated with heroin dependence severity in a sample of male, Caucasian chronic heroin users (n = 86). Results Results indicate that 118G allele carriers reported significantly more heroin use-related consequences and heroin-quit attempts, and were more likely to have sought treatment for their heroin use than 118AA homozygotes. Conclusions These preliminary findings, consistent with extant data, illustrate a role for OPRM1 allelic variation on heroin use characteristics, and provide support for considering genotype in heroin treatment and relapse prevention. PMID:25911999

  12. An early granulocyte colony-stimulating factor treatment attenuates neuropathic pain through activation of mu opioid receptors on the injured nerve

    PubMed Central

    Liao, Ming-Feng; Yeh, Shin-Rung; Lo, Ai-Lun; Chao, Po-Kuan; Lee, Yun-Lin; Hung, Yu-Hui; Lu, Kwok-Tung; Ro, Long-Sun

    2016-01-01

    Several studies have shown that the mu opioid receptor (MOR) located in the peripheral nerves can be activated after nerve injury and that it attenuates peripheral nociceptive signals to the spinal dorsal horn. Various cytokines and phosphorylated-p38 (p-p38) activation in the dorsal horn also play an important role in neuropathic pain development. Granulocyte-colony stimulating factor (GCSF) is a growth factor that can stimulate granulocyte formation and has been shown to exert an analgesic effect on neuropathic pain through recruiting opioid-containing leukocytes to the injured nerve. However, the underlying mechanisms are not well understood. Herein, the results of behavior tests in addition to MOR levels in the injured sciatic nerve and the levels of p-p38 and various cytokines in the spinal dorsal horn were studied in vehicle-treated or GCSF-treated chronic constriction injured (CCI) rats at different time points (i.e., 1, 3, and 7 days, respectively) after nerve injury. The results showed that a single early systemic GCSF treatment after nerve injury can up-regulate MORs in the injured nerve, which can decrease peripheral nociceptive signals. Thereafter, those changes suppress the pro-inflammatory cytokine IL-6 but enhance the anti-inflammatory cytokine IL-4, followed by decreases in p-p38 in the dorsal horn, and thus further attenuate neuropathic pain. PMID:27180600

  13. Post-Transcriptional Regulation of the Human Mu-Opioid Receptor (MOR) by Morphine-Induced RNA Binding Proteins hnRNP K and PCBP1

    PubMed Central

    Song, Kyu Young; Choi, Hack Sun; Law, Ping-Yee; Wei, Li-Na; Loh, Horace H.

    2016-01-01

    Expression of the mu-opioid receptor (MOR) protein is controlled by extensive transcriptional and post-transcriptional processing. MOR gene expression has previously been shown to be altered by a post-transcriptional mechanism involving the MOR mRNA untranslated region (UTR). Here, we demonstrate for the first time the role of heterogeneous nuclear ribonucleic acids (hnRNA)-binding protein (hnRNP) K and poly(C)-binding protein 1 (PCBP1) as post-transcriptional inducers in MOR gene regulation. In the absence of morphine, a significant level of MOR mRNA is sustained in its resting state and partitions in the translationally inactive polysomal fraction. Morphine stimulation activates the downstream targets hnRNP K and PCPB1 and induces partitioning of the MOR mRNA to the translationally active fraction. Using reporter and ligand binding assays, as well as RNA EMSA, we reveal potential RNP binding sites located in the 5′-untranslated region of human MOR mRNA. In addition, we also found that morphine-induced RNPs could regulate MOR expression. Our results establish the role of hnRNP K and PCPB1 in the translational control of morphine-induced MOR expression in human neuroblastoma (NMB) cells as well as cells stably expressing MOR (NMB1). PMID:27292014

  14. Post-Transcriptional Regulation of the Human Mu-Opioid Receptor (MOR) by Morphine-Induced RNA Binding Proteins hnRNP K and PCBP1.

    PubMed

    Song, Kyu Young; Choi, Hack Sun; Law, Ping-Yee; Wei, Li-Na; Loh, Horace H

    2017-03-01

    Expression of the mu-opioid receptor (MOR) protein is controlled by extensive transcriptional and post-transcriptional processing. MOR gene expression has previously been shown to be altered by a post-transcriptional mechanism involving the MOR mRNA untranslated region (UTR). Here, we demonstrate for the first time the role of heterogeneous nuclear ribonucleic acids (hnRNA)-binding protein (hnRNP) K and poly(C)-binding protein 1 (PCBP1) as post-transcriptional inducers in MOR gene regulation. In the absence of morphine, a significant level of MOR mRNA is sustained in its resting state and partitions in the translationally inactive polysomal fraction. Morphine stimulation activates the downstream targets hnRNP K and PCPB1 and induces partitioning of the MOR mRNA to the translationally active fraction. Using reporter and ligand binding assays, as well as RNA EMSA, we reveal potential RNP binding sites located in the 5'-untranslated region of human MOR mRNA. In addition, we also found that morphine-induced RNPs could regulate MOR expression. Our results establish the role of hnRNP K and PCPB1 in the translational control of morphine-induced MOR expression in human neuroblastoma (NMB) cells as well as cells stably expressing MOR (NMB1). J. Cell. Physiol. 232: 576-584, 2017. © 2016 Wiley Periodicals, Inc.

  15. Effects of the Novel, Selective and Low-Efficacy Mu Opioid Receptor Ligand NAQ on Intracranial Self-Stimulation in Rats

    PubMed Central

    Altarifi, Ahmad A.; Yuan, Yunyun; Zhang, Yan; Selley, Dana E.; Negus, S. Stevens

    2014-01-01

    Rationale Low-efficacy mu opioid receptor agonists may be useful for some clinical indications, but clinically available low-efficacy mu agonists also have low selectivity for mu vs. kappa opioid receptors. NAQ is a novel opioid receptor ligand with low-efficacy at mu receptors and greater mu-receptor selectivity than existing low-efficacy agonists. Objectives This study examined behavioral effects of NAQ in rats using an intracranial self-stimulation (ICSS) procedure that has been used previously to examine other opioids. NAQ effects were examined before, during and after chronic morphine treatment, and effects of NAQ were compared to effects of nalbuphine and naltrexone. Methods Adult male Sprague-Dawley rats were trained to respond for electrical brain stimulation delivered via electrodes implanted in the medial forebrain bundle. A range of brain stimulation frequencies maintained a wide range of baseline ICSS rates. Effects of NAQ (0.32-10 mg/kg), nalbuphine (1.0 mg/kg) and naltrexone (0.1 mg/kg) were determined before morphine treatment and during treatment with 3.2 and 18 mg/kg/day morphine. NAQ effects were also redetermined beginning two weeks after termination of morphine treatment. Results NAQ produced weak ICSS facilitation in morphine-naïve rats but more robust ICSS facilitation during and after morphine treatment and also reversed morphine withdrawal-associated depression of ICSS. These effects were similar to effects of nalbuphine. Conclusions These results agree with the in vitro characterization of NAQ as a low-efficacy mu agonist. Opioid exposure may enhance abuse-related effects of NAQ, but NAQ may also serve as a low-efficacy and relatively safe option for treatment of opioid withdrawal or dependence. PMID:25178814

  16. Methamphetamine-induced stereotypy correlates negatively with patch-enhanced prodynorphin and arc mRNA expression in the rat caudate putamen: the role of mu opioid receptor activation.

    PubMed

    Horner, Kristen A; Noble, Erika S; Gilbert, Yamiece E

    2010-06-01

    Amphetamines induce stereotypy, which correlates with patch-enhanced c-Fos expression the patch compartment of caudate putamen (CPu). Methamphetamine (METH) treatment also induces patch-enhanced expression of prodynorphin (PD), arc and zif/268 in the CPu. Whether patch-enhanced activation of any of these genes correlates with METH-induced stereotypy is unknown, and the factors that contribute to this pattern of expression are poorly understood. Activation of mu opioid receptors, which are expressed by the neurons of the patch compartment, may underlie METH-induced patch-enhanced gene expression and stereotypy. The current study examined whether striatal mu opioid receptor blockade altered METH-induced stereotypy and patch-enhanced gene expression, and if there was a correlation between the two responses. Animals were intrastriatally infused with the mu antagonist CTAP (10 microg/microl), treated with METH (7.5 mg/kg, s.c.), placed in activity chambers for 3h, and then sacrificed. CTAP pretreatment attenuated METH-induced increases in PD, arc and zif/268 mRNA expression and significantly reduced METH-induced stereotypy. Patch-enhanced PD and arc mRNA expression in the dorsolateral CPu correlated negatively with METH-induced stereotypy. These data indicate that mu opioid receptor activation contributes to METH-induced gene expression in the CPu and stereotypy, and that patch-enhanced PD and arc expression may be a homeostatic response to METH treatment. Copyright 2010 Elsevier Inc. All rights reserved.

  17. Methamphetamine-Induced Stereotypy Correlates Negatively with Patch-Enhanced Prodynorphin and ARC mRNA Expression in the Rat Caudate Putamen: The Role of Mu Opioid Receptor Activation

    PubMed Central

    Horner, Kristen A.; Noble, Erika S.; Gilbert, Yamiece E.

    2010-01-01

    Amphetamines induce stereotypy, which correlates with patch-enhanced c-Fos expression the patch compartment of caudate putamen (CPu). Methamphetamine (METH) treatment also induces patch-enhanced expression of prodynorphin (PD), arc and zif/268 in the CPu. Whether patch-enhanced activation of any of these genes correlates with METH-induced stereotypy is unknown, and the factors that contribute to this pattern of expression are poorly understood. Activation of mu opioid receptors, which are expressed by the neurons of the patch compartment, may underlie METH-induced patch-enhanced gene expression and stereotypy. The current study examined whether striatal mu opioid receptor blockade altered METH-induced stereotypy and patch-enhanced gene expression, and if there was a correlation between the two responses. Animals were intrastriatally infused with the mu antagonist CTAP (10 μg/μl), treated with METH (7.5 mg/kg, s.c.), placed in activity chambers for 3h, and then sacrificed. CTAP pretreatment attenuated METH-induced increases in PD, arc and zif/268 mRNA expression and significantly reduced METH-induced stereotypy. Patch-enhanced PD and arc mRNA expression in the dorsolateral CPu correlated negatively with METH-induced stereotypy. These data indicate that mu opioid receptor activation contributes to METH-induced gene expression in the CPu and stereotypy, and that patch-enhanced PD and arc expression may be a homeostatic response to METH treatment. PMID:20298714

  18. Type and location of fluorescent probes incorporated into the potent mu-opioid peptide [Dmt]DALDA affect potency, receptor selectivity and intrinsic efficacy.

    PubMed

    Schiller, P W; Berezowska, I; Weltrowska, G; Chen, H; Lemieux, C; Chung, N N

    2005-06-01

    The dermorphin-derived tetrapeptide H-Dmt-d-Arg-Phe-Lys-NH(2) (Dmt = 2',6'-dimethyltyrosine) ([Dmt(1)]DALDA) is a highly potent and selective mu-opioid agonist capable of crossing the blood-brain barrier and producing a potent, centrally mediated analgesic effect when given systemically. For the purpose of biodistribution studies by fluorescence techniques, [Dmt(1)]DALDA analogues containing various fluorescent labels [dansyl, anthraniloyl (atn), fluorescein, or 6-dimethylamino-2'-naphthoyl] in several different locations of the peptide were synthesized and characterized in vitro in the guinea-pig ileum and mouse vas deferens assays, and in mu-, delta- and kappa-opioid receptor-binding assays. The analogues showed various degrees of mu receptor-binding selectivity, but all of them were less mu-selective than the [Dmt(1)]DALDA parent peptide. Most analogues retained potent, full mu-agonist activity, except for one with fluorescein attached at the C-terminus (3a) (partial mu-agonist) and one containing beta-(6'-dimethylamino-2'-naphthoyl)alanine (aladan) in place of Phe(3) (4) (mu- and kappa-antagonist). The obtained data indicate that the receptor-binding affinity, receptor selectivity and intrinsic efficacy of the prepared analogues vary very significantly, depending on the type of fluorescent label used and on its location in the peptide. The results suggest that the biological activity profile of fluorescence-labeled peptide analogues should always be carefully determined prior to their use in biodistribution studies or other studies. One of the analogues containing the atn group (2a) proved highly useful in a study of cellular uptake and intracellular distribution by confocal laser scanning microscopy.

  19. The functional expression of mu opioid receptors on sensory neurons is developmentally regulated; morphine analgesia is less selective in the neonate.

    PubMed

    Nandi, Reema; Beacham, Daniel; Middleton, Jacqueta; Koltzenburg, Martin; Howard, Richard F; Fitzgerald, Maria

    2004-09-01

    Opioid requirements in neonatal patients are reported to be lower than older infants and this may be a reflection of the developmental regulation of opioid receptors. In this study we have investigated the postnatal regulation of Mu opioid receptor (MOR) function in both rat lumbar dorsal root ganglion (DRG) cultures and behavioural mechanical and thermal reflex tests in rat pups. Immunostaining with MOR and selective neurofilament (NF200) antibodies was combined with calcium imaging of MOR function in cultured neonatal and adult rat dorsal root ganglion cells. Calcium imaging showed that a significantly greater number of neonatal DRG neurons expressed functional MOR compared to adult (56.5+/-3.4 versus 39.9+/-1.5%, n=8, mean+/-SEM, P<0.001). This expression is confined to the large, neurofilament positive sensory neurons, while expression in small, nociceptive, neurofilament negative neurons remains unchanged. Sensory threshold testing in rat pups showed that the analgesic potency of systemic morphine to mechanical stimulation is significantly greater in the neonate and declines with postnatal age. Morphine analgesic potency in thermal nociceptive tests did not change with postnatal age. These experiments show that the MOR expressed on large DRG neurons in neonates are functional and are subject to postnatal developmental regulation. This changing functional receptor profile is consistent with greater morphine potency in mechanical, but not thermal, sensory tests in young animals. These results have important clinical implications for the use of morphine in neonates and provide a possible explanation for the differences in morphine requirements observed in the youngest patients.

  20. Persistent Inflammatory Pain Decreases the Antinociceptive Effects of the Mu Opioid Receptor Agonist DAMGO in the Locus Coeruleus of Male Rats

    PubMed Central

    Jongeling, Amy C.; Johns, Malcolm E.; Murphy, Anne Z.; Hammond, Donna L.

    2009-01-01

    Persistent inflammatory nociception increases levels of endogenous opioids with affinity for delta opioid receptors in the ventromedial medulla and enhances the antinociceptive effects of the mu opioid receptor (MOPr) agonist [D-Ala2-NMePhe4, Gly5-ol]enkephalin (DAMGO) (Hurley and Hammond, 2001). It also increases levels of endogenous opioids that act at MOPr elsewhere in the CNS (Zangen et al., 1998). This study tested the hypothesis that a sustained release of endogenous opioids leads to a downregulation of MOPr in the locus coeruleus (LC) and induces a state of endogenous opioid tolerance. Four days after injection of complete Freund’s adjuvant (CFA) in the left hindpaw of the rat, both the magnitude and duration of the antinociception produced by microinjection of DAMGO in the right LC were reduced. Saturation isotherms demonstrated a 50% decrease in MOPr Bmax in homogenates of the LC from CFA-treated rats; Kd was unchanged. Receptor autoradiography revealed that this decrease was bilateral. The decreased efficacy of DAMGO in CFA-treated rats most likely results from a decreased number of MOPr in the LC. Microinjection of the MOPr antagonist D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) in the LC did not exacerbate hyperalgesia in the ipsilateral hindpaw or produce hyperalgesia in the contralateral hindpaw of CFA-treated rats. The downregulation in MOPr is therefore unlikely to result from the induction of endogenous opioid tolerance in the LC. These results indicate that persistent inflammatory nociception alters the antinociceptive actions of MOPr agonists in the CNS by diverse mechanisms that are nucleus specific and likely to have different physiological implications. PMID:19265713

  1. A role for kappa-, but not mu-opioid, receptor activation in acute food deprivation-induced reinstatement of heroin seeking in rats.

    PubMed

    Sedki, Firas; Eigenmann, Karine; Gelinas, Jessica; Schouela, Nicholas; Courchesne, Shannon; Shalev, Uri

    2015-05-01

    Stress is considered to be one of the major triggers to drug relapse, even after prolonged periods of abstinence. In rats, the activation of stress-related brain systems, including corticotropin-releasing factor and norepinephrine, is critical for stress-induced reinstatement of extinguished drug seeking, an animal model for drug relapse. In addition, there are strong indications that activation of the endogenous opioid system is important for the effects of stress on drug seeking. More specifically, activation of the dynorphin/kappa opioid receptor (KOR) system is critically involved in the reinstatement of cocaine seeking following exposure to stressors, such as footshock, forced swimming or social stress. However, studies on the role of the dynorphin/KOR system in stress-induced reinstatement of heroin seeking are scarce. Here, rats were trained to self-administer heroin (0.1 mg/kg/infusion) for 10 days. Drug seeking was then extinguished and the rats were tested for acute (21 hours) food deprivation-induced reinstatement of heroin seeking. In two separate experiments, rats were injected with the mu-opioid receptor (MOR) antagonist, naltrexone (0.0, 1.0, 10.0 mg/kg; s.c.) or the KOR antagonist, norBNI (0.0, 1.0, 10.0 mg/kg; i.p.) before the reinstatement test. Naltrexone treatment did not affect stress-induced reinstatement. In contrast, treatment with norBNI dose-dependently attenuated food deprivation-induced reinstatement of heroin seeking. These results support the hypothesis that activation of KOR, but not MOR, is critically involved in stress-induced reinstatement of drug seeking.

  2. Region-dependent attenuation of mu opioid receptor-mediated G-protein activation in mouse CNS as a function of morphine tolerance.

    PubMed

    Sim-Selley, L J; Scoggins, K L; Cassidy, M P; Smith, L A; Dewey, W L; Smith, F L; Selley, D E

    2007-08-01

    Chronic morphine administration produces tolerance in vivo and attenuation of mu opioid receptor (MOR)-mediated G-protein activation measured in vitro, but the relationship between these adaptations is not clear. The present study examined MOR-mediated G-protein activation in the CNS of mice with different levels of morphine tolerance. Mice were implanted with morphine pellets, with or without supplemental morphine injections, to induce differing levels of tolerance as determined by a range of MOR-mediated behaviours. MOR function was measured using agonist-stimulated [(35)S]guanylyl-5'-O-(gamma-thio)-triphosphate ([(35)S]GTPgammaS) and receptor binding throughout the CNS. Morphine pellet implantation produced 6-12-fold tolerance in antinociceptive assays, hypothermia and Straub tail, as measured by the ratio of morphine ED(50) values between morphine-treated and control groups. Pellet implantation plus supplemental injections produced 25-50-fold tolerance in these tests. In morphine pellet-implanted mice, MOR-stimulated [(35)S]GTPgammaS binding was significantly reduced only in the nucleus tractus solitarius (NTS) and spinal cord dorsal horn in tissue sections from morphine pellet-implanted mice. In contrast, MOR-stimulated [(35)S]GTPgammaS binding was significantly decreased in most regions examined in morphine pellet+morphine injected mice, including nucleus accumbens, caudate-putamen, periaqueductal gray, parabrachial nucleus, NTS and spinal cord. Tolerance and the regional pattern of apparent MOR desensitization were influenced positively by the level of morphine exposure. These results indicate that desensitization of MOR-mediated G-protein activity is more regionally widespread upon induction of high levels of tolerance, suggesting that this response contributes more to high than low levels of tolerance to CNS-mediated effects of morphine.

  3. Differential modulation of methamphetamine-mediated behavioral sensitization by overexpression of Mu opioid receptors in nucleus accumbens and ventral tegmental area.

    PubMed

    Kuo, Chi-Chung; Shen, Hui; Harvey, Brandon K; Yu, Seong-Jin; Kopajtic, Theresa; Hinkle, Josh J; Kyrkanides, Stephanos; Katz, Jonathan L; Wang, Yun

    2016-02-01

    Repeated administration of methamphetamine (Meth) induces behavioral sensitization which is characterized by a progressive increase in locomotor response after each injection. Previous studies have shown that Mu opioid receptors (MORs) can regulate Meth-mediated behavioral sensitization. However, the reported interactions are controversial; systemic activation of MORs either enhanced or suppressed Meth sensitization. It is possible that alteration of Meth sensitization after systemic administration of MOR ligands reflects the sum of distinct MOR reactions in multiple brain regions. The purpose of the present study was to examine the actions of MORs on Meth sensitization after regionally selective overexpression of human MOR through an AAV6-based gene delivery system. We demonstrated that adeno-associated virus (AAV)-MOR increased MOR immunoreactivity and binding in vitro. AAV-MOR or AAV-green fluorescent protein (GFP) was injected into the nucleus accumbens (NAc) or ventral tegmental area (VTA) of adult mice. Two weeks after viral infection, animals received Meth or saline for five consecutive days. Locomotor behavior and striatal dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) level were determined. Repeated administration of Meth progressively increased locomotor activity; this sensitization reaction was attenuated by intra-NAc AAV-MOR microinjections. Infusion of AAV-MOR to VTA enhanced Meth sensitization. AAV-MOR significantly enhanced DA levels in VTA after VTA infection but reduced DOPAC/DA turnover in the NAc after NAc injection. Our data suggest a differential modulation of Meth sensitization by overexpression of MOR in NAc and VTA. Regional manipulation of MOR expression through AAV may be a novel approach to control Meth abuse and psychomimetic activity.

  4. Tolerance and sensitization to chronic escalating-dose heroin following extended withdrawal in Fischer rats: possible role of mu-opioid receptors

    PubMed Central

    Seip-Cammack, Katharine M.; Reed, Brian; Zhang, Yong; Ho, Ann; Kreek, Mary Jeanne

    2012-01-01

    Rationale/objectives Heroin addiction is characterized by recurrent cycles of drug use, abstinence and relapse. It is likely that neurobiological changes during chronic heroin exposure persist across withdrawal and impact behavioral responses to re-exposure. We hypothesized that, after extended withdrawal, heroin-withdrawn rats would express behavioral tolerance and/or sensitization in response to heroin re-exposure and that these responses might be associated with altered mu-opioid receptor (MOPr) activity. Methods Male Fischer rats were exposed chronically to escalating doses of heroin (7.5–75mg/kg/day), experienced acute spontaneous withdrawal and extended (10-day) abstinence, and were re-exposed chronically to heroin. Homecage behaviors and locomotor activity in response to heroin, as well as somatic withdrawal signs, were recorded. Separate groups of rats were sacrificed after extended abstinence and MOPr expression and G-protein coupling were analyzed using [3H]DAMGO and [35S]GTPγS assays. Results The depth of behavioral stupor was lower during the initial days of heroin re-exposure compared to the initial days of the first exposure period. Behavioral responses (e.g., stereotypy) and locomotion were elevated in response to heroin re-exposure at low doses. Rats conditioned for heroin place preference during the chronic re-exposure period expressed heroin preference during acute withdrawal; this preference was stronger than rats conditioned during chronic heroin exposure that followed chronic saline and injection-free periods. Extended withdrawal was associated with increased MOPr expression in the caudate-putamen and frontal and cingulate cortices. No changes in G-protein coupling were identified. Conclusions Aspects of tolerance/sensitization to heroin are present even after extended abstinence and may be associated with altered MOPr density. PMID:22829433

  5. Stress differentially alters mu opioid receptor density and trafficking in parvalbumin-containing interneurons in the female and male rat hippocampus.

    PubMed

    Milner, Teresa A; Burstein, Suzanne R; Marrone, Gina F; Khalid, Sana; Gonzalez, Andreina D; Williams, Tanya J; Schierberl, Kathryn C; Torres-Reveron, Annelyn; Gonzales, Keith L; McEwen, Bruce S; Waters, Elizabeth M

    2013-11-01

    Stress differentially affects hippocampal-dependent learning relevant to addiction and morphology in male and female rats. Mu opioid receptors (MORs), which are located in parvalbumin (PARV)-containing GABAergic interneurons and are trafficked in response to changes in the hormonal environment, play a critical role in promoting principal cell excitability and long-term potentiation. Here, we compared the effects of acute and chronic immobilization stress (AIS and CIS) on MOR trafficking in PARV-containing neurons in the hilus of the dentate gyrus in female and male rats using dual label immunoelectron microscopy. Following AIS, the density of MOR silver-intensified gold particles (SIGs) in the cytoplasm of PARV-labeled dendrites was significantly reduced in females (estrus stage). Conversely, AIS significantly increased the proportion of cytoplasmic MOR SIGs in PARV-labeled dendrites in male rats. CIS significantly reduced the number of PARV-labeled neurons in the dentate hilus of males but not females. However, MOR/PARV-labeled dendrites and terminals were significantly smaller in CIS females, but not males, compared with controls. Following CIS, the density of cytoplasmic MOR SIGs increased in PARV-labeled dendrites and terminals in females. Moreover, the proportion of near-plasmalemmal MOR SIGs relative to total decreased in large PARV-labeled dendrites in females. After CIS, no changes in the density or trafficking of MOR SIGs were seen in PARV-labeled dendrites or terminals in males. These data show that AIS and CIS differentially affect available MOR pools in PARV-containing interneurons in female and male rats. Furthermore, they suggest that CIS could affect principal cell excitability in a manner that maintains learning processes in females but not males.

  6. Naloxone can act as an analgesic agent without measurable chronic side effects in mice with a mutant mu-opioid receptor expressed in different sites of pain pathway

    PubMed Central

    Chou, Shu-Husan; Kao, Jen-Hsin; Tao, Pao-Luh; Law, Ping-Yee; Loh, Horace H.

    2012-01-01

    Midbrain periaqueductal gray (PAG) and spinal cord dorsal horn are major action sites of opioid analgesics in the pain pathway. Our previous study has shown that opioid antagonists activate MORS196A-CSTA (a mutant of mu-opioid receptor) as full agonists in vitro cell models and naloxone showed antinociceptive effects after the expression of MORS196A-CSTA in the spinal cord in mice. The purpose of this study is to investigate the site directed antinociceptive effects of naloxone in mice injected with dsAAV-MORS196A-CSTA-EGFP at spinal cord or at periaqueductal gray. MORS196A-CSTA-EGFP was administered to ICR mice using dsAAV as vector. We measured MORS196A-CSTA-EGFP expression by detecting the EGFP visualization with a fluorescence microscope. The antinociceptive effect of naloxone was determined by tail-flick test and hot plate test. Drug rewarding effect was evaluated by the conditioned place preference test. Naloxone (10 mg/kg, s.c.) elicited both supraspinal and spinal antinociceptive responses in mice injected with the virus at PAG while only spinal antinociceptive response was observed in mice injected with virus at dorsal horn region. Chronic naloxone treatment did not induce physical dependence or rewarding effect in mice injected with MORS196A-CSTA-EGFP in spinal cord or PAG. These data suggest that the observed naloxone-induced antinociceptive response is the consequence of the local expression of MORS196A-CSTA at specific sites of pain pathway. Injection of such MOR mutant and the systemic administration of naloxone can be a new strategy in the management of chronic pain without the various side effects associated with the use of morphine. PMID:22407757

  7. Effects of high-dose methadone maintenance on cocaine place conditioning, cocaine self-administration, and mu-opioid receptor mRNA expression in the rat brain.

    PubMed

    Leri, Francesco; Zhou, Yan; Goddard, Benjamin; Cummins, Erin; Kreek, Mary Jeanne

    2006-07-01

    Methadone maintenance at appropriate doses can effectively reduce cocaine abuse in heroin-dependent individuals. In the present studies, we investigated the effect of high-dose methadone maintenance cocaine conditioned place preference (CPP) and cocaine intravenous self-administration. Rats implanted with methadone-filled osmotic mini-pumps (20 and 55 mg/kg/day, SC) and conditioned with cocaine (1, 5, and 20 mg/kg, i.p.) did not express cocaine CPP. Similarly, rats implanted with methadone pumps (55 mg/kg/day) after cocaine conditioning (20 mg/kg) displayed neither spontaneous nor cocaine-precipitated (20 mg/kg, i.p.) CPP. In contrast, methadone maintenance (30 and 55 mg/kg/day, SC) did not alter the intravenous self-administration (continuous schedule of reinforcement) of various doses of cocaine (0.1, 0.5, and 2.0 mg/kg/inf). To explore neuropharmacological interactions between methadone maintenance and cocaine conditioning, we quantitatively measured mRNA levels of mu-opioid receptor (MOR) and proopiomelanocortin genes 10 days after methadone maintenance. MOR mRNA levels in both the nucleus accumbens core and frontal cortex were significantly elevated in rats exposed to cocaine during CPP conditioning. However, upregulation of MOR mRNA levels in the nucleus accumbens core were reduced by methadone maintenance in a dose-dependent manner. In conclusion, our results suggest that high-dose methadone maintenance does not alter the direct reinforcing effect of cocaine, but blocks spontaneous and cocaine-precipitated cocaine-seeking, possibly by preventing MOR alterations in the nucleus accumbens core induced by cocaine conditioning.

  8. Buprenorphine maintenance and mu-opioid receptor availability in the treatment of opioid use disorder: implications for clinical use and policy

    PubMed Central

    Greenwald, Mark K.; Comer, Sandra D.; Fiellin, David A.

    2014-01-01

    Background Sublingual formulations of buprenorphine (BUP) and BUP/naloxone have well-established pharmacokinetic and pharmacodynamic profiles, and are safe and effective for treating opioid use disorder. Since approvals of these formulations, their clinical use has increased. Yet, questions have arisen as to how BUP binding to mu-opioid receptors (μORs), the neurobiological target for this medication, relate to its clinical application. BUP produces dose- and time-related alterations of μOR availability but some clinicians express concern about whether doses higher than those needed to prevent opioid withdrawal symptoms are warranted, and policymakers consider limiting reimbursement for certain BUP dosing regimens. Methods We review scientific data concerning BUP-induced changes in μOR availability and their relationship to clinical efficacy. Results Withdrawal suppression appears to require ≤50% μOR availability, associated with BUP trough plasma concentrations ≥1 ng/mL; for most patients, this may require single daily BUP doses of 4-mg to defend against trough levels, or lower divided doses. Blockade of the reinforcing and subjective effects of typical doses of abused opioids require <20% μOR availability, associated with BUP trough plasma concentrations ≥3 ng/mL; for most individuals, this may require single daily BUP doses >16-mg, or lower divided doses. For individuals attempting to surmount this blockade with higher-than-usual doses of abused opioids, even larger BUP doses and <10% μOR availability would be required. Conclusion For these reasons, and given the complexities of studies on this issue and comorbid problems, we conclude that fixed, arbitrary limits on BUP doses in clinical care or limits on reimbursement for this care are unwarranted. PMID:25179217

  9. Increased serum IL-6 level time-dependently regulates hyperalgesia and spinal mu opioid receptor expression during CFA-induced arthritis.

    PubMed

    Tekieh, E; Zaringhalam, Jalal; Manaheji, H; Maghsoudi, N; Alani, B; Zardooz, H

    2011-01-01

    Interleukin (IL)-6 is known to cause pro- and anti-inflammatory effects during different stages of inflammation. Recent therapeutic investigations have focused on treatment of various inflammatory disorders with anti-cytokine substances. As a result, the aim of this study was to further elucidate the influence of IL-6 in hyperalgesia and edema during different stages of Complete Freund's Adjuvant (CFA)-induced arthritis (AA) in male Wistar rats. AA was induced by a single subcutaneous injection of CFA into the rats' hindpaw. Anti-IL-6 was administered either daily or weekly during the 21 days of study. Spinal mu opioid receptor (mOR) expression was detected by Western blotting. Daily and weekly treatment with an anti-IL-6 antibody significantly decreased paw edema in the AA group compared to the AA control group. Additionally, daily and weekly anti-IL-6 administration significantly reduced hyperalgesia on day 7 in the AA group compared to the AA control group; however, there were significant increases in hyperalgesia in the antibody-treated group on days 14 and 21 compared to the AA control group. IL-6 antibody-induced increases in hyperalgesia on the 14(th) and 21(st) days after CFA injection correlated with a time-dependent, significant reduction in spinal mOR expression during anti-IL-6 treatment. Our study confirmed the important time-dependent relationship between serum IL-6 levels and hyperalgesia during AA. These results suggest that the stages of inflammation in AA must be considered for anti-hyperalgesic and anti-inflammatory interventions via anti-IL-6 antibody treatment.

  10. Mu opioid receptor knockdown in the substantia nigra/ventral tegmental area by synthetic small interfering RNA blocks the rewarding and locomotor effects of heroin

    PubMed Central

    Zhang, Yong; Landthaler, Markus; Schlussman, Stefan D.; Yuferov, Vadim; Ho, Ann; Tuschl, Thomas; Kreek, Mary Jeanne

    2014-01-01

    Mu opioid receptors (MOP-r) play an important role in the rewarding and locomotor stimulatory effects of heroin. The aim of the current study was to determine whether infusion of small interfering RNAs (siRNA) targeting MOP-r into the midbrain could knock down MOP-r mRNA and affect heroin-induced locomotor activity or heroin-induced conditioned place preference. Ten week old male C57BL/6J mice were surgically implanted bilaterally with guide cannulae directed between the substantia nigra and ventral tegmental area. After 4 days recovery, mice were infused bilaterally with siRNAs that target the MOP-r (2mM × 0.75 μl/side/day for 3 days) or control siRNA. Seven days after the last infusion, a procedure for conditioned place preference was begun with four heroin (3mg/kg i.p.) administration sessions alternating with four saline sessions. While heroin induced an increase in locomotor activity in all groups, siRNAs targeting specific regions of MOP-r significantly attenuated this effect. Of particular interest, mice infused with specific siRNAs targeting the MOP-r failed to develop and express conditioned place preference to heroin, or showed a significantly attenuated preference. These alterations in reward related behaviors are likely due to the reduction in MOP-r mRNA and protein, shown in separate studies by in situ hybridization and autoradiography using the same MOP-r- siRNA infusions. Taken together, these studies demonstrate the utility of siRNA in the neurobiological study of specific components of the reward system and should contribute to the study of other complex behaviors. PMID:18938225

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

    PubMed

    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, P<0.05), but not at earlier time points (PND 1 and 7). MuR ligand binding was also reduced (by 22%, P<0.05) in the VTA of female JF offspring on PND 28. No effects of perinatal junk 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. The Effect of the [mu]-Opioid Receptor Antagonist Naloxone on Extinction of Conditioned Fear in the Developing Rat

    ERIC Educational Resources Information Center

    Kim, Jee Hyun; Richardson, Rick

    2009-01-01

    Several recent studies report that neurotransmitters that are critically involved in extinction in adult rats are not important for extinction in young rats. Specifically, pretest injection of the [gamma]-aminobutryic acid (GABA) receptor inverse agonist FG7142 has no effect on extinction in postnatal day (P)17 rats, although it reverses…

  13. The Effect of the [mu]-Opioid Receptor Antagonist Naloxone on Extinction of Conditioned Fear in the Developing Rat

    ERIC Educational Resources Information Center

    Kim, Jee Hyun; Richardson, Rick

    2009-01-01

    Several recent studies report that neurotransmitters that are critically involved in extinction in adult rats are not important for extinction in young rats. Specifically, pretest injection of the [gamma]-aminobutryic acid (GABA) receptor inverse agonist FG7142 has no effect on extinction in postnatal day (P)17 rats, although it reverses…

  14. Effects of the Mu opioid receptor polymorphism (OPRM1 A118G) on pain regulation, placebo effects and associated personality trait measures.

    PubMed

    Peciña, Marta; Love, Tiffany; Stohler, Christian S; Goldman, David; Zubieta, Jon-Kar

    2015-03-01

    Mu-opioid receptors (MOPRs) are critically involved in the modulation of pain and analgesia, and represent a candidate mechanism for the development of biomarkers of pain conditions and their responses to treatment. To further understand the human implications of genetic variation within the opioid system in pain and opioid-mediated placebo responses, we investigated the association between the functional single-nucleotide polymorphism (SNP) in the μ-opioid receptor gene (OPRM1), A118G, and psychophysical responses, personality traits, and neurotransmitter systems (dopamine (DA), opioid) related to pain and placebo analgesia. OPRM1 G carriers, compared with AA homozygotes, showed an overall reduction of baseline μ-opioid receptor availability in regions implicated in pain and affective regulation. In response to a sustained painful stimulus, we found no effect of A118G on pain-induced endogenous opioid release. Instead, AA homozygotes showed a blunted DA response in the nucleus accumbens (NAc) in response to the pain challenge. After placebo administration, G carriers showed more pronounced mood disturbances and lower placebo-induced μ-opioid system activation in the anterior insula (aINS), the amygdala (AMY), the NAc, the thalamus (THA), and the brainstem, as well as lower levels of DA D2/3 activation in the NAc. At a trait level, G carriers reported higher NEO-Neuroticism scores; a personality trait previously associated with increased pain and lower placebo responses, which were negatively correlated with baseline μ-opioid receptor availability in the aINS and subgenual anterior cingulate cortex (sgACC). Our results demonstrate that the A118G OPRM1 polymorphism contributes to interindividual variations in the function of neurotransmitters responsive to pain (endogenous opioid and dopamine), as well as their regulation through cognitive-emotional influences in the context of therapeutic expectations, the so-called placebo effect. These effects are relevant to

  15. Effects of the Mu Opioid Receptor Polymorphism (OPRM1 A118G) on Pain Regulation, Placebo Effects and Associated Personality Trait Measures

    PubMed Central

    Peciña, Marta; Love, Tiffany; Stohler, Christian S; Goldman, David; Zubieta, Jon-Kar

    2015-01-01

    Mu-opioid receptors (MOPRs) are critically involved in the modulation of pain and analgesia, and represent a candidate mechanism for the development of biomarkers of pain conditions and their responses to treatment. To further understand the human implications of genetic variation within the opioid system in pain and opioid-mediated placebo responses, we investigated the association between the functional single-nucleotide polymorphism (SNP) in the μ-opioid receptor gene (OPRM1), A118G, and psychophysical responses, personality traits, and neurotransmitter systems (dopamine (DA), opioid) related to pain and placebo analgesia. OPRM1 G carriers, compared with AA homozygotes, showed an overall reduction of baseline μ-opioid receptor availability in regions implicated in pain and affective regulation. In response to a sustained painful stimulus, we found no effect of A118G on pain-induced endogenous opioid release. Instead, AA homozygotes showed a blunted DA response in the nucleus accumbens (NAc) in response to the pain challenge. After placebo administration, G carriers showed more pronounced mood disturbances and lower placebo-induced μ-opioid system activation in the anterior insula (aINS), the amygdala (AMY), the NAc, the thalamus (THA), and the brainstem, as well as lower levels of DA D2/3 activation in the NAc. At a trait level, G carriers reported higher NEO-Neuroticism scores; a personality trait previously associated with increased pain and lower placebo responses, which were negatively correlated with baseline μ-opioid receptor availability in the aINS and subgenual anterior cingulate cortex (sgACC). Our results demonstrate that the A118G OPRM1 polymorphism contributes to interindividual variations in the function of neurotransmitters responsive to pain (endogenous opioid and dopamine), as well as their regulation through cognitive-emotional influences in the context of therapeutic expectations, the so-called placebo effect. These effects are relevant to

  16. Mu opioid receptor gene as a candidate for the study of obsessive compulsive disorder with and without tics.

    PubMed

    Urraca, N; Camarena, B; Gómez-Caudillo, L; Esmer, M C; Nicolini, H

    2004-05-15

    Obsessive compulsive disorder (OCD) is a complex psychiatric disease characterized by recurring obsessions or compulsions that cause significant distress to the patient. The etiology of this disorder remains largely unknown, although a genetic component has been suggested. Many candidates genes have been evaluated based on a possible serotoninergic and dopaminergic brain dysfunction. We postulate the micro opioid receptor (MOR) gene as a candidate because some observations support a role of the opioid system in OCD. The opioid antagonist, naloxone, rapidly exacerbates OCD symptoms and the opioid agonist, tramadol, was reported to be effective in the treatment of some patients. We studied two single nucleotide polymorphisms (C17T and A118G) in 51 trios with OCD. Genotyping was analyzed with transmission desequilibrium test (TDT). The allelic variant +17T of the C17T polymorphism had a low frequency (1%) in our population that did not allow for statistic analysis. However, for the allelic variant +G of the A118G polymorphism we were able to performed statistical comparisons. Our results showed a trend toward significance (chi(2) McNemar = 3.6, P = 0.065) for TDT in patients with comorbid tics. It is an interesting finding that should be tested in a larger sample of OCD patients with tics.

  17. Expression of delta- and mu-opioid receptors in the ventricular and subventricular zones of the developing human neocortex.

    PubMed

    Tripathi, Anushree; Khurshid, Nazia; Kumar, Praveen; Iyengar, Soumya

    2008-07-01

    Recent research has documented the involvement of the endogenous opioid system in neural development, including neurogenesis and neuronal differentiation. However, the expression of opioid receptors (ORs) in different cell types of the human ventricular and subventricular zones (VZ and SVZ) has not been studied during early gestation. In the present study, we have used immunohistochemistry and quantified the results to demonstrate that the levels of delta- and mu-OR subtypes were high in the VZ and SVZ between 11 and 16 gestation weeks (GW) and decreased by 20GW. These results have also been confirmed by studying OR mRNA expression in the VZ and SVZ. Both delta- and mu-OR subtypes were expressed by multipotential stem cells, newly differentiated neurons and developing glial cells to different extents. However, migrating neurons expressed negligible levels of both OR subtypes. Our results suggest that the opioid system may affect cellular proliferation and/or differentiation of stem cells into neurons and glia during the first and second trimesters of gestation in humans. Since layers II and III of the cerebral cortex are being formed during the second trimester, their development is most likely affected by the opioid system mediated through delta- and mu-ORs.

  18. Naloxegol: the first orally administered, peripherally acting, mu opioid receptor antagonist, approved for the treatment of opioid-induced constipation.

    PubMed

    Corsetti, M; Tack, J

    2015-08-01

    Treatment of opioid-induced constipation (OIC) is becoming a relevant clinical challenge as most of the treatments demonstrated to be more effective than placebo in treating OIC have safety issues limiting a broad clinical application. Naloxegol is the first orally administered, peripherally acting, µ opioid receptor antagonist approved by the FDA and EMA specifically for the treatment of noncancer patients with OIC. This review summarizes the results of the studies regarding the effects of naloxegol in OIC. Pharmacodynamic studies have demonstrated that naloxegol was able to inhibit gastrointestinal opioid effects while preserving central analgesic actions. Phase II and phase III studies in patients with noncancer OIC have confirmed the efficacy of naloxegol to inhibit OIC, and the most consistent efficacy was seen with the 25-mg dose once daily. Side effects were mainly gastrointestinal in origin (and usually transient and mild) and there were no signs of opioid withdrawal in the studies. Safety and tolerability were shown in a long-term safety study. Considering its efficacy, safety, route of administration and the limitations of most of the other available treatments, naloxegol has the potential to become the first-line treatment for noncancer patients with OIC.

  19. Nitric oxide and histone deacetylases modulate cocaine-induced mu-opioid receptor levels in PC12 cells

    PubMed Central

    2012-01-01

    Background Cocaine exposure has been reported to alter central μ-opioid receptor (MOR) expression in vivo. The present study employed an in vitro cellular model to explore possible mechanisms that may be involved in this action of cocaine. Methods To assess the effects of cocaine on MOR levels, two treatment regimens were tested in PC12 cells: single continuous or multiple intermittent. MOR protein levels were assessed by western blot analysis and quantitative PCR was used to determine relative MOR mRNA expression levels. To evaluate the role of nitric oxide (NO) and histone acetylation in cocaine-induced MOR expression, cells were pre-treated with the NO synthase inhibitor Nω-nitro-L-arginine methylester (L-NAME) or the non-selective histone acetyltransferase inhibitor curcumin. Results Both cocaine treatment regimens significantly increased MOR protein levels and protein stability, but only multiple intermittent treatments increased MOR mRNA levels as well as c-fos mRNA levels and activator protein 1 binding activity. Both regimens increased NO production, and pre-treatment with L-NAME prevented cocaine-induced increases in MOR protein and mRNA levels. Single and multiple cocaine treatment regimens inhibited histone deacetylase activity, and pre-treatment with curcumin prevented cocaine-induced up-regulation of MOR protein expression. Conclusions In the PC12 cell model, both NO and histone deacetylase activity regulate cocaine-induced MOR expression at both the transcriptional and post-transcriptional levels. Based on these novel findings, it is hypothesized that epigenetic mechanisms are implicated in cocaine’s action on MOR expression in neurons. PMID:23079001

  20. mu-Opioid receptor stimulation in the nucleus accumbens elevates fatty tastant intake by increasing palatability and suppressing satiety signals.

    PubMed

    Katsuura, Yoshihiro; Heckmann, Jennifer A; Taha, Sharif A

    2011-07-01

    Infusion of a μ-opioid receptor (MOR) agonist into the nucleus accumbens (NAcc) drives voracious food intake, an effect hypothesized to occur through increased tastant palatability. While intake of many palatable foods is elevated by MOR stimulation, this manipulation has a preferential effect on fatty food ingestion. Consumption of high-fat foods is increased by NAcc MOR stimulation even in rats that prefer a carbohydrate-rich alternative under baseline conditions. This suggests that NAcc MOR stimulation may not simply potentiate palatability signals and raises the possibility that mechanisms mediating fat intake may be distinct from those underlying intake of other tastants. The present study was conducted to investigate the physiological mechanisms underlying the effects of NAcc MOR stimulation on fatty food intake. In experiment 1, we analyzed lick microstructure in rats ingesting Intralipid to identify the changes underlying feeding induced by infusion of a MOR-specific agonist into the NAcc. MOR stimulation in the NAcc core, but not shell, increased burst duration and first-minute licks, while simultaneously increasing the rate and duration of Intralipid ingestion. These results suggest that MOR activation in the core increases Intralipid palatability and attenuates inhibitory postingestive feedback. In experiment 2, we measured the effects of MOR stimulation in the NAcc core on consumption of nonnutritive olestra. A MOR-specific agonist dose dependently increased olestra intake, demonstrating that caloric signaling is not required for hyperphagia induced by NAcc MOR stimulation. Feeding induced by drug infusion in both experiments 1 and 2 was blocked by a MOR antagonist. In experiment 3, we determined whether MOR activation in the NAcc core could attenuate satiety-related signaling caused by infusion of the melanocortin agonist MTII into the third ventricle. Suppression of intake caused by MTII was reversed by MOR stimulation. Together, our results suggest

  1. Prohormone convertase 2 (PC2) null mice have increased mu opioid receptor levels accompanied by altered morphine-induced antinociception, tolerance and dependence.

    PubMed

    Lutfy, K; Parikh, D; Lee, D L; Liu, Y; Ferrini, M G; Hamid, A; Friedman, T C

    2016-08-04

    Chronic morphine treatment increases the levels of prohormone convertase 2 (PC2) in brain regions involved in nociception, tolerance and dependence. Thus, we tested if PC2 null mice exhibit altered morphine-induced antinociception, tolerance and dependence. PC2 null mice and their wild-type controls were tested for baseline hot plate latency, injected with morphine (1.25-10mg/kg) and tested for antinociception 30min later. For tolerance studies, mice were tested in the hot plate test before and 30min following morphine (5mg/kg) on day 1. Mice then received an additional dose so that the final dose of morphine was 10mg/kg on this day. On days 2-4, mice received additional doses of morphine (20, 40 and 80mg/kg on days 1, 2, 3, and 4, respectively). On day 5, mice were tested in the hot plate test before and 30min following morphine (5mg/kg). For withdrawal studies, mice were treated with the escalating doses of morphine (10, 20, 40 and 80mg/kg) for 4days, implanted with a morphine pellet on day 5 and 3 days later injected with naloxone (1mg/kg) and signs of withdrawal were recorded. Morphine dose-dependently induced antinociception and the magnitude of this response was greater in PC2 null mice. Tolerance to morphine was observed in wild-type mice and this phenomenon was blunted in PC2 null mice. Withdrawal signs were also reduced in PC2 null mice. Immunohistochemical studies showed up-regulation of the mu opioid receptor (MOP) protein expression in the periaqueductal gray area, ventral tegmental area, lateral hypothalamus, medial hypothalamus, nucleus accumbens, and somatosensory cortex in PC2 null mice. Likewise, naloxone specific binding was increased in the brains of these mice compared to their wild-type controls. The results suggest that the PC2-derived peptides may play a functional role in morphine-induced antinociception, tolerance and dependence. Alternatively, lack of opioid peptides led to up-regulation of the MOP and altered morphine

  2. From the potent and selective mu opioid receptor agonist H-Dmt-d-Arg-Phe-Lys-NH(2) to the potent delta antagonist H-Dmt-Tic-Phe-Lys(Z)-OH.

    PubMed

    Balboni, Gianfranco; Cocco, Maria Teresa; Salvadori, Severo; Romagnoli, Romeo; Sasaki, Yusuke; Okada, Yoshio; Bryant, Sharon D; Jinsmaa, Yunden; Lazarus, Lawrence H

    2005-08-25

    H-Dmt-d-Arg-Phe-Lys-NH(2) ([Dmt(1)]DALDA) binds with high affinity and selectivity to the mu opioid receptor and is a potent and long-acting analgesic. Substitution of d-Arg in position 2 with Tic and masking of the lysine amine side chain by Z protection and of the C-terminal carboxylic function instead of the amide function transform a potent and selective mu agonist into a potent and selective delta antagonist H-Dmt-Tic-Phe-Lys(Z)-OH. Such a delta antagonist could be used as a pharmacological tool.

  3. Genetic Variation of the Mu Opioid Receptor (OPRM1) and Dopamine D2 Receptor (DRD2) is Related to Smoking Differences in Patients with Schizophrenia but not Bipolar Disorder.

    PubMed

    Hirasawa-Fujita, Mika; Bly, Michael J; Ellingrod, Vicki L; Dalack, Gregory W; Domino, Edward F

    It is not known why mentally ill persons smoke excessively. Inasmuch as endogenous opioid and dopaminergic systems are involved in smoking reinforcement, it is important to study mu opioid receptor (OPRM1) A118G (rs1799971), dopamine D2 receptor (DRD2) Taq1A (rs1800497) genotypes, and sex differences among patients with schizophrenia or bipolar disorder. Smokers and nonsmokers with schizophrenia (n=177) and bipolar disorder (n=113) were recruited and genotyped. They were classified into three groups: current smoker, former smoker, and never smoker by tobacco smoking status self-report. The number of cigarettes smoked per day was used as the major tobacco smoking parameter. In patients with schizophrenia, tobacco smoking prevalence was greater in males than in females as expected, but women had greater daily cigarette consumption (p<0.01). Subjects with schizophrenia who had the OPRM1 *G genotype smoked more cigarettes per day than the AA allele carriers with schizophrenia (p<0.05). DRD2 Taq1A genotype differences had no effect on the number of cigarettes smoked per day. However, female smokers with schizophrenia who were GG homozygous of the DRD2 receptor smoked more than the *A male smokers with schizophrenia (p<0.05). In bipolar patients, there were no OPRM1 and DRD2 Taq1A genotype differences in smoking status. There also were no sex differences for smoking behavior among the bipolar patients. The results of this study indicate that single nucleotide polymorphism (SNP) of the less functional mu opioid receptor increases tobacco smoking in patients with schizophrenia. Alteration of DRD2 receptor function also increased smoking behavior in females with schizophrenia.

  4. Genetic Variation of the Mu Opioid Receptor (OPRM1) and Dopamine D2 Receptor (DRD2) is Related to Smoking Differences in Patients with Schizophrenia but not Bipolar Disorder

    PubMed Central

    Hirasawa-Fujita, Mika; Bly, Michael J.; Ellingrod, Vicki L.; Dalack, Gregory W.; Domino, Edward F.

    2015-01-01

    It is not known why mentally ill persons smoke excessively. Inasmuch as endogenous opioid and dopaminergic systems are involved in smoking reinforcement, it is important to study mu opioid receptor (OPRM1) A118G (rs1799971), dopamine D2 receptor (DRD2) Taq1A (rs1800497) genotypes, and sex differences among patients with schizophrenia or bipolar disorder. Smokers and nonsmokers with schizophrenia (177) and bipolar disorder (113) were recruited and genotyped. They were classified into three groups: current smoker, former smoker, and never smoker by tobacco smoking status self-report. The number of cigarettes smoked per day was used as the major tobacco smoking parameter. In patients with schizophrenia, tobacco smoking prevalence was greater in males than in females as expected, but women had greater daily cigarette consumption (p<0.01). Subjects with schizophrenia who had the OPRM1 *G genotype smoked more cigarettes per day than the AA allele carriers with schizophrenia (p<0.05). DRD2 Taq1A genotype differences had no effect on the number of cigarettes smoked per day. However, female smokers with schizophrenia who were GG homozygous with the DRD2 receptor smoked more than the *A male smokers with schizophrenia (p<0.05). In bipolar patients, there were no OPRM1 and DRD2 Taq1A genotype differences in smoking status. There also were no sex differences for smoking behavior among the bipolar patients. The results of this study indicate that single nucleotide polymorphism (SNP) of the less functional mu opioid receptor increases tobacco smoking in patients with schizophrenia. Alteration of DRD2 receptor function also increased smoking behavior in females with schizophrenia. PMID:28548579

  5. Synthesis and characterization of potent and selective mu-opioid receptor antagonists, [Dmt(1), D-2-Nal(4)]endomorphin-1 (Antanal-1) and [Dmt(1), D-2-Nal(4)]endomorphin-2 (Antanal-2).

    PubMed

    Fichna, Jakub; do-Rego, Jean-Claude; Chung, Nga N; Lemieux, Carole; Schiller, Peter W; Poels, Jeroen; Broeck, Jozef Vanden; Costentin, Jean; Janecka, Anna

    2007-02-08

    To synthesize potent antagonists of the mu-opioid receptor, we prepared a series of endomorphin-1 and endomorphin-2 analogues with 3-(1-naphthyl)-d-alanine (d-1-Nal) or 3-(2-naphthyl)-d-alanine (d-2-Nal) in position 4. Some of these analogues displayed weak antagonist properties. We tried to strengthen these properties by introducing the structurally modified tyrosine residue 2,6-dimethyltyrosine (Dmt) in place of Tyr1. Among the synthesized compounds, [Dmt1, d-2-Nal4]endomorphin-1, designated antanal-1, and [Dmt1, d-2-Nal4]endomorphin-2, designated antanal-2, turned out to be highly potent and selective mu-opioid receptor antagonists, as judged on the basis of two functional assays, the receptor binding assay and the hot plate test of analgesia. Interestingly, another analogue of this series, [Dmt1, d-1-Nal4]endomorphin-1, turned out to be a moderately potent mixed mu-agonist/delta-antagonist.

  6. Dual single-scission event analysis of constitutive transferrin receptor (TfR) endocytosis and ligand-triggered β2-adrenergic receptor (β2AR) or Mu-opioid receptor (MOR) endocytosis

    PubMed Central

    Lampe, Marko; Pierre, Fabienne; Al-Sabah, Suleiman; Krasel, Cornelius; Merrifield, Christien J.

    2014-01-01

    The dynamic relationship between constitutive and ligand-triggered clathrin-mediated endocytosis is only poorly characterized, and it remains controversial whether clathrin-coated pits specialize to internalize particular receptor cargo. Here we analyzed the ligand-triggered endocytosis of the model G-protein–coupled receptors (GPCRs) β2-adrenergic receptor (β2AR) and Mu-opioid receptor (MOR) at the level of individual endocytic events using a total internal reflection fluorescence microscopy (TIRFM)–based assay. Similar to the constitutive endocytosis of transferrin receptor (TfR), ligand- triggered endocytosis of β2AR occurs via quantized scission events hosted by clathrin spots and plaques of variable size and persistence. To address whether clathrin-coated structures (CCSs) specialize to internalize particular GPCRs, we adapted the TIRFM imaging assay to simultaneously quantify the internalization of TfR and the ligand- triggered endocytosis of the β2AR or MOR. Agonist-triggered β2AR or MOR endocytosis extended the maturation time of CCSs, as shown previously, but did not affect the rate of constitutive TfR endocytosis or loading of TfR into individual endocytic vesicles. Both the β2AR and the MOR receptors entered cells in the same vesicles as TfR, and the overall evidence for CCS specialization was weak. These data support a simple model in which different cargoes internalize through common CCSs. PMID:25079691

  7. Dual single-scission event analysis of constitutive transferrin receptor (TfR) endocytosis and ligand-triggered β2-adrenergic receptor (β2AR) or Mu-opioid receptor (MOR) endocytosis.

    PubMed

    Lampe, Marko; Pierre, Fabienne; Al-Sabah, Suleiman; Krasel, Cornelius; Merrifield, Christien J

    2014-10-01

    The dynamic relationship between constitutive and ligand-triggered clathrin-mediated endocytosis is only poorly characterized, and it remains controversial whether clathrin-coated pits specialize to internalize particular receptor cargo. Here we analyzed the ligand-triggered endocytosis of the model G-protein-coupled receptors (GPCRs) β2-adrenergic receptor (β2AR) and Mu-opioid receptor (MOR) at the level of individual endocytic events using a total internal reflection fluorescence microscopy (TIRFM)-based assay. Similar to the constitutive endocytosis of transferrin receptor (TfR), ligand- triggered endocytosis of β2AR occurs via quantized scission events hosted by clathrin spots and plaques of variable size and persistence. To address whether clathrin-coated structures (CCSs) specialize to internalize particular GPCRs, we adapted the TIRFM imaging assay to simultaneously quantify the internalization of TfR and the ligand- triggered endocytosis of the β2AR or MOR. Agonist-triggered β2AR or MOR endocytosis extended the maturation time of CCSs, as shown previously, but did not affect the rate of constitutive TfR endocytosis or loading of TfR into individual endocytic vesicles. Both the β2AR and the MOR receptors entered cells in the same vesicles as TfR, and the overall evidence for CCS specialization was weak. These data support a simple model in which different cargoes internalize through common CCSs.

  8. Lack of association of a single-nucleotide polymorphism of the mu-opioid receptor gene with anxiety-related traits: results from a cross-sectional study of adults and a longitudinal study of children.

    PubMed

    Jorm, Anthony F; Prior, Margot; Sanson, Ann; Smart, Diana; Zhang, Yafei; Tan, Susan; Easteal, Simon

    2002-08-08

    There is evidence from animal experiments that the mu- and delta-opioid receptors may play a role in anxiety and depression. It might therefore be expected that functional polymorphisms of these genes in humans are associated with anxiety and depression. We investigated a single-nucleotide polymorphism (Asn40Asp) of the mu-opioid receptor gene (OPRM1). This association was investigated in two samples: 1) a cross-sectional survey of 867 community-living adults aged 18-79 years who were assessed for anxiety and depression symptoms and related personality traits; and 2) a longitudinal study of childhood temperament in which 660 children were followed from infancy to the mid-teens and assessed for anxiety-related temperament and behavior problems. The data did not support a role for the Asn40Asp polymorphism in anxiety and depression, despite adequate statistical power to detect small effects. Copyright 2002 Wiley-Liss, Inc.

  9. Buprenorphine-induced antinociception is mediated by mu-opioid receptors and compromised by concomitant activation of opioid receptor-like receptors.

    PubMed

    Lutfy, Kabirullah; Eitan, Shoshana; Bryant, Camron D; Yang, Yu C; Saliminejad, Nazli; Walwyn, Wendy; Kieffer, Brigitte L; Takeshima, Hiroshi; Carroll, F Ivy; Maidment, Nigel T; Evans, Christopher J

    2003-11-12

    Buprenorphine is a mixed opioid receptor agonist-antagonist used clinically for maintenance therapy in opiate addicts and pain management. Dose-response curves for buprenorphine-induced antinociception display ceiling effects or are bell shaped, which have been attributed to the partial agonist activity of buprenorphine at opioid receptors. Recently, buprenorphine has been shown to activate opioid receptor-like (ORL-1) receptors, also known as OP4 receptors. Here we demonstrate that buprenorphine, but not morphine, activates mitogen-activated protein kinase and Akt via ORL-1 receptors. Because the ORL-1 receptor agonist orphanin FQ/nociceptin blocks opioid-induced antinociception, we tested the hypothesis that buprenorphine-induced antinociception might be compromised by concomitant activation of ORL-1 receptors. In support of this hypothesis, the antinociceptive effect of buprenorphine, but not morphine, was markedly enhanced in mice lacking ORL-1 receptors using the tail-flick assay. Additional support for a modulatory role for ORL-1 receptors in buprenorphine-induced antinociception was that coadministration of J-113397, an ORL-1 receptor antagonist, enhanced the antinociceptive efficacy of buprenorphine in wild-type mice but not in mice lacking ORL-1 receptors. The ORL-1 antagonist also eliminated the bell-shaped dose-response curve for buprenorphine-induced antinociception in wild-type mice. Although buprenorphine has been shown to interact with multiple opioid receptors, mice lacking micro-opioid receptors failed to exhibit antinociception after buprenorphine administration. Our results indicate that the antinociceptive effect of buprenorphine in mice is micro-opioid receptor-mediated yet severely compromised by concomitant activation of ORL-1 receptors.

  10. Association of polymorphisms in nicotinic acetylcholine receptor alpha 4 subunit gene (CHRNA4), mu-opioid receptor gene (OPRM1), and ethanol-metabolizing enzyme genes with alcoholism in Korean patients.

    PubMed

    Kim, Soon Ae; Kim, Jong-Woo; Song, Ji-Young; Park, Sunny; Lee, Hee Jae; Chung, Joo-Ho

    2004-01-01

    Findings obtained from several studies indicate that ethanol enhances the activity of alpha4beta2 neuronal nicotinic acetylcholine receptor and support the possibility that a polymorphism of the nicotinic acetylcholine receptor alpha4 subunit gene (CHRNA4) modulates enhancement of nicotinic receptor function by ethanol. To identify the association between the CfoI polymorphism of the CHRNA4 and alcoholism, we examined distribution of genotypes and allele frequencies in Korean patients diagnosed with alcoholism (n = 127) and Korean control subjects without alcoholism (n = 185) with polymerase chain reaction-restriction fragment length polymorphism methods. We were able to detect the association between the CfoI polymorphism of the CHRNA4 and alcoholism in Korean patients (genotype P = .023; allele frequency P = .047). The genotypes and allele frequencies of known polymorphisms in other alcoholism candidate genes, such as alcohol metabolism-related genes [alcohol dehydrogenase 2 (ADH2), aldehyde dehydrogenase 2 (ALDH2), alcohol dehydrogenase 3 (ADH3), and cytochrome P450 2E1 (CYP2E1)] and mu-opioid receptor gene (OPRM1), were studied. The polymorphisms of ADH2, ALDH2, and CYP2E1 were significantly different in Korean patients with alcoholism and Korean control subjects without alcoholism, but ADH3 and OPRM1 did not differ between the two groups.

  11. 17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-(4'-pyridylcarboxamido)morphinan (NAP) Modulating the Mu Opioid Receptor in a Biased Fashion.

    PubMed

    Zhang, Yan; Williams, Dwight A; Zaidi, Saheem A; Yuan, Yunyun; Braithwaite, Amanda; Bilsky, Edward J; Dewey, William L; Akbarali, Hamid I; Streicher, John M; Selley, Dana E

    2016-03-16

    Mounting evidence has suggested that G protein-coupled receptors can be stabilized in multiple conformations in response to distinct ligands, which exert discrete functions through selective activation of various downstream signaling events. In accordance with this concept, we report biased signaling of one C6-heterocyclic substituted naltrexamine derivative, namely, 17-cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-(4'-pyridylcarboxamido)morphinan (NAP) at the mu opioid receptor (MOR). NAP acted as a low efficacy MOR partial agonist in the G protein-mediated [(35)S]GTPγS binding assay, whereas it did not significantly induce calcium flux or β-arrestin2 recruitment. In contrast, it potently blocked MOR full agonist-induced β-arrestin2 recruitment and translocation. Additionally, NAP dose-dependently antagonized MOR full agonist-induced intracellular calcium flux and β-arrestin2 recruitment. Further results in an isolated organ bath preparation confirmed that NAP reversed the morphine-induced reduction in colon motility. Ligand docking and dynamics simulation studies of NAP at the MOR provided more supporting evidence for biased signaling of NAP at an atomic level. Due to the fact that NAP is MOR selective and preferentially distributed peripherally upon systemic administration while β-arrestin2 is reportedly required for impairment of intestinal motility by morphine, biased antagonism of β-arrestin2 recruitment by NAP further supports its utility as a treatment for opioid-induced constipation.

  12. Design, Synthesis, and Biological Evaluation of 17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-[(4’-pyridyl)carboxamido]morphinan Derivatives as Peripheral Selective Mu Opioid Receptor Agents

    PubMed Central

    Yuan, Yunyun; Elbegdorj, Orgil; Chen, Jianyang; Akubathini, Shashidhar K.; Zhang, Feng; Stevens, David L.; Beletskaya, Irina O.; Scoggins, Krista L.; Zhang, Zhenxian; Gerk, Phillip M.; Selley, Dana E.; Akbarali, Hamid I.; Dewey, William L.; Zhang, Yan

    2012-01-01

    Peripheral selective mu opioid receptor (MOR) antagonists could alleviate the symptoms of opioid-induced constipation (OIC) without compromising the analgesic effect of opioids. However, a variety of adverse effects were associated with them, partially due to their relatively low MOR selectivity. NAP, a 6β-N-4'-pyridyl substituted naltrexamine derivative, was identified previously as a potent and highly selective MOR antagonist mainly acting within the peripheral nervous system. The noticeable diarrhea associated with it prompted the design and synthesis of its analogues in order to study its structure activity relationship. Among them, compound 8 showed improved pharmacological profiles compared to the original lead, acting mainly at peripheral while increasing the intestinal motility in morphine-pelleted mice (ED50=0.03 mg/kg). The slight decrease of the ED50 compared to the original lead was well compensated by the unobserved adverse effect. Hence, this compound seems to be a more promising lead to develop novel therapeutic agents toward OIC. PMID:23116124

  13. The mu-opioid receptor antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP) [but not D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP)] produces a nonopioid receptor-mediated increase in K+ conductance of rat locus ceruleus neurons.

    PubMed

    Chieng, B; Connor, M; Christie, M J

    1996-09-01

    The somatostatin analogues D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP) and D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) have been used widely as selective antagonists of mu-opioid receptors. Actions of CTOP and CTAP on the membrane properties of rat locus ceruleus neurons were studied using intracellular recordings of membrane currents in superfused brain slices. CTOP increased a K+ conductance with an EC50 of 560 nM. The maximal conductance increase produced by CTOP (10 microM) was similar to that produced by high concentrations of the mu-opioid agonists D-Ala-Met-enkephalinglyol (1 microM) and Met-enkephalin (10 microM), as well as an alpha 2-adrenoceptor agonist (UK14304, 3 microM) and somatostatin (1 microM). The K+ current produced by CTOP was not antagonized by naloxone (1 microM), suggesting it was not mediated by mu-opioid receptors. The K+ currents induced by high concentrations of CTOP desensitized to 42% of the initial maximum after prolonged superfusion (t1/2 = 247 sec). In the presence of fully desensitized CTOP responses, somatostatin (1 microM) still produced near-maximal K+ currents; i.e., there was no cross-desensitization, which suggests that CTOP might act on a receptor distinct from somatostatin receptors. However, the converse did not apply; high concentrations of CTOP (30 microM) did not produce any additional current in the presence of desensitized somatostatin responses. No cross-desensitization was observed between CTOP (10-30 microM) and Met-enkephalin (30 microM) or nociceptin (3 microM) regardless of the order of drug application. Cyclo-(7-aminoheptanoyl-Phe-D-Trp-Lys-Thr[Bzl], antagonized both somatostatin-(KD = 10 microM) and CTOP-(KD = 8 microM) induced K+ currents with similar potency. Concentrations of CTOP (100 nM) that produced a small K+ current partially antagonized the actions of Met-enkephalin (10 microM) on mu-opioid receptors. In contrast to CTOP, CTAP produced no K+ current at concentrations of 300 nM and 1 microM and

  14. /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

    SciTech Connect

    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.

  15. Behavioral and Cellular Pharmacology Characterization of 17-cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α-(isoquinoline-3′-carboxamido)morphinan (NAQ) as a Mu Opioid Receptor Selective Ligand

    PubMed Central

    Zhang, Yan; Braithwaite, Amanda; Yuan, Yunyun; Streicher, John M.; Bilsky, Edward J.

    2014-01-01

    Mu opioid receptor (MOR) selective antagonists and partial agonists have been used for the treatment of opioid abuse and addiction. Our recent efforts on the identification of MOR antagonists have provided several novel leads displaying interesting pharmacological profiles. Among them, 17-cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α-[(3'-isoquinolyl)acetamido]morphinan (NAQ) showed sub-nanomolar binding affinity to the MOR with significant selectivity over the delta opioid receptor (DOR) and the kappa opioid receptor (KOR). Its central nervous system penetration capacity together with marginal agonism in the MOR–GTPγS binding assay made it a very interesting molecule for developing novel opioid abuse and addiction therapeutic agents. Therefore, further pharmacological characterization was conducted to fully understand its biological profile. At the molecular and cellular level, NAQ not only induced no translocation of β-arrestin2 to the MOR, but also efficaciously antagonized the effect of DAMGO in MOR-βarr2eGFP-U2OS cells in the β-arrestin2 recruitment assay. At the in vivo level, NAQ displayed a potent inhibition of the analgesic effect of morphine in the tail-flick assay (ID50 = 1.19 mg/kg). NAQ (10 mg/kg) also significantly decreased the hyper-locomotion induced by acute morphine without inducing any vertical jumps. Meanwhile NAQ precipitated lesser withdrawal symptoms in morphine dependent mice than naloxone. In conclusion, NAQ may represent a new chemical entity for opioid abuse and addiction treatment. PMID:24815322

  16. Behavioral and cellular pharmacology characterization of 17-cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α-(isoquinoline-3'-carboxamido)morphinan (NAQ) as a mu opioid receptor selective ligand.

    PubMed

    Zhang, Yan; Braithwaite, Amanda; Yuan, Yunyun; Streicher, John M; Bilsky, Edward J

    2014-08-05

    Mu opioid receptor (MOR) selective antagonists and partial agonists have been used for the treatment of opioid abuse and addiction. Our recent efforts on the identification of MOR antagonists have provided several novel leads displaying interesting pharmacological profiles. Among them, 17-cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α-[(3'-isoquinolyl)acetamido]morphinan (NAQ) showed sub-nanomolar binding affinity to the MOR with significant selectivity over the delta opioid receptor (DOR) and the kappa opioid receptor (KOR). Its central nervous system penetration capacity together with marginal agonism in the MOR-GTPγS binding assay made it a very interesting molecule for developing novel opioid abuse and addiction therapeutic agents. Therefore, further pharmacological characterization was conducted to fully understand its biological profile. At the molecular and cellular level, NAQ not only induced no translocation of β-arrestin2 to the MOR, but also efficaciously antagonized the effect of DAMGO in MOR-βarr2eGFP-U2OS cells in the β-arrestin2 recruitment assay. At the in vivo level, NAQ displayed a potent inhibition of the analgesic effect of morphine in the tail-flick assay (ID50=1.19 mg/kg). NAQ (10 mg/kg) also significantly decreased the hyper-locomotion induced by acute morphine without inducing any vertical jumps. Meanwhile NAQ precipitated lesser withdrawal symptoms in morphine dependent mice than naloxone. In conclusion, NAQ may represent a new chemical entity for opioid abuse and addiction treatment.

  17. Beta-arrestin2 and c-Src regulate the constitutive activity and recycling of mu opioid receptors in dorsal root ganglion neurons.

    PubMed

    Walwyn, Wendy; Evans, Christopher J; Hales, Tim G

    2007-05-09

    Beta-arrestins bind to agonist-activated G-protein-coupled receptors regulating signaling events and initiating endocytosis. In beta-arrestin2-/- (beta arr2-/-) mice, a complex phenotype is observed that includes altered sensitivity to morphine. However, little is known of how beta-arrestin2 affects mu receptor signaling. We investigated the coupling of mu receptors to voltage-gated Ca2+ channels (VGCCs) in beta arr2+/+ and beta arr2-/- dorsal root ganglion neurons. A lack of beta-arrestin2 reduced the maximum inhibition of VGCCs by morphine and DAMGO (D-Ala2-N-Me-Phe4-glycol5-enkephalin) without affecting agonist potency, the onset of receptor desensitization, or the functional contribution of N-type VGCCs. The reduction in inhibition was accompanied by increased naltrexone-sensitive constitutive inhibitory coupling of mu receptors to VGCCs. Agonist-independent mu receptor inhibitory coupling was insensitive to CTAP (Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2), a neutral antagonist that inhibited the inverse agonist action of naltrexone. These functional changes were accompanied by diminished constitutive recycling and increased cell-surface mu receptor expression in beta arr2-/- compared with beta arr2+/+ neurons. Such changes could not be explained by the classical role of beta-arrestins in agonist-induced endocytosis. The localization of the nonreceptor tyrosine kinase c-Src appeared disrupted in beta arr2-/- neurons, and there was reduced activation of c-Src by DAMGO. Using the Src inhibitor PP2 [4-amino-5-(4-chlorophenyl)-(t-butyl)pyrazolo[3,4-d]pyrimidine], we demonstrated that defective Src signaling mimics the beta arr2-/- cellular phenotype of reduced mu agonist efficacy, increased constitutive mu receptor activity, and reduced constitutive recycling. We propose that beta-arrestin2 is required to target c-Src to constitutively active mu receptors, resulting in their internalization, providing another dimension to the complex role of beta-arrestin2 and c-Src in G

  18. Correlation of stable elevations in striatal mu-opioid receptor availability in detoxified alcoholic patients with alcohol craving: a positron emission tomography study using carbon 11-labeled carfentanil.

    PubMed

    Heinz, Andreas; Reimold, Matthias; Wrase, Jana; Hermann, Derik; Croissant, Bernhard; Mundle, Götz; Dohmen, Bernhard M; Braus, Dieter F; Braus, Dieter H; Schumann, Gunter; Machulla, Hans-Jürgen; Bares, Roland; Mann, Karl

    2005-01-01

    The pleasant effects of food and alcohol intake are partially mediated by mu-opiate receptors in the ventral striatum, a central area of the brain reward system. Blockade of mu-opiate receptors with naltrexone reduces the relapse risk among some but not all alcoholic individuals. To test the hypothesis that alcohol craving is pronounced among alcoholic individuals with a high availability of mu-opiate receptors in the brain reward system. Patients and comparison sample. The availability of central mu-opiate receptors was measured in vivo with positron emission tomography (PET) and the radioligand carbon 11-labeled carfentanil in the ventral striatum and compared with the severity of alcohol craving as assessed by the Obsessive Compulsive Drinking Scale (OCDS). Hospitalized care. Volunteer sample of 25 male alcohol-dependent inpatients assessed after detoxification of whom 12 underwent PET again 5 weeks later. Control group of 10 healthy men. After 1 to 3 weeks of abstinence, the availability of mu-opiate receptors in the ventral striatum, including the nucleus accumbens, was significantly elevated in alcoholic patients compared with healthy controls and remained elevated when 12 alcoholic patients had these levels measured 5 weeks later (P<.05 corrected for multiple testing). Higher availability of mu-opiate receptors in this brain area correlated significantly with the intensity of alcohol craving as assessed by the OCDS. Abstinent alcoholic patients displayed an increase in mu-opiate receptors in the ventral striatum, including the nucleus accumbens, which correlated with the severity of alcohol craving. These findings point to a neuronal correlate of alcohol urges.

  19. Association of time-dependent changes in mu opioid receptor mRNA, but not BDNF, TrkB, or MeCP2 mRNA and protein expression in the rat nucleus accumbens with incubation of heroin craving

    PubMed Central

    Theberge, Florence R. M.; Pickens, Charles L.; Goldart, Evan; Fanous, Sanya; Hope, Bruce T.; Liu, Qing-Rong

    2013-01-01

    Rationale and objectives Responding to heroin cues progressively increases after cessation of heroin self-administration (incubation of heroin craving). We investigated whether this incubation is associated with time-dependent changes in brain-derived neurotrophic factor (BDNF) and methyl-CpG binding protein 2 (MeCP2) signaling and mu opioid receptor (MOR) expression in nucleus accumbens (NAc), dorsal striatum (DS), and medial pre-frontal cortex (mPFC). We also investigated the effect of the preferential MOR antagonist naloxone on cue-induced heroin seeking during abstinence. Methods We trained rats to self-administer heroin or saline for 9–10 days and then dissected the NAc, DS, and mPFC at different abstinence days and measured mRNA and protein levels of BDNF, TrkB, and MeCP2, as well as MOR mRNA (Oprm1). In other groups, we assessed cue-induced heroin seeking in extinction tests after 1, 11, and 30 abstinence days, and naloxone’s (0–1.0 mg/kg) effect on extinction responding after 1 and 15 days. Results Cue-induced heroin seeking progressively increased or incubated during abstinence. This incubation was not associated with changes in BDNF, TrkB, or MeCP2 mRNA or protein levels in NAc, DS, or mPFC; additionally, no molecular changes were observed after extinction tests on day 11. In NAc, but not DS or mPFC, MOR mRNA decreased on abstinence day 1 and returned to basal levels over time. Naloxone significantly decreased cue-induced heroin seeking after 15 abstinence days but not 1 day. Conclusions Results suggest a role of MOR in incubation of heroin craving. As previous studies implicated NAc BDNF in incubation of cocaine craving, our data suggest that different mechanisms contribute to incubation of heroin versus cocaine craving. PMID:22790874

  20. Knockdown of ventral tegmental area mu-opioid receptors in rats prevents effects of social defeat stress: Implications for amphetamine cross-sensitization, social avoidance, weight regulation and expression of brain-derived neurotrophic factor

    PubMed Central

    Johnston, Caitlin E.; Herschel, Daniel; Lasek, Amy W.; Hammer, Ronald P.; Nikulina, Ella M.

    2014-01-01

    Social defeat stress causes social avoidance and long-lasting cross-sensitization to psychostimulants, both of which are associated with increased brain-derived neurotrophic factor (BDNF) expression in the ventral tegmental area (VTA). Moreover, social stress upregulates VTA mu-opioid receptor (MOR) mRNA. In the VTA, MOR activation inhibits GABA neurons to disinhibit VTA dopamine neurons, thus providing a role for VTA MORs in the regulation of psychostimulant sensitization. The present study determined the effect of lentivirus-mediated MOR knockdown in the VTA on the consequences of intermittent social defeat stress, a salient and profound stressor in humans and rodents. Social stress exposure induced social avoidance and attenuated weight gain in animals with non-manipulated VTA MORs, but both these effects were prevented by VTA MOR knockdown. Rats with non-manipulated VTA MOR expression exhibited cross-sensitization to amphetamine challenge (1.0 mg/kg, i.p.), evidenced by a significant augmentation of locomotion. By contrast, knockdown of VTA MORs prevented stress-induced cross-sensitization without blunting the locomotor-activating effects of amphetamine. At the time point corresponding to amphetamine challenge, immunohistochemical analysis was performed to examine the effect of stress on VTA BDNF expression. Prior stress exposure increased VTA BDNF expression in rats with non-manipulated VTA MOR expression, while VTA MOR knockdown prevented stress-induced expression of VTA BDNF. Taken together, these results suggest that upregulation of VTA MOR is necessary for the behavioral and biochemical changes induced by social defeat stress. Elucidating VTA MOR regulation of stress effects on the mesolimbic system may provide new therapeutic targets for treating stress-induced vulnerability to substance abuse. PMID:25446676

  1. Knockdown of ventral tegmental area mu-opioid receptors in rats prevents effects of social defeat stress: implications for amphetamine cross-sensitization, social avoidance, weight regulation and expression of brain-derived neurotrophic factor.

    PubMed

    Johnston, Caitlin E; Herschel, Daniel J; Lasek, Amy W; Hammer, Ronald P; Nikulina, Ella M

    2015-02-01

    Social defeat stress causes social avoidance and long-lasting cross-sensitization to psychostimulants, both of which are associated with increased brain-derived neurotrophic factor (BDNF) expression in the ventral tegmental area (VTA). Moreover, social stress upregulates VTA mu-opioid receptor (MOR) mRNA. In the VTA, MOR activation inhibits GABA neurons to disinhibit VTA dopamine neurons, thus providing a role for VTA MORs in the regulation of psychostimulant sensitization. The present study determined the effect of lentivirus-mediated MOR knockdown in the VTA on the consequences of intermittent social defeat stress, a salient and profound stressor in humans and rodents. Social stress exposure induced social avoidance and attenuated weight gain in animals with non-manipulated VTA MORs, but both these effects were prevented by VTA MOR knockdown. Rats with non-manipulated VTA MOR expression exhibited cross-sensitization to amphetamine challenge (1.0 mg/kg, i.p.), evidenced by a significant augmentation of locomotion. By contrast, knockdown of VTA MORs prevented stress-induced cross-sensitization without blunting the locomotor-activating effects of amphetamine. At the time point corresponding to amphetamine challenge, immunohistochemical analysis was performed to examine the effect of stress on VTA BDNF expression. Prior stress exposure increased VTA BDNF expression in rats with non-manipulated VTA MOR expression, while VTA MOR knockdown prevented stress-induced expression of VTA BDNF. Taken together, these results suggest that upregulation of VTA MOR is necessary for the behavioral and biochemical changes induced by social defeat stress. Elucidating VTA MOR regulation of stress effects on the mesolimbic system may provide new therapeutic targets for treating stress-induced vulnerability to substance abuse.

  2. Broad-spectrum analgesic efficacy of IBNtxA is mediated by exon 11-associated splice variants of the mu-opioid receptor gene.

    PubMed

    Wieskopf, Jeffrey S; Pan, Ying-Xian; Marcovitz, Jaclyn; Tuttle, Alexander H; Majumdar, Susruta; Pidakala, John; Pasternak, Gavril W; Mogil, Jeffrey S

    2014-10-01

    μ-Opioids remain vastly important for the treatment of pain, and would represent ideal analgesics if their analgesic effects could be separated from their many side effects. A recently synthesized compound, iodobenzoylnaltrexamide (IBNtxA), acting at 6-transmembrane (6-TM) splice variants of the μ-opioid receptor gene, was shown to have potent analgesic actions against acute, thermal pain accompanied by a vastly improved side-effect profile compared to 7-TM-acting drugs such as morphine. Whether such analgesia can be seen in longer-lasting and nonthermal algesiometric assays is not known. The current study demonstrates potent and efficacious IBNtxA inhibition of a wide variety of assays, including inflammatory and neuropathic hypersensitivity and spontaneous pain. We further demonstrate the dependence of such analgesia on 6-TM μ-opioid receptor variants using isobolographic analysis and the testing of Oprm1 (the μ-opioid receptor gene) exon 11 null mutant mice. Finally, the effect of nerve damage (spared nerve injury) and inflammatory injury (complete Freund's adjuvant) on expression of μ-opioid receptor variant genes in pain-relevant central nervous system loci was examined, revealing a downregulation of the mMOR-1D splice variant in the dorsal root ganglion after spared nerve injury. These findings are supportive of the potential value of 6-TM-acting drugs as novel analgesics.

  3. New 2',6'-dimethyl-L-tyrosine (Dmt) opioid peptidomimetics based on the Aba-Gly scaffold. Development of unique mu-opioid receptor ligands.

    PubMed

    Ballet, Steven; Salvadori, Severo; Trapella, Claudio; Bryant, Sharon D; Jinsmaa, Yunden; Lazarus, Lawrence H; Negri, Lucia; Giannini, Elisa; Lattanzi, Roberta; Tourwé, Dirk; Balboni, Gianfranco

    2006-06-29

    The Aba-Gly scaffold, incorporated into Dmt-Tic ligands (H-Dmt-Tic-Gly-NH-CH2-Ph, H-Dmt-Tic-Gly-NH-Ph, H-Dmt-Tic-NH-CH2-Bid), exhibited mixed micro/delta or delta opioid receptor activities with micro agonism. Substitution of Tic by Aba-Gly coupled to -NH-CH2-Ph (1), -NH-Ph (2), or -Bid (Bid=1H-benzimidazole-2-yl) (3) shifted affinity (Ki(micro)=0.46, 1.48, and 19.9 nM, respectively), selectivity, and bioactivity to micro-opioid receptors. These compounds represent templates for a new class of lead opioid agonists that are easily synthesized and suitable for therapeutic pain relief.

  4. Redoubling the ring size of an endomorphin-2 analog transforms a centrally acting mu-opioid receptor agonist into a pure peripheral analgesic.

    PubMed

    Piekielna, Justyna; De Marco, Rossella; Gentilucci, Luca; Cerlesi, Maria Camilla; Calo', Girolamo; Tömböly, Csaba; Artali, Roberto; Janecka, Anna

    2016-05-01

    The study reports the synthesis and biological evaluation of two opioid analogs, a monomer and a dimer, obtained as products of the solid-phase, side-chain to side-chain cyclization of the pentapeptide Tyr-d-Lys-Phe-Phe-AspNH2 . The binding affinities to the mu, delta, and kappa opioid receptors, as well as results obtained in a calcium mobilization functional assay are reported. Tyr-[d-Lys-Phe-Phe-Asp]2 -NH2 1 was a potent and selective full agonist of mu with sub-nanomolar affinity, while the dimer (Tyr-[d-Lys-Phe-Phe-Asp]2 -NH2 )2 2 showed a significant mixed mu/kappa affinity, acting as an agonist at the mu. Molecular docking computations were utilized to explain the ability of the dimeric cyclopeptide 2 to interact with the receptor. Interestingly, in spite of the increased ring size, the higher flexibility allowed 2 to fold and fit into the mu receptor binding pocket. Both cyclopeptides were shown to elicit strong antinociceptive activity after intraventricular injection but only cyclomonomer 1 was able to cross the blood-brain barrier. However, the cyclodimer 2 displayed a potent peripheral antinociceptive activity in a mouse model of visceral inflammatory pain. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 309-317, 2016.

  5. In vivo antinociception of potent mu opioid agonist tetrapeptide analogues and comparison with a compact opioid agonist - neurokinin 1 receptor antagonist chimera

    PubMed Central

    2012-01-01

    Background An important limiting factor in the development of centrally acting pharmaceuticals is the blood-brain barrier (BBB). Transport of therapeutic peptides through this highly protective physiological barrier remains a challenge for peptide drug delivery into the central nervous system (CNS). Because the most common strategy to treat moderate to severe pain consists of the activation of opioid receptors in the brain, the development of active opioid peptide analogues as potential analgesics requires compounds with a high resistance to enzymatic degradation and an ability to cross the BBB. Results Herein we report that tetrapeptide analogues of the type H-Dmt1-Xxx2-Yyy3-Gly4-NH2 are transported into the brain after intravenous and subcutaneous administration and are able to activate the μ- and δ opioid receptors more efficiently and over longer periods of time than morphine. Using the hot water tail flick test as the animal model for antinociception, a comparison in potency is presented between a side chain conformationally constrained analogue containing the benzazepine ring (BVD03, Yyy3: Aba), and a "ring opened" analogue (BVD02, Yyy3: Phe). The results show that in addition to the increased lipophilicity through amide bond N-methylation, the conformational constraint introduced at the level of the Phe3 side chain causes a prolonged antinociception. Further replacement of NMe-D-Ala2 by D-Arg2 in the tetrapeptide sequence led to an improved potency as demonstrated by a higher and maintained antinociception for AN81 (Xxx2: D-Arg) vs. BVD03 (Xxx2: NMe-D-Ala). A daily injection of the studied opioid ligands over a time period of 5 days did however result in a substantial decrease in antinociception on the fifth day of the experiment. The compact opioid agonist - NK1 antagonist hybrid SBCHM01 could not circumvent opioid induced tolerance. Conclusions We demonstrated that the introduction of a conformational constraint has an important impact on opioid receptor

  6. Robust age, but limited sex, differences in mu-opioid receptors in the rat brain: relevance for reward and drug-seeking behaviors in juveniles.

    PubMed

    Smith, Caroline J W; Ratnaseelan, Aarane M; Veenema, Alexa H

    2017-09-04

    In the brain, the µ-opioid receptor (MOR) is involved in reward-seeking behaviors and plays a pivotal role in the mediation of opioid use disorders. Furthermore, reward-seeking behaviors and susceptibility to opioid addiction are particularly evident during the juvenile period, with a higher incidence of opioid use in males and higher sensitivity to opioids in females. Despite these age and sex differences in MOR-mediated behaviors, little is known regarding potential age and sex differences in the expression of MORs in the brain. Here, we used receptor autoradiography to compare MOR binding densities between juvenile and adult male and female rats. Age differences were found in MOR binding density in 12 out of 33 brain regions analyzed, with 11 regions showing higher MOR binding density in juveniles than in adults. These include the lateral septum, as well as sub-regions of the bed nucleus of the stria terminalis, hippocampus, and thalamus. Sex differences in MOR binding density were observed in only two brain regions, namely, the lateral septum (higher in males) and the posterior cortical nucleus of the amygdala (higher in females). Overall, these findings provide an important foundation for the generation of hypotheses regarding differential functional roles of MOR activation in juveniles versus adults. Specifically, we discuss the possibility that higher MOR binding densities in juveniles may allow for higher MOR activation, which could facilitate behaviors that are heightened during the juvenile period, such as reward and drug-seeking behaviors.

  7. Involvement of Mu Opioid Receptor Signaling in the Protective Effect of Opioid against 6-Hydroxydopamine-Induced SH-SY5Y Human Neuroblastoma Cells Apoptosis

    PubMed Central

    Eftekhar-Vaghefi, Shahrzad; Esmaeili-Mahani, Saeed; Elyasi, Leila; Abbasnejad, Mehdi

    2015-01-01

    Introduction: The neuroprotective role of opioid morphine against 6-hydroxydopamine-induced cell death has been demonstrated. However, the exact mechanism(s) underlying such neuroprotection, especially the role of subtype receptors, has not yet been fully clarified. Methods: Here, we investigated the effects of different opioid agonists on 6-OHDA-induced neurotoxicity in human neuroblastoma SH-SY5Y cell line as an in vitro model of Parkinson’s disease. Cell damage was induced by 150 μM 6-OHDA and the cells viability was examined by MTT assay. Intracellular calcium, reactive oxygen species and mitochondrial membrane potential were assessed by fluorescence spectrophotometry method. Immunoblot technique was used to evaluate cytochrome-c and activated caspase-3 as biochemical markers of apoptosis induction. Results: The data showed that 6-OHDA caused significant cell damage, loss of mitochondrial membrane potential and increase in intracellular reactive oxygen species and calcium levels as well as activated caspase-3 and cytochrome-c release. Incubation of SH-SY5Y cells with μ-opioid agonists, morphine and DAMGO, but not with δ-opioid agonist, DADLE, elicited protective effect and reduced biochemical markers of cell damage and death. Discussion: The results suggest that μ-opioid receptors signaling participate in the opioid neuroprotective effects against 6-OHDA-induced neurotoxicity. PMID:26904174

  8. Measurement of central mu-opioid receptor binding in vivo with PET and [11C]carfentanil: a test-retest study in healthy subjects.

    PubMed

    Hirvonen, Jussi; Aalto, Sargo; Hagelberg, Nora; Maksimow, Anu; Ingman, Kimmo; Oikonen, Vesa; Virkkala, Jussi; Någren, Kjell; Scheinin, Harry

    2009-02-01

    [(11)C]Carfentanil has been widely used in positron emission tomography (PET) studies for measuring micro-opioid receptor binding in humans, but the reproducibility of the binding parameter estimates is unknown. Eight healthy volunteers were scanned twice during the same day with [(11)C]carfentanil PET, and binding to receptors was assessed with both reference tissue and arterial plasma input-based models using region of interest (ROI) and voxel-based quantification. The two-tissue compartmental model distribution volume (V(T)) was highly reproducible as indicated by low variability (VAR < 6%) and high intraclass correlation coefficients (ICC > 0.93). BP(ND) (BP relative to the nondisplaceable tissue compartment) was also highly reproducible (VAR < 10%, ICC > 0.90) both at ROI- and voxel-level, and reference tissue-based models provided stable estimates after 40 min. The reproducibility of [(11)C]carfentanil binding parameter estimates is excellent with outcome measures based on both arterial plasma and reference tissue input, and a scanning time of 40 min appears sufficient.

  9. [Dmt(1)]DALDA is highly selective and potent at mu opioid receptors, but is not cross-tolerant with systemic morphine.

    PubMed

    Riba, Pal; Ben, Yong; Nguyen, Thi M-D; Furst, Susanna; Schiller, Peter W; Lee, Nancy M

    2002-01-01

    The clinical effectiveness of morphine is limited by several side effects, including the development of tolerance and dependence. Most of these side effects are believed to be mediated by central opioid receptors; therefore, hydrophilic opioids, which don't cross the blood-brain barrier, may have advantages over morphine in some clinical applications. We recently synthesized several analogues of DALDA (Tyr-D-Arg-Phe-Lys-NH2), a highly hydrophilic peptide derived from the endogenous opioid peptide dermorphin; all of them, particularly [Dmt(1)] DALDA (Dmt - 2',6'-dimethyl tyrosine), had high potency and selectivity at mu receptors, the target of morphine, in activity assays. Here we report the pharmacological characterization of [Dmt(1)] DALDA in the whole animal. [Dmt(1)]DALDA was 40 times more potent than morphine in inducing antinociception in mice when both drugs were given s.c., and 6-14 times more potent than DAMGO, a selective m agonist, when both drugs were given it. However, [Dmt(1)]DALDA showed poor cross-tolerance to morphine; thus chronic morphine treatment of animals increased the antinociceptive AD(50) of systemic [Dmt(1)]DALDA two fold or less, as compared to an 8-9-fold increase for morphine and a 4-5-fold increase for DAMGO. The antinociceptive activity of [Dmt(1)]DALDA (i.t) was blocked by CTAP, a selective mu antagonist, but not by TIPP psi, a selective delta antagonist, nor by nor-BNI, a selective kappa antagonist. [Dmt(1)]DALDA-induced antinociception was also blocked by naloxone methiodide, an antagonist that does not cross the blood-brain barrier, when agonist and antagonist were given i.t. or i.c.v., but not when they were given s.c. We conclude that [Dmt(1)] DALDA is a highly potent analgesic acting at mu receptors. Though it appears to penetrate the blood-brain barrier, it exhibits low cross-tolerance to morphine, suggesting that it may have advantages over the latter in certain clinical applications.

  10. Alvimopan: an oral, peripherally acting, mu-opioid receptor antagonist for the treatment of opioid-induced bowel dysfunction--a 21-day treatment-randomized clinical trial.

    PubMed

    Paulson, Daniel M; Kennedy, Daniel T; Donovick, Roger A; Carpenter, Randall L; Cherubini, Maryann; Techner, Lee; Du, Wei; Ma, Yuju; Schmidt, William K; Wallin, Bruce; Jackson, David

    2005-03-01

    Alvimopan has been shown to reverse the inhibitory effect of opioids on gastrointestinal transit without affecting analgesia. We evaluated oral alvimopan, 0.5 or 1 mg, versus placebo, once daily for 21 days, in 168 patients with opioid-induced bowel dysfunction (OBD) who were receiving chronic opioid therapy (minimum, 1 month) for nonmalignant pain (n = 148) or opioid dependence (n = 20). The primary outcome was the proportion of patients having at least one bowel movement (BM) within 8 hours of study drug on each day during the 21-day treatment period. Averaged over the 21-day treatment period, 54%, 43%, and 29% of patients had a BM within 8 hours after alvimopan 1 mg, 0.5 mg, or placebo, respectively (P < .001). Secondary outcomes of median times to first BM were 3, 7, and 21 hours after initial doses of 1 mg, 0.5 mg, and placebo, respectively (P < .001; 1 mg vs placebo). Weekly BMs and overall patient satisfaction were increased after the 1-mg dose (P < .001 at weeks 1 and 2 vs placebo, and P = .046, respectively). Treatment-emergent adverse events were primarily bowel-related, occurred during the first week of treatment, and were of mild to moderate severity. Alvimopan was generally well tolerated and did not antagonize opioid analgesia. Patients treated with chronic opioid therapy often experience opioid-induced bowel dysfunction as a result of undesirable effects on peripheral opioid receptors located in the gastrointestinal tract. Alvimopan, a novel peripheral opioid mu-receptor antagonist, has demonstrated significant efficacy for the management of opioid-induced bowel dysfunction without compromise of centrally mediated opioid-induced analgesia.

  11. Anti-nociceptive action of peripheral mu-opioid receptors by G-beta-gamma protein-mediated inhibition of TRPM3 channels.

    PubMed

    Dembla, Sandeep; Behrendt, Marc; Mohr, Florian; Goecke, Christian; Sondermann, Julia; Schneider, Franziska M; Schmidt, Marlene; Stab, Julia; Enzeroth, Raissa; Leitner, Michael G; Nuñez-Badinez, Paulina; Schwenk, Jochen; Nürnberg, Bernd; Cohen, Alejandro; Philipp, Stephan E; Greffrath, Wolfgang; Bünemann, Moritz; Oliver, Dominik; Zakharian, Eleonora; Schmidt, Manuela; Oberwinkler, Johannes

    2017-08-15

    Opioids, agonists of µ-opioid receptors (µORs), are the strongest pain killers clinically available. Their action includes a strong central component, which also causes important adverse effects. However, µORs are also found on the peripheral endings of nociceptors and their activation there produces meaningful analgesia. The cellular mechanisms downstream of peripheral µORs are not well understood. Here we show in neurons of murine dorsal root ganglion cells that pro-nociceptive TRPM3 channels, present in the peripheral parts of nociceptors, are strongly inhibited by µOR activation, much more than other TRP channels in the same compartment, like TRPV1 and TRPA1. Inhibition of TRPM3 channels occurs via a short signaling cascade involving Gβγ proteins, which form a complex with TRPM3. Accordingly, activation of peripheral µORs in vivo strongly attenuates TRPM3-dependent pain. Our data establish TRPM3 inhibition as important consequence of peripheral µOR activation indicating that pharmacologically antagonizing TRPM3 may be a useful analgesic strategy.

  12. Dual growth of adolescent smoking and drinking: evidence for an interaction between the mu-opioid receptor (OPRM1) A118G polymorphism and sex.

    PubMed

    Kleinjan, Marloes; Poelen, Evelien A; Engels, Rutger C M E; Verhagen, Maaike

    2013-11-01

    Smoking and alcohol use often co-occur during adolescence, but little is known about the codevelopment of these substances. In the search for etiological factors that help to explain the development of adolescent substance use patterns, studies have revealed substantial heritability for both alcohol use and smoking. In this regard, the µ-opioid receptor gene (OPRM1, chromosome 6q24-q25) has been linked to both substances. This study examined the predictive relationships between initial level and growth of smoking and drinking in 311 early adolescents (13-15 years old) over a 4-year period. In addition, the effects of the A118G polymorphism of the OPRM1 gene on the initial values and the development over time of alcohol use and smoking were assessed. Finally, as prevalence and heritability estimates for both alcohol- and smoking-related behaviors differ between males and females, OPRM1 by sex interactions were tested. We found that high initial levels of early adolescent alcohol consumption were related to a stronger increase in smoking levels over time. In contrast, high initial levels of smoking were not related to growth of alcohol use. No main OPRM1 effects were found, but sex-specificity of the gene was found for smoking development. Male A-allele carriers showed a faster development in smoking behavior, whereas in females, the G-allele led to a faster development in smoking. Thus, in addition to high levels of alcohol as a risk factor for the development of smoking behavior, sex-specific effects exist for OPRM1, which may additionally have consequences for the development of adolescent smoking.

  13. Elevated mu-opioid receptor expression in the nucleus of the solitary tract accompanies attenuated withdrawal signs after chronic low dose naltrexone in opiate-dependent rats.

    PubMed

    Van Bockstaele, E J; Rudoy, C; Mannelli, P; Oropeza, V; Qian, Y

    2006-02-15

    We previously described a decrease in withdrawal behaviors in opiate-dependent rats that were chronically treated with very low doses of naltrexone in their drinking water. Attenuated expression of withdrawal behaviors correlated with decreased c-Fos expression and intracellular signal transduction elements [protein kinase A regulatory subunit II (PKA) and phosphorylated cAMP response element binding protein (pCREB)] in brainstem noradrenergic nuclei. In this study, to determine whether similar cellular changes occurred in forebrain nuclei associated with drug reward, expressions of PKA and pCREB were analyzed in the ventral tegmental area, frontal cortex, striatum, and amygdala of opiate-treated rats that received low doses of naltrexone in their drinking water. No significant difference in PKA or pCREB was detected in these regions following drug treatment. To examine further the cellular mechanisms in noradrenergic nuclei that could underlie attenuated withdrawal behaviors following low dose naltrexone administration, the nucleus of the solitary tract (NTS) and locus coeruleus (LC) were examined for opioid receptor (OR) protein expression. Results showed a significant increase in muOR expression in the NTS of morphine-dependent rats that received low doses of naltrexone in their drinking water, and increases in muOR expression were also found to be dose dependent. Protein expression of muOR in the LC and deltaOR in either brain region remained unchanged. In conclusion, our previously reported decreases in c-Fos and PKA expression in the NTS following pretreatment with low doses of naltrexone may be partially explained by a greater inhibition of NTS neurons resulting from increased muOR expression in this region.

  14. Melatonin enhances antinociceptive effects of delta-, but not mu-opioid agonist in mice.

    PubMed

    Li, Shi-rong; Wang, Ting; Wang, Rui; Dai, Xu; Chen, Qiang; Li, Ren-de

    2005-05-10

    This present study examines the effect of melatonin on antinociceptive action induced by opioid agonists in mice using the tail-flick test. When injected either by intraperitoneal (i.p.) (1, 5, 25 mg/kg) or by intracerebroventricular (i.c.v.) (0.25, 0.5, 1 mg/kg) routes, melatonin significantly enhanced the delta-opioid agonist deltorphin I induced antinociception, but not mu-opioid agonist endomorphin-1. Further investigation showed that i.c.v. luzindole (0.5 mg/kg) (an antagonist of melatonin receptor) significantly antagonized the enhanced antinociceptive effect of i.c.v. melatonin. These results demonstrated that melatonin can specifically enhance the antinociception induced by specific opioid receptor agonist (i.e., delta opioid agonist) acting on melatonin receptor and that melatonin may have augmentation effect on analgesia with delta-, but not mu-opioid agonists in mice.

  15. Mu Opioid Mediated Discriminative-Stimulus Effects of Tramadol: An Individual Subjects Analysis

    PubMed Central

    Strickland, Justin C.; Rush, Craig R.; Stoops, William W.

    2015-01-01

    Drug discrimination procedures use dose-dependent generalization, substitution, and pretreatment with selective agonists and antagonists to evaluate receptor systems mediating interoceptive effects of drugs. Despite the extensive use of these techniques in the nonhuman animal literature, few studies have used human subjects. Specifically, human studies have not routinely used antagonist administration as a pharmacological tool to elucidate the mechanisms mediating the discriminative stimulus effects of drugs. This study evaluated the discriminative-stimulus effects of tramadol, an atypical analgesic with monoamine and mu opioid activity. Three human subjects first learned to discriminate 100 mg tramadol from placebo. A range of tramadol doses (25 to 150 mg) and hydromorphone (4 mg) with and without naltrexone pretreatment (50 mg) were then administered to subjects after acquiring the discrimination. Tramadol produced dose-dependent increases in drug-appropriate responding and hydromorphone partially or fully substituted for tramadol in all subjects. These effects were attenuated by naltrexone. Individual subject records indicated a relationship between mu opioid activity (i.e., miosis) and drug discrimination performance. Our findings indicate that mu opioid activity may mediate the discriminative-stimulus effects of tramadol in humans. The correspondence of generalization, substitution, and pretreatment findings with the animal literature supports the neuropharmacological specificity of the drug discrimination procedure. PMID:25664525

  16. Mu opioid mediated discriminative-stimulus effects of tramadol: an individual subjects analysis.

    PubMed

    Strickland, Justin C; Rush, Craig R; Stoops, William W

    2015-03-01

    Drug discrimination procedures use dose-dependent generalization, substitution, and pretreatment with selective agonists and antagonists to evaluate receptor systems mediating interoceptive effects of drugs. Despite the extensive use of these techniques in the nonhuman animal literature, few studies have used human participants. Specifically, human studies have not routinely used antagonist administration as a pharmacological tool to elucidate the mechanisms mediating the discriminative stimulus effects of drugs. This study evaluated the discriminative-stimulus effects of tramadol, an atypical analgesic with monoamine and mu opioid activity. Three human participants first learned to discriminate 100 mg tramadol from placebo. A range of tramadol doses (25 to 150 mg) and hydromorphone (4 mg) with and without naltrexone pretreatment (50 mg) were then administered to participants after they acquired the discrimination. Tramadol produced dose-dependent increases in drug-appropriate responding and hydromorphone partially or fully substituted for tramadol in all participants. These effects were attenuated by naltrexone. Individual participant records indicated a relationship between mu opioid activity (i.e., miosis) and drug discrimination performance. Our findings indicate that mu opioid activity may mediate the discriminative-stimulus effects of tramadol in humans. The correspondence of generalization, substitution, and pretreatment findings with the animal literature supports the neuropharmacological specificity of the drug discrimination procedure. © Society for the Experimental Analysis of Behavior.

  17. Truncated mu opioid GPCR variant involvement in opioid-dependent and opioid-independent pain modulatory systems within the CNS

    PubMed Central

    Marrone, Gina F.; Grinnell, Steven G.; Lu, Zhigang; Rossi, Grace C.; Le Rouzic, Valerie; Xu, Jin; Majumdar, Susruta; Pan, Ying-Xian; Pasternak, Gavril W.

    2016-01-01

    The clinical management of severe pain depends heavily on opioids acting through mu opioid receptors encoded by the Oprm1 gene, which undergoes extensive alternative splicing. In addition to generating a series of prototypic seven transmembrane domain (7TM) G protein-coupled receptors (GPCRs), Oprm1 also produces a set of truncated splice variants containing only six transmembrane domains (6TM) through which selected opioids such as IBNtxA (3′-iodobenzoyl-6β-naltrexamide) mediate a potent analgesia without many undesirable effects. Although morphine analgesia is independent of these 6TM mu receptor isoforms, we now show that the selective loss of the 6TM variants in a knockout model eliminates the analgesic actions of delta and kappa opioids and of α2-adrenergic compounds, but not cannabinoid, neurotensin, or muscarinic drugs. These observations were confirmed by using antisense paradigms. Despite their role in analgesia, loss of the 6TM variants were not involved with delta opioid-induced seizure activity, aversion to the kappa drug U50,488H, or α2-mediated hypolocomotion. These observations support the existence of parallel opioid and nonopioid pain modulatory systems and highlight the ability to dissociate unwanted delta, kappa1, and α2 actions from analgesia. PMID:26976581

  18. Truncated mu opioid GPCR variant involvement in opioid-dependent and opioid-independent pain modulatory systems within the CNS.

    PubMed

    Marrone, Gina F; Grinnell, Steven G; Lu, Zhigang; Rossi, Grace C; Le Rouzic, Valerie; Xu, Jin; Majumdar, Susruta; Pan, Ying-Xian; Pasternak, Gavril W

    2016-03-29

    The clinical management of severe pain depends heavily on opioids acting through mu opioid receptors encoded by the Oprm1 gene, which undergoes extensive alternative splicing. In addition to generating a series of prototypic seven transmembrane domain (7TM) G protein-coupled receptors (GPCRs), Oprm1 also produces a set of truncated splice variants containing only six transmembrane domains (6TM) through which selected opioids such as IBNtxA (3'-iodobenzoyl-6β-naltrexamide) mediate a potent analgesia without many undesirable effects. Although morphine analgesia is independent of these 6TM mu receptor isoforms, we now show that the selective loss of the 6TM variants in a knockout model eliminates the analgesic actions of delta and kappa opioids and of α2-adrenergic compounds, but not cannabinoid, neurotensin, or muscarinic drugs. These observations were confirmed by using antisense paradigms. Despite their role in analgesia, loss of the 6TM variants were not involved with delta opioid-induced seizure activity, aversion to the kappa drug U50, 488H, or α2-mediated hypolocomotion. These observations support the existence of parallel opioid and nonopioid pain modulatory systems and highlight the ability to dissociate unwanted delta, kappa1, and α2 actions from analgesia.

  19. (/sup 3/H)(D-Ala2,NMePhe4,Gly-ol5)-enkephalin (mu-opioid) binding in beige-J mice

    SciTech Connect

    Raffa, R.B.; Baldy, W.J. Jr.; Shank, R.P.; Mathiasen, J.R.; Vaught, J.L.

    1988-05-01

    Tritiated (D-Ala2,NMePhe4,Gly-ol5)-enkephalin ((3H)DAGO) was used to examine mu-opioid receptor number and mu-ligand binding in brain synaptic membranes (P2 fraction) from C57BL/6J-bgJ/bgJ (beige-J) mice, a strain with combined deficiencies in immunological function (resembling Chediak-Higashi syndrome) and analgesic response to mu-opioid agonists such as morphine and DAGO. As controls, white mice, beige-J littermates (normally responsive to mu-opioid agonists), and a known mu-deficient strain (CXBK) were also examined. Neither the KD (0.47 to 0.49 nM) nor the Bmax (153 to 168 fmol/mg protein) determined for beige-J mice was significantly different from values determined for littermates or white mice. In contrast, the Bmax of CXBK mice (66 fmol/mg protein) was clearly less than that of the other strains. The analgesic defect of beige-J mice, therefore, is not likely due to an insufficient number of mu-opioid receptors, as it presumably is in CXBK mice. Carbachol (200 micrograms/ml), which partly corrects the analgesic defect of beige-J mice, had no effect on (3H)DAGO binding either acutely in vitro or chronically ex vivo after administration to beige-J mice for three weeks. Hence, the analgesic defect of beige-J mice appears to be due to some defect in the mu-opioid receptor-effector coupling mechanism or to some endogenous substance that inhibits binding of mu-opioid ligands to otherwise functional receptors.

  20. Detection of mu opioid receptor (MOPR) and its glycosylation in rat and mouse brains by western blot with anti-μC, an affinity-purified polyclonal anti-MOPR antibody.

    PubMed

    Huang, Peng; Chen, Chongguang; Liu-Chen, Lee-Yuan

    2015-01-01

    Our experience demonstrates that it is difficult to identify MOPR in rat and mouse brains by western blot, in part due to low abundance of the receptor and a wide relative molecular mass (Mr) range of the receptor associated with its heterogeneous glycosylation states. Here, we describe generation and purification of anti-μC (a rabbit polyclonal anti-MOPR antibody), characterization of its specificity in immunoblotting of HA-tagged MOPR expressed in a cell line, and ultimately, unequivocal detection of the MOPR in brain tissues by western blot with multiple rigorous controls. In particular, using brain tissues from MOPR knockout (K/O) mice as the negative controls allowed unambiguous identification of the MOPR band, since the anti-MOPR antibody, even after affinity purification, recognizes nonspecific protein bands. The MOPR was resolved as a faint, broad, and diffuse band with a wide Mr range of 58-84 kDa depending on brain regions and species. Upon deglycosylation to remove N-linked glycans by PNGase F (but not Endo H), the MOPR became a dense and sharp band with Mr of ~43 kDa, close to the theoretical Mr of its deduced amino acid sequences. Thus, MOPRs in rodent brains are differentially glycosylated by complex type of N-linked glycans in brain region- and species-specific manners. Furthermore, we characterized the MOPR in an A112G/N38D-MOPR knockin mouse model that possesses the equivalent substitution of the A118G/N40D SNP in the human MOPR gene. The substitution removes one of the four and five N-linked consensus glycosylation sites of the mouse and human MOPR, respectively. We demonstrated that the Mr of the MOPR in A112G mouse brains was lower than that in wild-type mouse brains, and that the difference was due to lower degrees of N-linked glycosylation.

  1. Phosphorylation of poly(rC) binding protein 1 (PCBP1) contributes to stabilization of mu opioid receptor (MOR) mRNA via interaction with AU-rich element RNA-binding protein 1 (AUF1) and poly A binding protein (PABP)

    PubMed Central

    Hwang, Cheol Kyu; Wagley, Yadav; Law, Ping-Yee; Wei, Li-Na; Loh, Horace H.

    2016-01-01

    Gene regulation at the post-transcriptional level is frequently based on cis- and trans-acting factors on target mRNAs. We found a C-rich element (CRE) in mu-opioid receptor (MOR) 3′-untranslated region (UTR) to which poly (rC) binding protein 1 (PCBP1) binds, resulting in MOR mRNA stabilization. RNA immunoprecipitation and RNA EMSA revealed the formation of PCBP1-RNA complexes at the element. Knockdown of PCBP1 decreased MOR mRNA half-life and protein expression. Stimulation by forskolin increased cytoplasmic localization of PCBP1 and PCBP1/MOR 3′-UTR interactions via increased serine phosphorylation that was blocked by protein kinase A (PKA) or (phosphatidyl inositol-3) PI3-kinase inhibitors. The forskolin treatment also enhanced serine- and tyrosine-phosphorylation of AU-rich element binding protein (AUF1), concurrent with its increased binding to the CRE, and led to an increased interaction of poly A binding protein (PABP) with the CRE and poly(A) sites. AUF1 phosphorylation also led to an increased interaction with PCBP1. These findings suggest that a single co-regulator, PCBP1, plays a crucial role in stabilizing MOR mRNA, and is induced by PKA signaling by conforming to AUF1 and PABP. PMID:27836661

  2. Alvimopan, a peripherally acting mu-opioid receptor (PAM-OR) antagonist for the treatment of opioid-induced bowel dysfunction: results from a randomized, double-blind, placebo-controlled, dose-finding study in subjects taking opioids for chronic non-cancer pain.

    PubMed

    Webster, Lynn; Jansen, Jan Peter; Peppin, John; Lasko, Ben; Irving, Gordon; Morlion, Bart; Snidow, Jerry; Pierce, Amy; Mortensen, Eric; Kleoudis, Christi; Carter, Eric

    2008-07-15

    Our objective was to investigate the efficacy and safety of alvimopan, a peripherally acting mu-opioid receptor (PAM-OR) antagonist, in subjects with non-cancer pain and opioid-induced bowel dysfunction (OBD), and to identify at least one treatment regimen that improves OBD. Following a 2-week baseline period, 522 subjects reporting <3 spontaneous bowel movements (SBMs)/week (with >or=25% accompanied by a sensation of incomplete evacuation, straining, or lumpy hard stools), requiring analgesia equivalent to >or=30 mg oral morphine/day were randomized to alvimopan 0.5mg twice daily (BID), 1mg once daily (QD), 1mg BID, or placebo for 6 weeks. Compared with placebo, there was a statistically and clinically significant increase in mean weekly SBM frequency over the initial 3 weeks of treatment (primary endpoint) with alvimopan 0.5mg BID (+1.71 mean SBMs/week), alvimopan 1mg QD (+1.64) and alvimopan 1mg BID (+2.52); P<0.001 for all comparisons. Increased SBM frequency and additional treatment effects, including improvements in symptoms such as straining, stool consistency, incomplete evacuation, abdominal bloating/discomfort, and decreased appetite, were sustained over 6 weeks. The most frequently reported adverse events were abdominal pain, nausea, and diarrhea, occurring more frequently in the higher dosage groups. The alvimopan 0.5mg BID regimen demonstrated the best benefit-to-risk profile for managing OBD with alvimopan in this study population, with a side effect profile similar to that of placebo. There was no evidence of opioid analgesia antagonism. Competitive peripheral antagonism of opioids with alvimopan can restore GI function and relieve OBD without compromising analgesia.

  3. Repeated Mu-Opioid Exposure Induces a Novel Form of the Hyperalgesic Priming Model for Transition to Chronic Pain

    PubMed Central

    Araldi, Dioneia; Ferrari, Luiz F.

    2015-01-01

    The primary afferent nociceptor was used as a model system to study mechanisms of pain induced by chronic opioid administration. Repeated intradermal injection of the selective mu-opioid receptor (MOR) agonist DAMGO induced mechanical hyperalgesia and marked prolongation of prostaglandin E2 (PGE2) hyperalgesia, a key feature of hyperalgesic priming. However, in contrast to prior studies of priming induced by receptor-mediated (i.e., TNFα, NGF, or IL-6 receptor) or direct activation of protein kinase Cε (PKCε), the pronociceptive effects of PGE2 in DAMGO-treated rats demonstrated the following: (1) rapid induction (4 h compared with 3 d); (2) protein kinase A (PKA), rather than PKCε, dependence; (3) prolongation of hyperalgesia induced by an activator of PKA, 8-bromo cAMP; (4) failure to be reversed by a protein translation inhibitor; (5) priming in females as well as in males; and (6) lack of dependence on the isolectin B4-positive nociceptor. These studies demonstrate a novel form of hyperalgesic priming induced by repeated administration of an agonist at the Gi-protein-coupled MOR to the peripheral terminal of the nociceptor. SIGNIFICANCE STATEMENT The current study demonstrates the molecular mechanisms involved in the sensitization of nociceptors produced by repeated activation of mu-opioid receptors and contributes to our understanding of the painful condition observed in patients submitted to chronic use of opioids. PMID:26354917

  4. Differential mechanism of G-protein activation induced by endogenous mu-opioid peptides, endomorphin and beta-endorphin.

    PubMed

    Mizoguchi, Hirokazu; Tseng, Leon F; Suzuki, Tsutomu; Sora, Ichiro; Narita, Minoru

    2002-07-01

    It is well documented that the mu-opioid receptor (MOP-R) is expressed by neurons in several central nervous system regions. Its occupancy with agonist drugs modulate a variety of physiological processes including pain, reward, stress, immune responses, neuroendocrine functions, and cardiovascular control. Based on the receptor binding assay, endomorphin-1 and endomorphin-2 have the highest specificity and affinity for the MOP-R of any endogenous substance so far described in the mammalian nervous system. In contrast, beta-endorphin exhibits the strongest actions among endogenous opioid peptides mainly through the MOP-R; however, it also shows the distinct pharmacological actions. Recent cloning and expression studies have indicated that MOP-Rs are seven-transmembrane domain receptors whose actions are mediated through activation of heterotrimeric guanine nucleotide binding proteins (G-proteins). The activation of G-proteins by MOP-Rs can be measured by assessing agonist-induced stimulation of membrane binding of guanosine-5'-o-(3-[35S]thio)triphosphate ([35S]GTPgammaS). The subject of the present review is to focus on the differential mechanism underlying G-protein activation induced by these mu-opioid peptides using the [35S]GTPgammaS binding assay.

  5. Systemic and spinal administration of the mu opioid, remifentanil, produces antinociception in amphibians.

    PubMed

    Mohan, Shekher; Stevens, Craig W

    2006-03-18

    Remifentanil is a relatively new opioid analgesic related to the fentanyl family of mu opioid receptor agonists and is used clinically for its unique property of having an ultra-short duration of action. However, there is little preclinical data on the analgesic (antinociceptive) effects of remifentanil and none obtained in non-mammalian animal models. The antinociceptive effects of remifentanil were assessed by using the acetic acid test in amphibians. Systemic and spinal administration of remifentanil was made by subcutaneous and intraspinal injections in the Northern grass frog, Rana pipiens. After administration, remifentanil produced dose-dependent and long-lasting antinociceptive effects which persisted for five hours after systemic administration but gave a shorter duration of action after spinal delivery. The antinociceptive effects of remifentanil were significantly blocked by pretreatment with systemic naltrexone. Systemic and spinal administration of remifentanil produced log dose-response curves which yielded ED50 values of 7.1 nmol/g and 3.2 nmol/animal respectively. The relative antinociceptive potency of remifentanil compared to other opioids administered to amphibians is similar to that found in mammalian models.

  6. Soymorphins, novel mu opioid peptides derived from soy beta-conglycinin beta-subunit, have anxiolytic activities.

    PubMed

    Ohinata, Kousaku; Agui, Shun; Yoshikawa, Masaaki

    2007-10-01

    Based on the amino acid sequence YPFV found in the soy beta-conglycinin beta-subunit, which is common to an opioid peptide human beta-casomorphin-4, peptides YPFVV, YPFVVN, and YPFVVNA were synthesized according to their primary structure. On guinea pig ileum (GPI) assay, they showed opioid activity (IC50 = 6.0, 9.2 and 13 microM respectively) more potent than human beta-casomorphins, and were named soymorphins-5, -6, and -7, respectively. Their opioid activities on mouse vas deferens (MVD) assay were less potent than on GPI assay, suggesting that they are selective for the mu opioid receptor. Human beta-casomorphin-4 and soymorphin-5 were released from the soy 7S fraction (beta-conglycinin) by the action of gastrointestinal proteases. Soymorphins-5, -6, and -7 had anxiolytic activities after oral administration at doses of 10-30 mg/kg in the elevated plus-maze test in mice.

  7. Central effects of ethanol interact with endogenous mu opioid activity to control isolation-induced analgesia in maternally separated infant rats

    PubMed Central

    Nizhnikov, Michael E.; Kozlov, Andrey P.; Kramskaya, Tatiana. A.; Varlinskaya, Elena I.; Spear, Norman E.

    2014-01-01

    Endogenous opioid activity plays an important role in ethanol consumption and reinforcement in infant rats. Opioid systems are also involved in mediation and regulation of stress responses. Social isolation is a stressful experience for preweanling rats and changes the effects of ethanol through opioid-dependent mechanisms. The present study assessed effects of intracisternal (i.c.) administration of a selective mu-opioid antagonist (CTOP) and i.p. administration of a nonspecific opioid antagonist (naloxone) on voluntary intake and behavior in socially isolated 12–day-old (P12) pups treated with 0.5 g/kg ethanol. Voluntary intake of 0.1% saccharin or water, locomotion, rearing activity, paw licking and grooming were assessed during short-term isolation from littermates (STSI; 8-min duration). Thermal nociceptive reactivity was measured before and after this intake test, with normalized differences between pre- and post-test latencies of paw withdrawal from a hot plate (49°C) used as an index of isolation-induced analgesia (IIA). Results indicated several effects of social isolation and ethanol mediated through the mu-opioid system. Effects of low dose ethanol (0.5 g/kg) and voluntary consumption of saccharin interacted with endogenous mu-opioid activity associated with STSI. Blockade of mu-opioid receptors on saccharin consumption and paw licking-grooming affected intoxicated animals. Low dose ethanol and ingestion of saccharin blunted effects of CTOP on rearing behavior and nociceptive reactivity. Central injections of CTOP stimulated paw licking and grooming dependent on ethanol dose and type of fluid ingested. Ethanol selectively increased saccharin intake during STSI in females, naloxone and CTOP blocked ethanol–mediated enhancement of saccharin intake. We suggest that enhancement of saccharin intake by ethanol during STSI is the product of synergism between isolation-induced mu- opioid activity that increases the pup’s sensitivity to appetitive taste

  8. Central effects of ethanol interact with endogenous mu-opioid activity to control isolation-induced analgesia in maternally separated infant rats.

    PubMed

    Nizhnikov, Michael E; Kozlov, Andrey P; Kramskaya, Tatiana A; Varlinskaya, Elena I; Spear, Norman E

    2014-03-01

    Endogenous opioid activity plays an important role in ethanol consumption and reinforcement in infant rats. Opioid systems are also involved in mediation and regulation of stress responses. Social isolation is a stressful experience for preweanling rats and changes the effects of ethanol through opioid-dependent mechanisms. The present study assessed effects of intracisternal (i.c.) administration of a selective mu-opioid antagonist (CTOP) and i.p. administration of a nonspecific opioid antagonist (naloxone) on voluntary intake and behavior in socially isolated 12-day-old (P12) pups treated with 0.5 g/kg ethanol. Voluntary intake of 0.1% saccharin or water, locomotion, rearing activity, paw licking and grooming were assessed during short-term isolation from littermates (STSI; 8-min duration). Thermal nociceptive reactivity was measured before and after this intake test, with normalized differences between pre- and post-test latencies of paw withdrawal from a hot plate (49°C) used as an index of isolation-induced analgesia (IIA). Results indicated several effects of social isolation and ethanol mediated through the mu-opioid system. Effects of low dose ethanol (0.5 g/kg) and voluntary consumption of saccharin interacted with endogenous mu-opioid activity associated with STSI. Blockade of mu-opioid receptors on saccharin consumption and paw licking-grooming affected intoxicated animals. Low dose ethanol and ingestion of saccharin blunted effects of CTOP on rearing behavior and nociceptive reactivity. Central injections of CTOP stimulated paw licking and grooming dependent on ethanol dose and type of fluid ingested. Ethanol selectively increased saccharin intake during STSI in females, naloxone and CTOP blocked ethanol-mediated enhancement of saccharin intake. We suggest that enhancement of saccharin intake by ethanol during STSI is the product of synergism between isolation-induced mu-opioid activity that increases the pup's sensitivity to appetitive taste

  9. Mu-opioids activate phospholipase C in SH-SY5Y human neuroblastoma cells via calcium-channel opening.

    PubMed Central

    Smart, D; Smith, G; Lambert, D G

    1995-01-01

    We have recently reported that, in SH-SY5Y cells, mu-opioid receptor occupancy activates phospholipase C via a pertussis toxin-sensitive G-protein. In the present study we have further characterized the mechanisms involved in this process. Fentanyl (0.1 microM) caused a monophasic increase in inositol 1,4,5-trisphosphate mass formation, with a peak (20.5 +/- 3.6 pmol/mg of protein) at 15 s. Incubation in Ca(2+)-free buffer abolished this response, while Ca2+ replacement 1 min later restored the stimulation of inositol 1,4,5-trisphosphate formation (20.1 +/- 0.6 pmol/mg of protein). In addition, nifedipine (1 nM-0.1 mM), an L-type Ca(2+)-channel antagonist, caused a dose-dependent inhibition of inositol 1,4,5-trisphosphate formation, with an IC50 of 60.3 +/- 1.1 nM. Elevation of endogenous beta/gamma subunits by selective activation of delta-opioid and alpha 2 adrenoceptors failed to stimulate phospholipase C. Fentanyl also caused a dose-dependent (EC50 of 16.2 +/- 1.0 nM), additive enhancement of carbachol-induced inositol 1,4,5-trisphosphate formation. In summary, we have demonstrated that in SH-SY5Y cells activation of the mu-opioid receptor allows Ca2+ influx to activate phospholipase C. However, the possible role of this mechanism in the process of analgesia remains to be elucidated. PMID:7832776

  10. Pharmacological characterization of the dermorphin analog [Dmt(1)]DALDA, a highly potent and selective mu-opioid peptide.

    PubMed

    Neilan, C L; Nguyen, T M; Schiller, P W; Pasternak, G W

    2001-05-04

    The dermorphin-derived peptide [Dmt(1)]DALDA (H-Dmt-D-Arg-Phe-Lys-NH(2)), labels mu-opioid receptors with high affinity and selectivity in receptor binding assays. In mouse, radiant heat tail-flick assay [Dmt(1)]DALDA produced profound spinal and supraspinal analgesia, being approximately 5000- and 100-fold more potent than morphine on a molar basis, respectively. When administered systemically, [Dmt(1)]DALDA was over 200-fold more potent than morphine. Pharmacologically, [Dmt(1)]DALDA was distinct from morphine. [Dmt(1)]DALDA displayed no cross-tolerance to morphine in the model used and it retained supraspinal analgesic activity in morphine-insensitive CXBK mice. Supraspinally, it also differed from morphine in its lack of sensitivity towards naloxonazine. Finally, in antisense mapping studies, [Dmt(1)]DALDA was insensitive to MOR-1 exon probes that reduced morphine analgesia, implying a distinct receptor mechanism of action. Thus, [Dmt(1)]DALDA is an interesting and extraordinarily potent, systemically active peptide analgesic, raising the possibility of novel approaches in the design of clinically useful drugs.

  11. Hepatic changes in metabolic gene expression in old ghrelin and ghrelin receptor knockout mice

    USDA-ARS?s Scientific Manuscript database

    Ghrelin knockout (GKO) and ghrelin receptor (growth hormone secretagogue receptor) knockout (GHSRKO) mice exhibit enhanced insulin sensitivity, but the mechanism is unclear. Insulin sensitivity declines with age and is inversely associated with accumulation of lipid in liver, a key glucoregulatory ...

  12. Endogenous regulators of G protein signaling differentially modulate full and partial mu-opioid agonists at adenylyl cyclase as predicted by a collision coupling model.

    PubMed

    Clark, M J; Linderman, J J; Traynor, J R

    2008-05-01

    Regulator of G protein signaling (RGS) proteins accelerate the endogenous GTPase activity of Galpha(i/o) proteins to increase the rate of deactivation of active Galpha-GTP and Gbetagamma signaling molecules. Previous studies have suggested that RGS proteins are more effective on less efficiently coupled systems such as with partial agonist responses. To determine the role of endogenous RGS proteins in functional responses to mu-opioid agonists of different intrinsic efficacy, Galpha(i/o) subunits with a mutation at the pertussis toxin (PTX)-sensitive cysteine (C351I) and with or without a mutation at the RGS binding site (G184S) were stably expressed in C6 glioma cells expressing a mu-opioid receptor. Cells were treated overnight with PTX to inactivate endogenous G proteins. Maximal inhibition of forskolin-stimulated adenylyl cyclase by the low-efficacy partial agonists buprenorphine and nalbuphine was increased in cells expressing RGS-insensitive Galpha(o)(CIGS), Galpha(i2)(CIGS), or Galpha(i3)(CIGS) compared with their Galpha(CI) counterparts, but the RGS-insensitive mutation had little or no effect on the maximal inhibition by the higher efficacy agonists DAMGO and morphine. The potency of all the agonists to inhibit forskolin-stimulated adenylyl cyclase was increased in cells expressing RGS-insensitive Galpha(o)(CIGS), Galpha(i2)(CIGS), or Galpha(i3)(CIGS), regardless of efficacy. These data are comparable with predictions based on a collision coupling model. In this model, the rate of G protein inactivation, which is modulated by RGS proteins, and the rate of G protein activation, which is affected by agonist intrinsic efficacy, determine the maximal agonist response and potency at adenylyl cyclase under steady state conditions.

  13. Neuroendocrine effects of dexmedetomidine: evidence of cross-tolerance between a mu-opioid agonist and an alpha 2-adrenoceptor agonist in growth hormone secretion of the male rat.

    PubMed

    Idänpään-Heikkilä, J J; Rauhala, P; Männistö, P T

    1996-03-01

    The role of alpha 2-adrenergic receptors (adrenoceptors) in the secretion of growth hormone, prolactin and thyrotropin was studied using highly selective agonists and antagonists of the alpha 2-adrenoceptor. The interplay between opiates and alpha 2-adrenergic drugs in the acute secretion of growth hormone and prolactin, as well as the possible cross-tolerance between morphine (mu-opioid receptor agonist) and dexmedetomidine (alpha 2-adrenoceptor agonist) in growth hormone secretion were also evaluated. Dexmedetomidine dose-dependently increased plasma growth hormone and prolactin levels and decreased thyrotropin levels. The enhanced secretion of both growth hormone and prolactin was antagonized by atipamezole (an alpha 2-adrenoceptor antagonist) but not by prazosin (an alpha 1-adrenoceptor antagonist). Morphine (5 mg/kg)-induced stimulation of growth hormone secretion was antagonized by both naloxone (mu-opioid antagonist) and atipamezole. Naloxone, but not atipamezole, antagonized the morphine-induced increase in prolactin secretion. Dexmedetomidine increased growth hormone secretion in the saline pretreated rats, but did not do so in the morphine-tolerant rats. The stimulation of alpha 2-adrenoceptor enhances secretion of both growth hormone and prolactin. The adrenergic regulation of thyrotropin secretion still remains unclear. Evidently, adrenergic mechanisms are involved in the morphine-induced stimulation of growth hormone secretion, but not in the morphine-induced stimulation of prolactin secretion. In addition, there is a clear cross-tolerance between dexmedetomidine and morphine in growth hormone secretion of the rat.

  14. Selective postsynaptic inhibition of tonic-firing neurons in substantia gelatinosa by mu-opioid agonist.

    PubMed

    Santos, Sónia F A; Melnick, Igor V; Safronov, Boris V

    2004-11-01

    Spinal substantia gelatinosa (SG) is a site of action of administered and endogenous opioid agonists and is an important element in the system of antinociception. However, little is known about the types of neurons serving as specific postsynaptic targets for opioid action within the SG. To study the spinal mechanisms of opioidergic analgesia, the authors compared the action of mu-opioid agonist [D-Ala, N-Me-Phe, Gly-ol]-enkephalin (DAMGO) on SG neurons with different intrinsic firing properties. Whole cell patch clamp recordings from spinal cord slices of Wistar rats were used to study the sensitivity of SG neurons to DAMGO. Three groups of neurons with distinct distributions in SG were classified: tonic-, adapting-, and delayed-firing neurons. DAMGO at 1 microm concentration selectively hyperpolarized all tonic-firing neurons tested, whereas none of the adapting- or delayed-firing neurons were affected. The effect of DAMGO on tonic-firing neurons was due to activation of G protein-coupled inward-rectifier K conductance, which could be blocked by 500 microm Ba and 500 microm Cs but increased by 50 microm baclofen. As a functional consequence of DAMGO action, a majority of tonic-firing neurons changed their pattern of intrinsic firing from tonic to adapting. It is suggested that tonic-firing neurons, presumably functioning as excitatory interneurons, are primary postsynaptic targets for administered and endogenous opioid agonists in spinal SG. Functional transition of cells in this group from tonic to adapting firing mode may represent an important mechanism facilitating opioidergic analgesia.

  15. Sensorimotor development in neonatal progesterone receptor knockout mice.

    PubMed

    Willing, Jari; Wagner, Christine K

    2014-01-01

    Early exposure to steroid hormones can permanently and dramatically alter neural development. This is best understood in the organizational effects of hormones during development of brain regions involved in reproductive behaviors or neuroendocrine function. However, recent evidence strongly suggests that steroid hormones play a vital role in shaping brain regions involved in cognitive behavior such as the cerebral cortex. The most abundantly expressed steroid hormone receptor in the developing rodent cortex is the progesterone receptor (PR). In the rat, PR is initially expressed in the developmentally-critical subplate at E18, and subsequently in laminas V and II/III through the first three postnatal weeks (Quadros et al. [2007] J Comp Neurol 504:42-56; Lopez & Wagner [2009]: J Comp Neurol 512:124-139), coinciding with significant periods of dendritic maturation, the arrival of afferents and synaptogenesis. In the present study, we investigated PR expression in the neonatal mouse somatosensory cortex. Additionally, to investigate the potential role of PR in developing cortex, we examined sensorimotor function in the first two postnatal weeks in PR knockout mice and their wildtype (WT) and heterozygous (HZ) counterparts. While the three genotypes were similar in most regards, PRKO and HZ mice lost the rooting reflex 2-3 days earlier than WT mice. These studies represent the first developmental behavioral assessment of PRKO mice and suggest PR expression may play an important role in the maturation of cortical connectivity and sensorimotor integration. Copyright © 2013 Wiley Periodicals, Inc.

  16. Mu-Opioid Stimulation in Rat Prefrontal Cortex Engages Hypothalamic Orexin/Hypocretin-Containing Neurons, and Reveals Dissociable Roles of Nucleus Accumbens and Hypothalamus in Cortically Driven Feeding

    PubMed Central

    Mena, Jesus D.; Selleck, Ryan A.

    2013-01-01

    Mu-opioid receptor (μOR) stimulation within ventral medial prefrontal cortex (vmPFC) induces feeding and hyperactivity, resulting possibly from recruitment of glutamate signaling in multiple vmPFC projection targets. We tested this hypothesis by analyzing Fos expression in vmPFC terminal fields after intra-vmPFC μOR stimulation, and by examining of the impact of glutamate receptor blockade in two feeding-related targets of vmPFC, the lateral-perifornical hypothalamic area (LH-PeF) and nucleus accumbens shell (Acb shell), upon behavioral effects elicited by intra-vmPFC μOR stimulation in rats. Intra-vmPFC infusion of the μOR agonist, DAMGO, provoked Fos expression in the dorsomedial sector of tuberal hypothalamus (including the perifornical area) and increased the percentage of Fos-expressing hypocretin/orexin-immunoreactive neurons in these zones. NMDA receptor blockade in the LH-PeF nearly eliminated intra-vmPFC DAMGO-induced food intake without altering DAMGO-induced hyperactivity. In contrast, blocking AMPA-type glutamate receptors within the Acb shell (the feeding-relevant subtype in this structure) antagonized intra-vmPFC DAMGO-induced hyperlocomotion but enhanced food intake. Intra-vmPFC DAMGO also elevated the breakpoint for sucrose-reinforced progressive-ratio responding; this effect was significantly enhanced by concomitant AMPA blockade in the Acb shell. Conversely, intra-Acb shell AMPA stimulation reduced breakpoint and increased nonspecific responding on the inactive lever. These data indicate intra-vmPFC μOR signaling jointly modulates appetitive motivation and generalized motoric activation through functionally dissociable vmPFC projection targets. These findings may shed light on the circuitry underlying disorganized appetitive responses in psychopathology; e.g., binge eating and opiate or alcohol abuse, disorders in which μORs and aberrant cortical activation have been implicated. PMID:24259576

  17. Bone growth and turnover in progesterone receptor knockout mice.

    SciTech Connect

    Rickard, David J.; Iwaniec, Urszula T.; Evans, Glenda; Hefferan, Theresa E.; Hunter, Jaime C.; Waters, Katrina M.; Lydon, John P.; O'Malley, Bert W.; Khosla, Sundeep; Spelsberg, Thomas C.; Turner, Russell T.

    2008-05-01

    The role of progesterone receptor (PR) signaling in skeletal metabolism is controversial. To address whether signaling through the PR is necessary for normal bone growth and turnover, we performed histomorphometric and mCT analyses of bone from homozygous female PR knockout (PRKO) mice at 6, 12, and 26 weeks of age. These mice possess a null mutation of the PR locus, which blocks the gene expression of A and B isoforms of PR. Body weight gain, uterine weight gain and tibia longitudinal bone growth was normal in PRKO mice. In contrast, total and cortical bone mass were increased in long bones of post-pubertal (12 and 26-week-old) PRKO mice, whereas cancellous bone mass was normal in the tibia but increased in the humerus. The striking 57% decrease in cancellous bone from the proximal tibia metaphysis which occurred between 6 and 26 weeks in WT mice was abolished in PRKO mice. The improved bone balance in aging PRKO mice was associated with elevated bone formation and a tendency toward reduced osteoclast perimeter. Taken together, these findings suggest that PR signaling in mice attenuates the accumulation of cortical bone mass during adolescence and is required for early age-related loss of cancellous bone.

  18. INDUCTION OF MAMMARY GLAND DEVELOPMENT IN ESTROGEN RECEPTOR-ALPHA KNOCKOUT MICE

    EPA Science Inventory

    Mammary glands from the estrogen receptor knockout ( ERKO) mouse do not undergo ductal morphogenesis or alveolar development. Disrupted Er signaling may result in reduced estrogen-responsive gene products in the mammary gland or reduced mammotropic hormones that contribute t...

  19. INDUCTION OF MAMMARY GLAND DEVELOPMENT IN ESTROGEN RECEPTOR-ALPHA KNOCKOUT MICE

    EPA Science Inventory

    Mammary glands from the estrogen receptor knockout ( ERKO) mouse do not undergo ductal morphogenesis or alveolar development. Disrupted Er signaling may result in reduced estrogen-responsive gene products in the mammary gland or reduced mammotropic hormones that contribute t...

  20. Altered Morphine-Induced Analgesia in Neurotensin Type 1 Receptor Null Mice

    PubMed Central

    Roussy, Geneviève; Beaudry, Hélène; Lafrance, Mylène; Belleville, Karine; Beaudet, Nicolas; Wada, Keiji; Gendron, Louis; Sarret, Philippe

    2013-01-01

    Both neurotensin (NT) and opioid agonists have been shown to induce antinociception in rodents after central administration. Besides, previous studies have revealed the existence of functional interactions between NT and opioid systems in the regulation of pain processing. We recently demonstrated that NTS1 receptors play a key role in the mediation of the analgesic effects of NT in long-lasting pain. In the present study, we therefore investigated whether NTS1 gene deletion affected the antinociceptive action of mu opioid drugs. To this end, pain behavioral responses to formalin were determined following systemic administration of morphine in both male and female NTS1 knockout mice. Acute injection of morphine (2 or 5 mg/kg) produced strong antinociceptive effects in both male and female wild-type littermates, with no significant sex differences. On the other hand, morphine analgesia was considerably reduced in NTS1-deficient mice of both sexes compared to their respective controls, indicating that the NTS1 receptor actively participates in mu opioid alleviating pain. By examining specifically the flinching, licking and biting nociceptive behaviors, we also showed that the functional crosstalk between NTS1 and mu opioid receptors influences the supraspinally-mediated behaviors. Interestingly, sexual dimorphic action of morphine-induced pain inhibition was found in NTS1 null mice in the formalin test, suggesting that the endogenous NT system interacts differently with the opioid network in male and female mice. Altogether, these results demonstrated that NTS1 receptor activation operates downstream to the opioidergic transmission and that NTS1-selective agonists combined with morphine may act synergistically to reduce persistent pain. PMID:20727387

  1. The antinociceptive effect of the mu-opioid fentanyl is reduced in the presence of the alpha(2)-adrenergic antagonist idazoxan in inflammation.

    PubMed

    Herrero, J F; Solano, R E

    1999-09-04

    Interactions between alpha(2)-adrenergic and mu-opioid systems play an important role in the modulation of hyperalgesic states. The antinociceptive effects of alpha(2)-adrenergic agonists and mu-opioids are potentiated when co-administered; however, attempts to induce cross reversal of the antinociceptive effects of alpha(2)-adrenergic and mu-opioid systems have produced contradictory results. We have studied the possible endogenous tonic control of the alpha(2)-adrenergic systems in the modulation of pain in inflammation, and the interactions between the two antinociceptive systems in rat spinal cord nociceptive reflexes activated by both natural and electrical stimulation. The facilitatory actions of the alpha(2)-adrenergic antagonist idazoxan were compared in control rats and in animals with carrageenan-induced paw inflammation. The antinociceptive effect of the mu-opioid fentanyl was tested alone and in the presence of idazoxan. In agreement with some previous observations, idazoxan i.v. produced no change in responses to natural and electrical stimulation in normal animals. In animals with inflammation, idazoxan only induced facilitation of responses evoked by noxious thermal stimulation but not by mechanical or electrical stimulation. Fentanyl reduced the responses to either stimuli with lower potency in the presence of idazoxan, but only in animals with inflammation. Its dose-response curve was shifted to the right between 1.8- and 3. 5-fold depending on the stimulus used. It is concluded that the increase of thermal responses by idazoxan in animals with inflammation is probably due to changes in the peripheral blood flow. Nevertheless, since an interaction with mu-opioids is clear in inflammation, endogenous alpha(2)-adrenergic systems play an important role in the modulation of the effectiveness of opioids during inflammation.

  2. Mu-opioid blockade reduces ethanol effects on intake and behavior of the infant rat during short-term but not long-term social isolation

    PubMed Central

    Kozlov, Andrey P.; Nizhnikov, Michael E.; Kramskaya, Tatiana. A.; Varlinskaya, Elena I.; Spear, Norman E.

    2013-01-01

    Numerous findings in adult and infant rats have shown that the endogenous opioid system is involved in control of ethanol consumption and its reinforcing effects. Opioid systems are also involved in reactivity to social isolation with several factors (age, duration, and type of isolation) affecting this modulation. The present study investigated the effects of a selective mu – opioid antagonist CTOP (0, 0.1, 0.5 mg/kg), ethanol (0, 0.5 g/kg), and the interaction of the two drugs on the behavioral consequences of two types of social isolation given to preweanling rats: 1) short–term social isolation from littermates (STSI, duration 8 minutes) and 2) relatively long-term (5 hours) isolation (LTSI) from the dam and littermates. Voluntary intake of saccharin, locomotion, rearing activity, paw licking, and grooming were assessed during an 8 – min. intake test. Thermal nociceptive reactivity was also measured before and after the testing session with normalized differences in pre- and post-test latencies of paw withdrawal from a hot plate (49 °C) used as an index of isolation-induced analgesia (IIA). The results indicate that pharmacological blockade of mu-opioid receptors by CTOP substantially attenuated ethanol’s anxiolytic effects on the developing rat’s reactions to social isolation. Some of these stress-attenuating effects of CTOP were observed only in animals exposed to short-term isolation (STSI) but not in pups isolated for 5 hours (LTSI). Ethanol selectively increased saccharin intake during STSI in females and CTOP blocked this effect. Ethanol decreased the magnitude of analgesia associated with STSI but had no effect on pain reactivity during LTSI. CTOP by itself did not affect IIA or saccharin intake in sober animals. The findings of the present experiments indicate that the anxiolytic effects of 0.5 g/kg ethanol on pups exposed to STSI are modulated by endogenous opioid activity. PMID:23182856

  3. Inflammatory pain is enhanced in delta opioid receptor-knockout mice

    PubMed Central

    Gavériaux-Ruff, Claire; Karchewski, Laurie A.; Hever, Xavier; Matifas, Audrey; Kieffer, Brigitte L.

    2015-01-01

    To examine the involvement of opioid receptors in inflammatory pain, we compared Complete Freund’s Adjuvant-induced hyperalgesia in mice lacking mu, delta or kappa receptors under the same experimental conditions. Mechanical allodynia and thermal hyperalgesia were measured using von Frey filaments and the plantar test, respectively. All three receptor-knockout mice, as well as wild-type animals, developed inflammatory hyperalgesia following Complete Freund’s Adjuvant administration. Mu-receptor mutants showed similar hyperalgesia to wild-types in the two tests. Kappa-receptor mutants exhibited enhanced mechanical allodynia compared with wild-type mice but similar thermal hyperalgesia. In contrast, mechanical allodynia and thermal hyperalgesia were both markedly augmented in delta-receptor mutants, indicating a role for an endogenous delta-receptor tone in the control of inflammatory pain. Treatment with the delta-selective agonist SNC80 produced antihyperalgesia, and this effect was abolished in the delta-receptor knockout mice. Altogether, these data demonstrate that delta receptors inhibit inflammatory pain when activated either endogenously or exogenously. We have previously shown enhanced neuropathic pain in delta-receptor knockout mice. The delta receptor definitely represents a promising target for treating chronic pain conditions. PMID:18513322

  4. Effect of vitamin D receptor knockout on cornea epithelium wound healing and tight junctions.

    PubMed

    Elizondo, Rodolfo A; Yin, Zhaohong; Lu, Xiaowen; Watsky, Mitchell A

    2014-07-24

    Our laboratory previously determined that vitamin D3, the vitamin D receptor (VDR), and 1α hydroxylase are present and active in the eye. In this study, we examined the effects of VDR knockout on wound healing, the tight junction-associated proteins occludin and ZO-1, and tight junction numbers in mouse corneas. Epithelial wounds (2-mm) were made with an agar brush on 4-week-old and 10-week-old wild-type, heterozygous, and VDR knockout mouse corneas. Mice were on a normal or high lactose, Ca(2+), and PO₄(-) diet. Wound-healing area was measured over time. Real-time PCR was used to quantify occludin and ZO-1 message expression. Western blot was used for protein expression. Transmission electron microscopy was used to examine corneal epithelium and endothelium tight junctions. Immunofluorescence was used to examine epithelial ZO-1 distribution. Results showed a decreased healing rate in 10-week-old VDR knockout mice compared with wild-types. Vitamin D receptor knockout mice on the special diet had no difference in healing rate compared with wild-types. Real-time PCR showed decreased expression of occludin and ZO-1 in 10-week-old VDR knockout mice compared with wild-types. Western blot of 10-week-old knockout mouse corneas showed decreased occludin expression compared with wild-types. Transmission electron microscopy showed a significant difference in tight junction numbers in VDR knockouts versus wild-types. Immunofluorescence showed a change in ZO-1 distribution among genotypes. Vitamin D receptor knockout affects mouse corneal epithelium wound healing and tight junction integrity. Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.

  5. [Roles of histamine receptors in pain perception: a study using receptors gene knockout mice].

    PubMed

    Yanai, Kazuhiko; Mobarakeh, Jalal Izadi; Kuramasu, Atsuo; Sakurada, Shinobu

    2003-11-01

    To study the participation of histamine H1- and H2-receptors in pain perception, H1 and H2 receptor knockout (KO) mice were examined for pain threshold by means of three kinds of nociceptive tasks. These included assays for thermal, mechanical, and chemical nociception. H1KO mice showed significantly fewer nociceptive responses to the hot-plate, tail-flick, tail-pressure, paw-withdrawal, formalin, capsaicin, and abdominal constriction tests. Sensitivity to noxious stimuli in H1KO mice was significantly decreased when compared to wild-type mice. The antinociceptive phenotypes of H2KO were relatively less prominent when compared to H1KO mice. We also examined the antinociceptive effects of intrathecally-, intracerebroventricularly-, and subcutaneously-administered morphine in H1KO and H2KO mice. In these nociceptive assays, the antinociceptive effects produced by morphine were more enhanced in both H1KO and H2KO mice. The effects of histamine H1- and H2-receptor antagonists on morphine-induced antinociception were studied in ICR mice. The intrathecal, intracerebroventricular and subcutaneous co-administrations of d-chlorpheniramine enhanced the effects of morphine in all nociceptive assays examined. In addition, intrathecal co-administrations of cimetidine enhanced the antinociception of morphine in the hot plate tests. These results suggest that existing H1 and H2 receptors play an inhibitory role in morphine-induced antinociception in the spinal and supra-spinal levels.

  6. Normal maternal behavior, but increased pup mortality, in conditional oxytocin receptor knockout females.

    PubMed

    Macbeth, Abbe H; Stepp, Jennifer E; Lee, Heon-Jin; Young, W Scott; Caldwell, Heather K

    2010-10-01

    Oxytocin (Oxt) and the Oxt receptor (Oxtr) are implicated in the onset of maternal behavior in a variety of species. Recently, we developed two Oxtr knockout lines: a total body knockout (Oxtr-/-) and a conditional Oxtr knockout (OxtrFB/FB) in which the Oxtr is lacking only in regions of the forebrain, allowing knockout females to potentially nurse and care for their biological offspring. In the current study, we assessed maternal behavior of postpartum OxtrFB/FB females toward their own pups and maternal behavior of virgin Oxtr-/- females toward foster pups and compared knockouts of both lines to wildtype (Oxtr+/+) littermates. We found that both Oxtr-/- and OxtrFB/FB females appear to have largely normal maternal behaviors. However, with first litters, approximately 40% of the OxtrFB/FB knockout dams experienced high pup mortality, compared to fewer than 10% of the Oxtr+/+ dams. We then went on to test whether or not this phenotype occurred in subsequent litters or when the dams were exposed to an environmental disturbance. We found that regardless of the degree of external disturbance, OxtrFB/FB females lost more pups on their first and second litters compared to wildtype females. Possible reasons for higher pup mortality in OxtrFB/FB females are discussed.

  7. Postnatal handling reverses social anxiety in serotonin receptor 1A knockout mice.

    PubMed

    Zanettini, C; Carola, V; Lo Iacono, L; Moles, A; Gross, C; D'Amato, F R

    2010-02-01

    Mice lacking the serotonin receptor 1A (Htr1a knockout, Htr1a(KO)) show increased innate and conditioned anxiety. This phenotype depends on functional receptor activity during the third through fifth weeks of life and thus appears to be the result of long-term changes in brain function as a consequence of an early deficit in serotonin signaling. To evaluate whether this phenotype can be influenced by early environmental factors, we subjected Htr1a knockout mice to postnatal handling, a procedure known to reduce anxiety-like behavior and stress responses in adulthood. Offspring of heterozygous Htr1a knockout mice were separated from their mother and exposed 15 min each day from postnatal day 1 (PD1) to PD14 to clean bedding. Control animals were left undisturbed. Maternal behavior was observed during the first 13 days of life. Adult male offspring were tested in the open field, social approach and resident-intruder tests and assessed for corticosterone response to restraint stress. Knockout mice showed increased anxiety in the open field and in the social approach test as well as an enhanced corticosterone response to stress. However, while no effect of postnatal handling was seen in wild-type mice, handling reduced anxiety-like behavior in the social interaction test and the corticosterone response to stress in knockout mice. These findings extend the anxiety phenotype of Htr1a(KO) mice to include social anxiety and demonstrate that this phenotype can be moderated by early environmental factors.

  8. Acute and Chronic Mu Opioids Differentially Regulate Thrombospondins 1 and 2 Isoforms in Astrocytes

    PubMed Central

    2013-01-01

    Chronic opioids induce synaptic plasticity, a major neuronal adaptation. Astrocyte activation in synaptogenesis may play a critical role in opioid tolerance, withdrawal, and dependence. Thrombospondins 1 and 2 (TSP1/2) are astrocyte-secreted matricellular glycoproteins that promote neurite outgrowth as well as dendritic spine and synapse formation, all of which are inhibited by chronic μ opioids. In prior studies, we discovered that the mechanism of TSP1 regulation by μ opioids in astrocytes involves crosstalk between three different classes of receptors, μ opioid receptor, EGFR and TGFβR. Moreover, TGFβ1 stimulated TSP1 expression via EGFR and ERK/MAPK activation, indicating that EGFR is a signaling hub for opioid and TGFβ1 actions. Using various selective antagonists, and inhibitors, here we compared the mechanisms of chronic opioid regulation of TSP1/2 isoform expression in vivo and in immortalized rat cortical astrocytes. TSP1/2 release from astrocytes was also monitored. Acute and chronic μ opioids, morphine, and the prototypic μ ligand, DAMGO, modulated TSP2 protein levels. TSP2 but not TSP1 protein content was up-regulated by acute (3 h) morphine or DAMGO by an ERK/MAPK dependent mechanism. Paradoxically, TSP2 protein levels were altered neither by TGFβ1 nor by astrocytic neurotrophic factors, EGF, CNTF, and BMP4. TSP1/2 immunofluorescence was increased in astrocytes subjected to scratch-wounding, suggesting TSPs may be useful markers for the “reactive” state of these cells and potentially for different types of injury. Previously, we determined that chronic morphine attenuated both neurite outgrowth and synapse formation in cocultures of primary astrocytes and neurons under similar temporal conditions that μ opioids reduced TSP1 protein levels in astrocytes. Here we found that, after the same 8 day treatment, morphine or DAMGO diminished TSP2 protein levels in astrocytes. Therefore, μ opioids may deter synaptogenesis via both TSP1/2 isoforms

  9. Acute and chronic mu opioids differentially regulate thrombospondins 1 and 2 isoforms in astrocytes.

    PubMed

    Phamduong, Ellen; Rathore, Maanjot K; Crews, Nicholas R; D'Angelo, Alexander S; Leinweber, Andrew L; Kappera, Pranay; Krenning, Thomas M; Rendell, Victoria R; Belcheva, Mariana M; Coscia, Carmine J

    2014-02-19

    Chronic opioids induce synaptic plasticity, a major neuronal adaptation. Astrocyte activation in synaptogenesis may play a critical role in opioid tolerance, withdrawal, and dependence. Thrombospondins 1 and 2 (TSP1/2) are astrocyte-secreted matricellular glycoproteins that promote neurite outgrowth as well as dendritic spine and synapse formation, all of which are inhibited by chronic μ opioids. In prior studies, we discovered that the mechanism of TSP1 regulation by μ opioids in astrocytes involves crosstalk between three different classes of receptors, μ opioid receptor, EGFR and TGFβR. Moreover, TGFβ1 stimulated TSP1 expression via EGFR and ERK/MAPK activation, indicating that EGFR is a signaling hub for opioid and TGFβ1 actions. Using various selective antagonists, and inhibitors, here we compared the mechanisms of chronic opioid regulation of TSP1/2 isoform expression in vivo and in immortalized rat cortical astrocytes. TSP1/2 release from astrocytes was also monitored. Acute and chronic μ opioids, morphine, and the prototypic μ ligand, DAMGO, modulated TSP2 protein levels. TSP2 but not TSP1 protein content was up-regulated by acute (3 h) morphine or DAMGO by an ERK/MAPK dependent mechanism. Paradoxically, TSP2 protein levels were altered neither by TGFβ1 nor by astrocytic neurotrophic factors, EGF, CNTF, and BMP4. TSP1/2 immunofluorescence was increased in astrocytes subjected to scratch-wounding, suggesting TSPs may be useful markers for the "reactive" state of these cells and potentially for different types of injury. Previously, we determined that chronic morphine attenuated both neurite outgrowth and synapse formation in cocultures of primary astrocytes and neurons under similar temporal conditions that μ opioids reduced TSP1 protein levels in astrocytes. Here we found that, after the same 8 day treatment, morphine or DAMGO diminished TSP2 protein levels in astrocytes. Therefore, μ opioids may deter synaptogenesis via both TSP1/2 isoforms, but

  10. Altered Sleep and Affect in the Neurotensin Receptor 1 Knockout Mouse

    PubMed Central

    Fitzpatrick, Karrie; Winrow, Christopher J.; Gotter, Anthony L.; Millstein, Joshua; Arbuzova, Janna; Brunner, Joseph; Kasarskis, Andrew; Vitaterna, Martha H.; Renger, John J.; Turek, Fred W.

    2012-01-01

    Study Objective: Sleep and mood disorders have long been understood to have strong genetic components, and there is considerable comorbidity of sleep abnormalities and mood disorders, suggesting the involvement of common genetic pathways. Here, we examine a candidate gene implicated in the regulation of both sleep and affective behavior using a knockout mouse model. Design: Previously, we identified a quantitative trait locus (QTL) for REM sleep amount, REM sleep bout number, and wake amount in a genetically segregating population of mice. Here, we show that traits mapping to this QTL correlated with an expression QTL for neurotensin receptor 1 (Ntsr1), a receptor for neurotensin, a ligand known to be involved in several psychiatric disorders. We examined sleep as well as behaviors indicative of anxiety and depression in the NTSR1 knockout mouse. Measurements and Results: NTSR1 knockouts had a lower percentage of sleep time spent in REM sleep in the dark phase and a larger diurnal variation in REM sleep duration than wild types under baseline conditions. Following sleep deprivation, NTSR1 knockouts exhibited more wake and less NREM rebound sleep. NTSR1 knockouts also showed increased anxious and despair behaviors. Conclusions: Here we illustrate a link between expression of the Ntsr1 gene and sleep traits previously associated with a particular QTL. We also demonstrate a relationship between Ntsr1 and anxiety and despair behaviors. Given the considerable evidence that anxiety and depression are closely linked with abnormalities in sleep, the data presented here provide further evidence that neurotensin and Ntsr1 may be a component of a pathway involved in both sleep and mood disorders. Citation: Fitzpatrick K; Winrow CJ; Gotter AL; Millstein J; Arbuzova J; Brunner J; Kasarskis A; Vitaterna MH; Renger JJ; Turek FW. Altered sleep and affect in the neurotensin receptor 1 knockout mouse. SLEEP 2012;35(7):949-956. PMID:22754041

  11. Wheel running reduces high-fat diet intake, preference and mu-opioid agonist stimulated intake.

    PubMed

    Liang, Nu-Chu; Bello, Nicholas T; Moran, Timothy H

    2015-05-01

    The ranges of mechanisms by which exercise affects energy balance remain unclear. One potential mechanism may be that exercise reduces intake and preference for highly palatable, energy dense fatty foods. The current study used a rodent wheel running model to determine whether and how physical activity affects HF diet intake/preference and reward signaling. Experiment 1 examined whether wheel running affected the ability of intracerebroventricular (ICV) μ opioid receptor agonist D-Ala2, NMe-Phe4, Glyol5-enkephalin (DAMGO) to increase HF diet intake. Experiment 2 examined the effects of wheel running on the intake of and preference for a previously preferred HF diet. We also assessed the effects of wheel running and diet choice on mesolimbic dopaminergic and opioidergic gene expression. Experiment 1 revealed that wheel running decreased the ability of ICV DAMGO administration to stimulate HF diet intake. Experiment 2 showed that wheel running suppressed weight gain and reduced intake and preference for a previously preferred HF diet. Furthermore, the mesolimbic gene expression profile of wheel running rats was different from that of their sedentary paired-fed controls but similar to that of sedentary rats with large HF diet consumption. These data suggest that alterations in preference for palatable, energy dense foods play a role in the effects of exercise on energy homeostasis. The gene expression results also suggest that the hedonic effects of exercise may substitute for food reward to limit food intake and suppress weight gain. Published by Elsevier B.V.

  12. Wheel running reduces high-fat diet intake, preference and mu-opioid agonist stimulated intake

    PubMed Central

    Liang, Nu-Chu; Bello, Nicholas T.; Moran, Timothy H.

    2015-01-01

    The ranges of mechanisms by which exercise affects energy balance remain unclear. One potential mechanism may be that exercise reduces intake and preference for highly palatable, energy dense fatty foods. The current study used a rodent wheel running model to determine whether and how physical activity affects HF diet intake/preference and reward signaling. Experiment 1 examined whether wheel running affected the ability of intracerebroventricular (ICV) µ opioid receptor agonist D-Ala2, NMe-Phe4, Glyol5-enkephalin (DAMGO) to increase HF diet intake. Experiment 2 examined the effects of wheel running on the intake of and preference for a previously preferred HF diet. We also assessed the effects of wheel running and diet choice on mesolimbic dopaminergic and opioidergic gene expression. Experiment 1 revealed that wheel running decreased the ability of ICV DAMGO administration to stimulate HF diet intake. Experiment 2 showed that wheel running suppressed weight gain and reduced intake and preference for a previously preferred HF diet. Furthermore, the mesolimbic gene expression profile of wheel running rats was different from that of their sedentary paired-fed controls but similar to that of sedentary rats with large HF diet consumption. These data suggest that alterations in preference for palatable, energy dense foods play a role in the effects of exercise on energy homeostasis. The gene expression results also suggest that the hedonic effects of exercise may substitute for food reward to limit food intake and suppress weight gain. PMID:25668514

  13. Mu opioid modulation of oxytocin secretion in late pregnant and parturient rats. Involvement of noradrenergic neurotransmission.

    PubMed

    Kutlu, Selim; Yilmaz, Bayram; Canpolat, Sinan; Sandal, Suleyman; Ozcan, Mete; Kumru, Selahattin; Kelestimur, Haluk

    2004-01-01

    We have investigated effects of micro- and kappa-opioid agonists and antagonists on plasma oxytocin levels and noradrenaline content in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) of 20-day pregnant rats. beta-Endorphin, oxytocin, estrogen and progesterone profiles in late pregnant and parturient rats were also sought. Stage of estrous cycle was monitored by vaginal smear, and pro-estrous animals were left overnight with male. In the first set of experiments, pregnant rats were monitored and decapitated on days 20 and 21 and after the delivery of second pup. In the second set, 20-day pregnant rats were intracerebroventricularly infused with morphine (50 microg/10 microl), U50,488H (kappa-agonist; 50 microg/10 microl), clocinnamox (micro-antagonist; 50 microg/10 microl) and norbinaltorphimine (kappa-antagonist; 50 microg/10 microl). Controls received saline alone. Serum estrogen and progesterone levels were measured by enzyme immunoassay, and plasma oxytocin and beta-endorphin by radioimmunoassay. Noradrenaline and its metabolite (3,4-dihydroxyphenylglycol) were determined in micropunched hypothalamic nuclei by HPLC-ECD. In parturient rats, oxytocin levels were increased (p < 0.05) and beta-endorphin decreased (p < 0.01) compared to 20-day pregnant animals. Serum progesterone concentrations progressively declined towards parturition (p < 0.001). Clocinnamox raised oxytocin levels (p < 0.01) while U50,488H caused decreases (p < 0.05). Noradrenaline content was elevated by clocinnamox in the SON (p < 0.01) and PVN (p < 0.05) compared to control values. Other agonists and antagonists had no significant effect on the noradrenergic neurotransmission or oxytocin secretion. We suggest that noradrenaline may mediate the inhibitory effects of micro-opioids on oxytocin release. Our findings have also shown that kappa-opioid receptors are not involved in modulation of oxytocin neurons in late pregnant rats. Copyright 2004 S. Karger AG, Basel

  14. Synergistic regulation of serotonin and opioid signaling contributes to pain insensitivity in Nav1.7 knockout mice.

    PubMed

    Isensee, Jörg; Krahé, Leonhardt; Moeller, Katharina; Pereira, Vanessa; Sexton, Jane E; Sun, Xiaohui; Emery, Edward; Wood, John N; Hucho, Tim

    2017-01-10

    Genetic loss of the voltage-gated sodium channel Nav1.7 (Nav1.7(-/-)) results in lifelong insensitivity to pain in mice and humans. One underlying cause is an increase in the production of endogenous opioids in sensory neurons. We analyzed whether Nav1.7 deficiency altered nociceptive heterotrimeric guanine nucleotide-binding protein-coupled receptor (GPCR) signaling, such as initiated by GPCRs that respond to serotonin (pronociceptive) or opioids (antinociceptive), in sensory neurons. We found that the nociceptive neurons of Nav1.7 knockout (Nav1.7(-/-)) mice, but not those of Nav1.8 knockout (Nav1.8(-/-)) mice, exhibited decreased pronociceptive serotonergic signaling through the 5-HT4 receptors, which are Gαs-coupled GPCRs that stimulate the production of cyclic adenosine monophosphate resulting in protein kinase A (PKA) activity, as well as reduced abundance of the RIIβ regulatory subunit of PKA. Simultaneously, the efficacy of antinociceptive opioid signaling mediated by the Gαi-coupled mu opioid receptors was increased. Consequently, opioids inhibited more efficiently tetrodotoxin-resistant sodium currents, which are important for pain-initiating neuronal activity in nociceptive neurons. Thus, Nav1.7 controls the efficacy and balance of GPCR-mediated pro- and antinociceptive intracellular signaling, such that without Nav1.7, the balance is shifted toward antinociception, resulting in lifelong endogenous analgesia.

  15. Neuron-specific (pro)renin receptor knockout prevents the development of salt-sensitive hypertension.

    PubMed

    Li, Wencheng; Peng, Hua; Mehaffey, Eamonn P; Kimball, Christie D; Grobe, Justin L; van Gool, Jeanette M G; Sullivan, Michelle N; Earley, Scott; Danser, A H Jan; Ichihara, Atsuhiro; Feng, Yumei

    2014-02-01

    The (pro)renin receptor (PRR), which binds both renin and prorenin, is a newly discovered component of the renin-angiotensin system that is highly expressed in the central nervous system. The significance of brain PRRs in mediating local angiotensin II formation and regulating blood pressure remains unclear. The current study was performed to test the hypothesis that PRR-mediated, nonproteolytic activation of prorenin is the main source of angiotensin II in the brain. Thus, PRR knockout in the brain is expected to prevent angiotensin II formation and development of deoxycorticosterone acetate-salt-induced hypertension. A neuron-specific PRR (ATP6AP2) knockout mouse model was generated using the Cre-LoxP system. Physiological parameters were recorded by telemetry. PRR expression, detected by immunostaining and reverse transcription-polymerase chain reaction, was significantly decreased in the brains of knockout mice compared with wild-type mice. Intracerebroventricular infusion of mouse prorenin increased blood pressure and angiotensin II formation in wild-type mice. This hypertensive response was abolished in PRR-knockout mice in association with a reduction in angiotensin II levels. Deoxycorticosterone acetate-salt increased PRR expression and angiotensin II formation in the brains of wild-type mice, an effect that was attenuated in PRR-knockout mice. PRR knockout in neurons prevented the development of deoxycorticosterone acetate-salt-induced hypertension as well as activation of cardiac and vasomotor sympathetic tone. In conclusion, nonproteolytic activation of prorenin through binding to the PRR mediates angiotensin II formation in the brain. Neuron-specific PRR knockout prevents the development of deoxycorticosterone acetate-salt-induced hypertension, possibly through diminished angiotensin II formation.

  16. Knockout of the vascular endothelial glucocorticoid receptor abrogates dexamethasone-induced hypertension

    PubMed Central

    GOODWIN, Julie E.; ZHANG, Junhui; GONZALEZ, David; ALBINSSON, Sebastian; GELLER, David S.

    2012-01-01

    Glucocorticoid-mediated hypertension is incompletely understood. Recent studies have suggested the primary mechanism of this form of hypertension may be through the effects of glucocorticoids on vascular tissues and not to excess sodium and water reabsorption as traditionally believed. Objective The goal of this study was to better understand the role of the vasculature in the generation and maintenance of glucocorticoid-mediated hypertension. Methods We created a mouse model with a tissue-specific knockout of the glucocorticoid receptor in the vascular endothelium. Results We show that these mice are relatively resistant to dexamethasone-induced hypertension. After one week of dexamethasone treatment, control animals have a mean blood pressure increase of 13.1 mm Hg while knockout animals have only a 2.7 mm Hg increase (p<0.001). Interestingly, the knockout mice have slightly elevated baseline BP compared to the controls (112.2 ± 2.5 mm Hg vs. 104.6 ± 1.2 mm Hg, p = 0.04), a finding which is not entirely explained by our data. Furthermore, we demonstrate that the knockout resistance arterioles have a decreased contractile response to dexamethasone with only 6.6% contraction in knockout vessels compared to 13.4% contraction in control vessels (p=0.034). Finally, we show that in contrast to control animals, the knockout animals are able to recover a significant portion of their normal circadian blood pressure rhythm suggesting that the vascular endothelial glucocorticoid receptor may function as a peripheral circadian clock. Conclusions Our study highlights the importance of the vascular endothelial GR in several fundamental physiologic processes, namely blood pressure homeostasis and circadian rhythm. PMID:21659825

  17. Enhanced morphine-induced antinociception in histamine H3 receptor gene knockout mice.

    PubMed

    Mobarakeh, Jalal Izadi; Takahashi, Kazuhiro; Yanai, Kazuhiko

    2009-09-01

    Previous studies have implicated a potential role for histamine H3 receptor in pain processing. There have been conflicting data, however, on the roles of H3 receptors in pain perception, and little information is available about the role of spinal histamine H3 receptors in morphine-induced antinociception. In the present study we examined the role of histamine H3 receptor in morphine-induced antinociception using histamine H3 receptor knockout mice and a histamine H3 receptor antagonist. Anitinociception was evaluated by assays for four nociceptive stimuli: hot-plate, tail-flick, paw-withdrawal, and formalin tests. Antinociception induced by morphine (0.125 nmol/5 microl, i.t.) was significantly augmented in histamine H3 receptor knockout (-/-) mice compared to the wild-type (+/+) mice in all four assays of pain. Furthermore, the effect of intrathecally administered morphine with thioperamide, a histamine H3 antagonist, was examined in C57BL/6J mice. A low dose of i.t. administered thioperamide (0.125 nmol/5 microl) alone had no significant effect on the nociceptive response. In contrast, the combination of morphine (0.125 nmol/5 microl, i.t.) with the same dose of thioperamide resulted in a significant reduction in the pain-related behaviors in all four nociceptive tests. These results suggest that histamine exerts inhibitory effects on morphine-induced antinociception through H3 receptors at the spinal level.

  18. Enhanced antinociceptive effects of morphine in histamine H2 receptor gene knockout mice.

    PubMed

    Mobarakeh, Jalal Izadi; Takahashi, Kazuhiro; Sakurada, Shinobu; Kuramasu, Atsuo; Yanai, Kazuhiko

    2006-09-01

    We have previously shown that antinociceptive effects of morphine are enhanced in histamine H1 receptor gene knockout mice. In the present study, involvement of supraspinal histamine H2 receptor in antinociception by morphine was examined using histamine H2 receptor gene knockout (H2KO) mice and histamine H2 receptor antagonists. Antinociception was evaluated by assays for thermal (hot-plate, tail-flick and paw-withdrawal tests), mechanical (tail-pressure test) and chemical (formalin and capsaicin tests) stimuli. Thresholds for pain perception in H2KO mice were higher than wild-type mice. Antinociceptive effects of intracerebroventricularly administered morphine were enhanced in the H2KO mice compared to wild-type mice. Intracerebroventricular co-administration of morphine and cimetidine produced significant antinociceptive effects in the wild-type mice when compared to morphine or cimetidine alone. Furthermore, zolantidine, a selective and hydrophobic H2 receptor antagonist, enhanced the effects of morphine in all nociceptive assays examined. These results suggest that histamine exerts inhibitory effects on morphine-induced antinociception through H2 receptors at the supraspinal level. Our present and previous studies suggest that H1 and H2 receptors cooperatively function to modulate pain perception in the central nervous system.

  19. Collecting duct-specific knockout of the endothelin B receptor causes hypertension and sodium retention.

    PubMed

    Ge, Yuqiang; Bagnall, Alan; Stricklett, Peter K; Strait, Kevin; Webb, David J; Kotelevtsev, Yuri; Kohan, Donald E

    2006-12-01

    Collecting duct (CD)-derived endothelin-1 (ET-1) inhibits renal Na reabsorption and its deficiency increases blood pressure (BP). The role of CD endothelin B (ETB) receptors in mediating these effects is unknown. CD-specific knockout of the ETB receptor was achieved using an aquaporin-2 promoter-Cre recombinase transgene and the loxP-flanked ETB receptor gene (CD ETB KO). Systolic BP in mice with CD-specific knockout of the ETB receptor, ETA receptor (CD ETA KO) and ET-1 (CD ET-1 KO), and their respective controls were compared during normal- and high-salt diet. On a normal-sodium diet, CD ETB KO mice had elevated BP, which increased further during high salt feeding. However, the degree of hypertension in CD ETB KO mice and the further increase in BP during salt feeding were lower than that of CD ET-1 KO mice, whereas CD ETA KO mice were normotensive. CD ETB KO mice had impaired sodium excretion following acute sodium loading. Aldosterone and plasma renin activity were decreased in CD ETB KO mice on normal- and high-sodium diets, while plasma and urinary ET-1 levels did not differ from controls. In conclusion, the CD ETB receptor partially mediates the antihypertensive and natriuretic effects of ET-1. CD ETA and ETB receptors do not fully account for the antihypertensive and natriuretic effects of CD-derived ET-1, suggesting paracrine effects of this peptide.

  20. Changes in signaling pathways regulating neuroplasticity induced by neurokinin 1 receptor knockout.

    PubMed

    Musazzi, Laura; Perez, Jorge; Hunt, Stephen P; Racagni, Giorgio; Popoli, Maurizio

    2005-03-01

    Neurokinin 1 (NK-1) receptor knockout mice showed behavioral responses similar to animals chronically treated with antidepressants. The aim of this study was to analyse, in NK-1 receptor knockout, the molecular modifications of signaling pathways involved in the pathophysiology of depression and antidepressant mechanism. We found, in total cell cytosol from the prefrontal/frontal cortex, hippocampus and striatum, a marked up-regulation of Ca(2+)-independent enzymatic activity and Thr(286) autophosphorylation of Ca(2+)/calmodulin-dependent protein kinase (CaMK) II. Similar changes in CaMKII regulation were previously observed in rats chronically treated with antidepressants. In striatum, up-regulation of the activity and phosphorylation of CaMKII was also found in the homogenate and synaptosomes. No major changes were observed in the Ca(2+)-dependent kinase activity, with the exception of homogenate from the prefrontal/frontal cortex. We also analysed the expression and phosphorylation of presynaptic proteins, which modulate synaptic vesicle trafficking and exocytosis, and found a marked decrease in synapsin I total expression and basal phosphorylation of Ser(603) (the phosphorylation site for CaMKII) in the prefrontal/frontal cortex. Accordingly, the Ca(2+)/calmodulin-dependent posthoc endogenous phosphorylation of synapsin I in the same area was increased. The knockout of NK-1 receptor had no consequences on the expression or phosphorylation levels of the transcription factor cAMP-responsive element-binding protein and its regulating kinase CaMKIV. However, phosphorylation of ERK1/2-mitogen-activated protein kinases was reduced in the hippocampus and striatum, again resembling an effect previously observed in antidepressant-treated rats. These results show similarities between NK-1 knockouts and animals chronically treated with antidepressants and support the putative antidepressant activity of NK-1 receptor antagonists.

  1. Development of osteoarthritic features in estrogen receptor knockout mice.

    PubMed

    Sniekers, Y H; van Osch, G J V M; Ederveen, A G H; Inzunza, J; Gustafsson, J-A; van Leeuwen, J P T M; Weinans, H

    2009-10-01

    Estrogens are suggested to play a role in the development of osteoarthritis as indicated by the increased prevalence in women after menopause. We studied whether deletion of the estrogen receptor (ER) alpha, beta, or both in female mice results in cartilage damage, osteophytosis, and changes in subchondral bone of skeletally mature animals. We studied knee joints of 6-month-old female ERalpha-/-, ERbeta-/-, and (double) ERalpha-/-beta-/- mice and their wild type (wt) littermates. The presence and size of osteophytes and osteoarthritic changes in cartilage were analyzed using histology. Changes in subchondral plate and trabecular bone were studied using micro-CT. In ERalpha-/-beta-/- mice, we observed an increase in number and/or size of osteophytes and thinning of the lateral subchondral plate. However, cartilage damage was not different from wt. In ERalpha-/- or ERbeta-/- mice, no significant differences in cartilage damage score, osteophyte formation, or subchondral plate thickness were found. The bone volume fraction of the epiphyseal trabecular bone was unchanged in ERalpha-/- mice, increased in ERbeta-/- mice, and decreased in ERalpha-/-beta-/- mice. We conclude that deletion of both ERs leads to increased osteophytosis, but deletion of one or both ERs does not lead to overt cartilage damage in 6-month-old mice.

  2. Absence of the SP/SP receptor circuitry in the substance P-precursor knockout mice or SP receptor, neurokinin (NK)1 knockout mice leads to an inhibited cytokine response in granulomas associated with murine Taenia crassiceps infection.

    PubMed

    Garza, Armandina; Weinstock, Joel; Robinson, Prema

    2008-12-01

    Neurocysticercosis, caused by the cestode Taenia solium, is the most common parasitic infection of the human central nervous system that leads to seizures. Taenia crassiceps cysticercosis in mice is an experimental model for Taenia solium cysticercosis. Similar to the human infection, live parasites cause little or no granulomatous inflammation. Dying parasites initiate a granulomatous reaction. The neuropeptide, substance P (SP), stimulates T-helper (TH) 1 cytokine production. In the current studies, we determined whether absence of SP/SP receptor circuitry in the SP-precursor, preprotachykinin, knockout or SP-receptor, neurokinin (NK) 1, knockout mice affected granuloma cytokine production. We hence compared the levels of Th1 cytokines interleukin (IL)-2 and interferon (IFN)-gamma, and levels of Th2/immunoregulatory cytokines IL-4 and IL-10, by enzyme-linked immunosorbent assay in T. crassiceps-induced granulomas derived from infected C57BL/6 wild type (WT) versus SP-precursor knockout and NK1 knockout mice. We found that mean levels of IL-2, IFN-gamma, IL-4, and IL-10 in infected WT-derived granulomas were significantly higher than those of granulomas derived from infected SP-precursor knockout or the NK1 receptor (NKIR)knockout mice. Levels of Th2/immunoregulatory cytokines, IL-4 and IL-10 were higher in early stage granulomas (histologically-staged on basis of evidence of parasite remnants) versus late stage granulomas (no parasite-remnants) of both knockouts, whereas the reverse was noted in WT-derived granulomas. These study established that the absence of an SP/SP receptor circuitry in the SP precursor knockout mice or NK1 receptor knockout mice led to an inhibited cytokine response.

  3. Caffeine reverses antinociception by oxcarbazepine by inhibition of adenosine A1 receptors: insights using knockout mice.

    PubMed

    Sawynok, Jana; Reid, Allison R; Fredholm, Bertil B

    2010-04-12

    Oxcarbazepine is an anticonvulsant drug that has been explored as a novel therapeutic agent to treat neuropathic pain in humans. It produces antinociception in several preclinical models of pain, and these actions are blocked by methylxanthine adenosine receptor antagonists which implicates adenosine it its actions. In this study, the antinociceptive effect of oxcarbazepine, and the ability of caffeine to reverse its actions, were examined using the formalin test (2%) in wild-type mice and in mice lacking adenosine A(1) receptors by way of further exploring the involvement of adenosine in its actions. Oxcarbazepine produced dose-related suppression of formalin-evoked flinching responses in wild-type mice following both systemic and intraplantar administration, and this action was reversed by systemic and intraplantar administration of caffeine, respectively. The ability of oxcarbazepine to inhibit flinching after systemic and intraplantar administration was unaltered in homozygous (-/-) and heterozygous (+/-) adenosine A(1) receptor knockout mice. However, caffeine no longer reversed this antinociception. Our results indicate that, while adenosine A(1) receptors are not required for oxcarbazepine to produce antinociception in knockout mice, such receptors are essential in order to see caffeine reversal of this antinociceptive effect. Copyright (c) 2010 Elsevier Ireland Ltd. All rights reserved.

  4. Co-Expression of Regulator of G Protein Signaling 4 (RGS4) and the MU Opioid Receptor in Regions of Rat Brain: Evidence That RGS4 Attenuates MU Opioid Receptor Signaling

    DTIC Science & Technology

    2003-01-01

    BamH1 and EcoR1 endonucleases and ligated into a linearized fusion protein expression vector (pGEX-2T). Competent JM109 E . coli bacteria (Promega...25 kDa protein on a 12% sodium dodecyl sulfate polyacrylamide gel. Recombinant RGS4 was produced in E coli . Bacteria were transformed with the pGEX...endonucleases and ligated into linearized pGEX-2T as previously described (18). Competent JM109 E . coli bacteria (Promega; Madison, WI) were transformed with

  5. Cardiac-Specific Knockout of ETA Receptor Mitigates Paraquat-Induced Cardiac Contractile Dysfunction.

    PubMed

    Wang, Jiaxing; Lu, Songhe; Zheng, Qijun; Hu, Nan; Yu, Wenjun; Li, Na; Liu, Min; Gao, Beilei; Zhang, Guoyong; Zhang, Yingmei; Wang, Haichang

    2016-07-01

    Paraquat (1,1'-dim ethyl-4-4'-bipyridinium dichloride), a highly toxic quaternary ammonium herbicide widely used in agriculture, exerts potent toxic prooxidant effects resulting in multi-organ failure including the lung and heart although the underlying mechanism remains elusive. Recent evidence suggests possible involvement of endothelin system in paraquat-induced acute lung injury. This study was designed to examine the role of endothelin receptor A (ETA) in paraquat-induced cardiac contractile and mitochondrial injury. Wild-type (WT) and cardiac-specific ETA receptor knockout mice were challenged to paraquat (45 mg/kg, i.p.) for 48 h prior to the assessment of echocardiographic, cardiomyocyte contractile and intracellular Ca(2+) properties, as well as apoptosis and mitochondrial damage. Levels of the mitochondrial proteins for biogenesis and oxidative phosphorylation including UCP2, HSP90 and PGC1α were evaluated. Our results revealed that paraquat elicited cardiac enlargement, mechanical anomalies including compromised echocardiographic parameters (elevated left ventricular end-systolic and end-diastolic diameters as well as reduced factional shortening), suppressed cardiomyocyte contractile function, intracellular Ca(2+) handling, overt apoptosis and mitochondrial damage. ETA receptor knockout itself failed to affect myocardial function, apoptosis, mitochondrial integrity and mitochondrial protein expression. However, ETA receptor knockout ablated or significantly attenuated paraquat-induced cardiac contractile and intracellular Ca(2+) defect, apoptosis and mitochondrial damage. Taken together, these findings revealed that endothelin system in particular the ETA receptor may be involved in paraquat-induced toxic myocardial contractile anomalies possibly related to apoptosis and mitochondrial damage.

  6. Assessment of 5-HT7 Receptor Agonists Selectivity Using Nociceptive and Thermoregulation Tests in Knockout versus Wild-Type Mice

    PubMed Central

    Brenchat, Alex; Rocasalbas, Maria; Zamanillo, Daniel; Hamon, Michel; Vela, José Miguel; Romero, Luz

    2012-01-01

    No study has ever examined the effect of 5-HT7 receptor agonists on nociception by using 5-HT7 receptor knockout mice. Basal sensitivity to noxious heat stimuli and formalin-induced nociception in both phase I and II of the formalin test did not differ in 5-HT7 receptor knockout mice and paired wild-type controls. Similarly, there was no significant difference in basal body temperature between both genotypes. Subcutaneous administration of 5-HT7 receptor agonists AS-19 (10 mg/kg), E-57431 (10 mg/kg), and E-55888 (20 mg/kg) significantly reduced formalin-induced licking/biting behavior during the phase II of the test in wild-type but not in 5-HT7 receptor knockout mice. At these active analgesic doses, none of the three 5-HT7 receptor agonists modified the basal body temperature neither in wild-type nor in 5-HT7 receptor knockout mice. However, a significant decrease in body temperature was observed at a higher dose (20 mg/kg) of AS-19 and E-57431 in both genotypes. Our data strongly suggest that the 5-HT7 receptor agonists AS-19, E-57431, and E-55888 produce antinociception in the formalin test by activating 5-HT7 receptors. These results also strengthen the idea that the 5-HT7 receptor plays a role in thermoregulation, but by acting in concert with other receptors. PMID:22761612

  7. Acetylcholine receptors in the retinas of the α7 nicotinic acetylcholine receptor knockout mouse

    PubMed Central

    Souza, Fred G. Oliveira; Bruce, Kady S.; Strang, Christianne E.; Morley, Barbara J.; Keyser, Kent T.

    2014-01-01

    Purpose The α7 nicotinic acetylcholine receptor (nAChR) is widely expressed in the nervous system, including in the inner retinal neurons in all species studied to date. Although reductions in the expression of α7 nAChRs are thought to contribute to the memory and visual deficits reported in Alzheimer’s disease (AD) and schizophrenia , the α7 nAChR knockout (KO) mouse is viable and has only slight visual dysfunction. The absence of a major phenotypic abnormality may be attributable to developmental mechanisms that serve to compensate for α7 nAChR loss. We hypothesized that the upregulation of genes encoding other nAChR subunits or muscarinic acetylcholine receptor (mAChR) subtypes during development partially accounts for the absence of major deficiencies in the α7 nAChR KO mouse. The purpose of this study was to determine whether the deletion of the α7 nAChR subunit in a mouse model resulted in changes in the regulation of other cholinergic receptors or other ion channels in an α7 nAChR KO mouse when compared to a wild-type (WT) mouse. Methods To examine gene expression changes, we employed a quantitative real-time polymerase chain reaction (qPCR) using whole retina RNA extracts as well as RNA extracted from selected regions of the retina. These extracts were collected using laser capture microdissection (LCM). The presence of acetylcholine receptor (AChR) subunit and subtype proteins was determined via western blotting. To determine any differences in the number and distribution of choline acetyltransferase (ChAT) amacrine cells, we employed wholemount and vertical immunohistochemistry (IHC) and cell counting. Additionally, in both WT and α7 nAChR KO mouse retinas, the distribution of the nAChR subunit and mAChR subtype proteins were determined via IHC for those KO mice that experienced mRNA changes. Results In the whole retina, there was a statistically significant upregulation of α2, α9, α10, β4, nAChR subunit, and m1 and m4 mAChR subtype

  8. Quantification of beta adrenergic receptor subtypes in beta-arrestin knockout mouse airways.

    PubMed

    Hegde, Akhil; Strachan, Ryan T; Walker, Julia K L

    2015-01-01

    In allergic asthma Beta 2 adrenergic receptors (β2ARs) are important mediators of bronchorelaxation and, paradoxically, asthma development. This contradiction is likely due to the activation of dual signaling pathways that are downstream of G proteins or β-arrestins. Our group has recently shown that β-arrestin-2 acts in its classical role to desensitize and constrain β2AR-induced relaxation of both human and murine airway smooth muscle. To assess the role of β-arrestins in regulating β2AR function in asthma, we and others have utilized β-arrestin-1 and -2 knockout mice. However, it is unknown if genetic deletion of β-arrestins in these mice influences β2AR expression in the airways. Furthermore, there is lack of data on compensatory expression of βAR subtypes when either of the β-arrestins is genetically deleted, thus necessitating a detailed βAR subtype expression study in these β-arrestin knockout mice. Here we standardized a radioligand binding methodology to characterize and quantitate βAR subtype distribution in the airway smooth muscle of wild-type C57BL/6J and β-arrestin-1 and β-arrestin-2 knockout mice. Using complementary competition and single-point saturation binding assays we found that β2ARs predominate over β1ARs in the whole lung and epithelium-denuded tracheobronchial smooth muscle of C57BL/6J mice. Quantification of βAR subtypes in β-arrestin-1 and β-arrestin-2 knockout mouse lung and epithelium-denuded tracheobronchial tissue showed that, similar to the C57BL/6J mice, both knockouts display a predominance of β2AR expression. These data provide further evidence that β2ARs are expressed in greater abundance than β1ARs in the tracheobronchial smooth muscle and that loss of either β-arrestin does not significantly affect the expression or relative proportions of βAR subtypes. As β-arrestins are known to modulate β2AR function, our analysis of βAR subtype expression in β-arrestin knockout mice airways sets a reference

  9. The Kappa Opioid Agonist U-50, 488H Antagonizes Respiratory Effects of Mu Opioid Receptor Agonists in Conscious Rats

    DTIC Science & Technology

    1993-01-01

    Laborator , Animals of the Institute of status is somewhat ambiguous due to the limited selectivities Labhoratory Animal Resources, National Research...Medical Neurosciences, D~ivisiotn of RerpvlitrWler 1 d regmilitorv role of dynorphin on morphine- and si-endorphin-influced analgesial . Arms

  10. Development and Characterization of Uterine Glandular Epithelium Specific Androgen Receptor Knockout Mouse Model.

    PubMed

    Choi, Jaesung Peter; Zheng, Yu; Skulte, Katherine A; Handelsman, David J; Simanainen, Ulla

    2015-11-01

    While estrogen action is the major driver of uterine development, androgens acting via the androgen receptor (AR) may also promote uterine growth as suggested by uterine phenotypes in global AR knockout (ARKO) female mice. Because AR is expressed in uterine endometrial glands, we generated (Cre/loxP) uterine gland epithelium-specific ARKO (ugeARKO) to determine the role of endometrial gland-specific androgen actions. However, AR in uterine gland epithelium may not be required for normal uterine development and function because ugeARKO females had normal uterine development and fertility. To determine if exogenous androgens acting via AR can fully support uterine growth in the absence of estrogens, the ARKO and ugeARKO females were ovariectomized and treated with supraphysiological doses of testosterone or dihydrotestosterone (nonaromatizable androgen). Both dihydrotestosterone and testosterone supported full uterine regrowth in wild-type females while ARKO females had no regrowth (comparable to ovariectomized only). These findings suggest that androgens acting via AR can promote full uterine regrowth in the absence of estrogens. The ugeARKO had 50% regrowth when compared to intact uterine glands, and histomorphologically, both the endometrial and myometrial areas were significantly (P < 0.05) reduced, suggesting glandular epithelial AR located in the endometrium may indirectly modify myometrial development. Additionally, to confirm Cre function in endometrial glands, we generated uge-specific PTEN knockout mouse model. The ugePTEN knockout females developed severe endometrial hyperplasia and therefore present a novel model for future research.

  11. Retinoid-related orphan receptor γ (RORγ) adult induced knockout mice develop lymphoblastic lymphoma.

    PubMed

    Liljevald, Maria; Rehnberg, Maria; Söderberg, Magnus; Ramnegård, Marie; Börjesson, Jenny; Luciani, Donatella; Krutrök, Nina; Brändén, Lena; Johansson, Camilla; Xu, Xiufeng; Bjursell, Mikael; Sjögren, Anna-Karin; Hornberg, Jorrit; Andersson, Ulf; Keeling, David; Jirholt, Johan

    2016-11-01

    RORγ is a nuclear hormone receptor which controls polarization of naive CD4(+) T-cells into proinflammatory Th17 cells. Pharmacological antagonism of RORγ has therapeutic potential for autoimmune diseases; however, this mechanism may potentially carry target-related safety risks, as mice deficient in Rorc, the gene encoding RORγ, develop T-cell lymphoma with 50% frequency. Due to the requirement of RORγ during development, the Rorc knockout (KO) animals lack secondary lymphoid organs and have a dysregulation in the generation of CD4+ and CD8+ T cells. We wanted to extend the evaluation of RORγ deficiency to address the question whether lymphomas, similar to those observed in the Rorc KO, would develop in an animal with an otherwise intact adult immune system. Accordingly, we designed a conditional RORγ knockout mouse (Rorc CKO) where the Rorc locus could be deleted in adult animals. Based on these studies we can confirm that these animals also develop lymphoma in a similar time frame as embryonic Rorc knockouts. This study also suggests that in animals where the gene deletion is incomplete, the thymus undergoes a rapid selection process replacing Rorc deficient cells with remnant thymocytes carrying a functional Rorc locus and that subsequently, these animals do not develop lymphoblastic lymphoma.

  12. Axonal regeneration of optic nerve after crush in Nogo66 receptor knockout mice.

    PubMed

    Su, Ying; Wang, Feng; Teng, Yan; Zhao, Shi-Guang; Cui, Hao; Pan, Shang-Ha

    2009-09-04

    Mature retinal ganglion cells (RGCs) cannot regenerate injured axons because some neurite growth inhibitors, including the C-terminal of Nogo-A (Nogo66), myelin-associated glycoprotein (MAG) and Omgp, exert their effects on neuron regeneration through the Nogo receptor (NgR). In this study, the axonal regeneration of retinal ganglion cells (RGCs) after optic nerve (ON) crush was investigated both in vivo and in vitro in NgR knockout mice. We used NgR knockout mice as the experimental group, and C57BL/6 mice as the control group. Partial ON injury was induced by using a specially designed ON clip to pinch the ON 1mm behind the mouse eyeball with 40g pressure for 9s. NgR mRNA was studied by in situ hybridization (ISH). NgR protein was studied by Western blot. Growth Associated Protein 43 (GAP-43), a plasticity protein expressed highly during axon regeneration, was studied by immunofluorescence staining on the frozen sections. RGCs were cultured and purified. The axonal growth of RGCs was calculated by a computerized image analyzer. We found that compared with the control group, the GAP-43 expression was significantly higher and the axonal growth was significantly more active at every observation time point in the experimental group. These results indicate that NgR genes play an important role in the axonal regeneration after ON injury, while knockout of NgR is effective for eliminating this inhibition and enhancing axonal regeneration.

  13. Impairments in the initiation of maternal behavior in oxytocin receptor knockout mice.

    PubMed

    Rich, Megan E; deCárdenas, Emily J; Lee, Heon-Jin; Caldwell, Heather K

    2014-01-01

    Oxytocin (Oxt) acting through its single receptor subtype, the Oxtr, is important for the coordination of physiology and behavior associated with parturition and maternal care. Knockout mouse models have been helpful in exploring the contributions of Oxt to maternal behavior, including total body Oxt knockout (Oxt -/-) mice, forebrain conditional Oxtr knockout (Oxtr FB/FB) mice, and total body Oxtr knockout (Oxtr -/-) mice. Since Oxtr -/- mice are unable to lactate, maternal behavior has only been examined in virgin females, or in dams within a few hours of parturition, and there have been no studies that have examined their anxiety-like and depression-like behavior following parturition. To improve our understanding of how the absence of Oxt signaling affects maternal behavior, mood and anxiety, we designed a study using Oxtr -/- mice that separated nursing behavior from other aspects of maternal care, such as licking and grooming by thelectomizing (i.e. removing the nipples) of Oxtr +/+ mice and sham-thelectomizing Oxtr -/- mice, and pairing both genotypes with a wet nurse. We then measured pup abandonment, maternal behavior, and postpartum anxiety-like and depression-like behaviors. We hypothesized that genetic disruption of the Oxtr would impact maternal care, mood and anxiety. Specifically, we predicted that Oxtr -/- dams would have impaired maternal care and increased anxiety-like and depression-like behaviors in the postpartum period. We found that Oxtr -/- dams had significantly higher levels of pup abandonment compared to controls, which is consistent with previous work in Oxtr FB/FB mice. Interestingly, Oxtr -/- dams that initiated maternal care did not differ from wildtype controls in measures of maternal behavior. We also did not find any evidence of altered anxiety-like or depressive-like behavior in the postpartum period of Oxtr -/- dams. Thus, our data suggest that Oxt lowers the threshold for the initiation of maternal behavior.

  14. Control of excitatory synaptic transmission by capsaicin is unaltered in TRPV1 vanilloid receptor knockout mice

    PubMed Central

    Benninger, Felix; Freund, Tamás F.; Hájos, Norbert

    2008-01-01

    Several studies have shown that capsaicin could effectively regulate excitatory synaptic transmission in the central nervous system, but the assumption that this effect is mediated by TRPV1 vanilloid receptors (TRPV1Rs) has not been tested directly. To provide direct evidence, we compared the effect of capsaicin on excitatory synapses in wild type mice and TRPV1R knockouts. Using whole-cell patch-clamp techniques, excitatory postsynaptic currents (EPSCs) were recorded in granule cells of the dentate gyrus. First, we investigated the effect of capsaicin on EPSCs evoked by focal stimulation of fibers in the stratum moleculare. Bath application of 10 μM capsaicin reduced the amplitude of evoked EPSCs both in wild type and TRPV1R knockout animals to a similar extent. Treatment of the slices with the TRPV1R antagonist capsazepine (10 μM) alone, or together with the agonist capsaicin, also caused a decrease in the EPSC amplitude both in wild type and TRPV1R knockout animals. Both drugs appeared to affect the efficacy of excitatory synapses at presynaptic sites, since a significant increase was observed in paired-pulse ratio of EPSC amplitude after drug treatment. Next we examined the effect of capsaicin on spontaneously occurring EPSCs. This prototypic vanilloid ligand increased the frequency of events without changing their amplitude in wild type mice. Similar enhancement in the frequency without altering the amplitude of spontaneous EPSCs was observed in TRPV1R knockout mice. These data strongly argue against the hypothesis that capsaicin modulates excitatory synaptic transmission by activating TRPV1Rs, at least in the hippocampal network. PMID:17651868

  15. μ-Opioid Receptor Knockout Mice Are Insensitive to Methamphetamine-Induced Behavioral Sensitization

    PubMed Central

    Shen, Xine; Purser, Chris; Tien, Lu-Tai; Chiu, Chi-Tso; Paul, Ian A.; Baker, Rodney; Loh, Horace H.; Ho, Ing K.; Ma, Tangeng

    2011-01-01

    Repeated administration of psychostimulants to rodents can lead to behavioral sensitization. Previous studies, using nonspecific opioid receptor (OR) antagonists, revealed that ORs were involved in modulation of behavioral sensitization to methamphetamine (METH). However, the contribution of OR subtypes remains unclear. In the present study, using μ-OR knockout mice, we examined the role of μ-OR in the development of METH sensitization. Mice received daily intraperitoneal injection of drug or saline for 7 consecutive days to initiate sensitization. To express sensitization, animals received one injection of drug (the same as for initiation) or saline on day 11. Animal locomotor activity and stereotypy were monitored during the periods of initiation and expression of sensitization. Also, the concentrations of METH and its active metabolite amphetamine in the blood were measured after single and repeated administrations of METH. METH promoted significant locomotor hyperactivity at low doses and stereotyped behaviors at relative high doses (2.5 mg/kg and above). Repeated administration of METH led to the initiation and expression of behavioral sensitization in wild-type mice. METH-induced behavioral responses were attenuated in the μ-OR knockout mice. Haloperidol (a dopamine receptor antagonist) showed a more potent effect in counteracting METH-induced stereotypy in the μ-OR knockout mice. Saline did not induce behavioral sensitization in either genotype. No significant difference was observed in disposition of METH and amphetamine between the two genotypes. Our study indicated that the μ-opioid system is involved in modulating the development of behavioral sensitization to METH. PMID:20209629

  16. Mu-opioid induction of monocyte chemoattractant protein-1, RANTES, and IFN-gamma-inducible protein-10 expression in human peripheral blood mononuclear cells.

    PubMed

    Wetzel, M A; Steele, A D; Eisenstein, T K; Adler, M W; Henderson, E E; Rogers, T J

    2000-12-01

    Strong evidence for the direct modulation of the immune system by opioids is well documented. Mu-opioids have been shown to alter the release of cytokines important for both host defense and the inflammatory response. Proinflammatory chemokines monocyte chemoattractant protein-1 (MCP-1), RANTES, and IFN-gamma-inducible protein-10 (IP-10) play crucial roles in cell-mediated immune responses, proinflammatory reactions, and viral infections. In this report, we show that [D-Ala(2),N:-Me-Phe(4),Gly-ol(5)]enkephalin (DAMGO), a mu-opioid-selective agonist, augments the expression in human PBMCs of MCP-1, RANTES, and IP-10 at both the mRNA and protein levels. Because of the proposed relationship between opioid abuse and HIV-1 infection, we also examined the impact of DAMGO on chemokine expression in HIV-infected cells. Our results show that DAMGO administration induces a significant increase in RANTES and IP-10 expression, while MCP-1 protein levels remain unaffected in PBMCs infected with the HIV-1 strain. In contrast, we show a dichotomous effect of DAMGO treatment on IP-10 protein levels expressed by T- and M-tropic HIV-infected PBMCs. The differential modulation of chemokine expression in T- and M-tropic HIV-1-infected PBMCs by opioids supports a detrimental role for opioids during HIV-1 infection. Modulation of chemokine expression may enhance trafficking of potential noninfected target cells to the site of active infection, thus directly contributing to HIV-1 replication and disease progression to AIDS.

  17. Multiple CNS nicotinic receptors mediate L-dopa-induced dyskinesias: studies with parkinsonian nicotinic receptor knockout mice.

    PubMed

    Quik, Maryka; Campos, Carla; Grady, Sharon R

    2013-10-15

    Accumulating evidence supports the idea that drugs acting at nicotinic acetylcholine receptors (nAChRs) may be beneficial for Parkinson's disease, a neurodegenerative movement disorder characterized by a loss of nigrostriatal dopaminergic neurons. Nicotine administration to parkinsonian animals protects against nigrostriatal damage. In addition, nicotine and nAChR drugs improve L-dopa-induced dyskinesias, a debilitating side effect of L-dopa therapy which remains the gold-standard treatment for Parkinson's disease. Nicotine exerts its antidyskinetic effect by interacting with multiple nAChRs. One approach to identify the subtypes specifically involved in L-dopa-induced dyskinesias is through the use of nAChR subunit null mutant mice. Previous work with β2 and α6 nAChR knockout mice has shown that α6β2* nAChRs were necessary for the development/maintenance of L-dopa-induced abnormal involuntary movements (AIMs). The present results in parkinsonian α4 nAChR knockout mice indicate that α4β2* nAChRs also play an essential role since nicotine did not reduce L-dopa-induced AIMs in such mice. Combined analyses of the data from α4 and α6 knockout mice suggest that the α6α4β2β3 subtype may be critical. In contrast to the studies with α4 and α6 knockout mice, nicotine treatment did reduce L-dopa-induced AIMs in parkinsonian α7 nAChR knockout mice. However, α7 nAChR subunit deletion alone increased baseline AIMs, suggesting that α7 receptors exert an inhibitory influence on L-dopa-induced AIMs. In conclusion, α6β2*, α4β2* and α7 nAChRs all modulate L-dopa-induced AIMs, although their mode of regulation varies. Thus drugs targeting one or multiple nAChRs may be optimal for reducing L-dopa-induced dyskinesias in Parkinson's disease.

  18. Raphe serotonin neuron-specific oxytocin receptor knockout reduces aggression without affecting anxiety-like behavior in male mice only

    PubMed Central

    Pagani, Jerome H.; Williams Avram, Sarah K.; Cui, Zhenzhong; Song, June; Mezey, Éva; Senerth, Julia M.; Baumann, Michael H.; Young, W. Scott

    2015-01-01

    Serotonin and oxytocin influence aggressive and anxiety-like behaviors, though it is unclear how the two may interact. That the oxytocin receptor is expressed in the serotonergic raphe nuclei suggests a mechanism by which the two neurotransmitters may cooperatively influence behavior. We hypothesized that oxytocin acts on raphe neurons to influence serotonergically-mediated anxiety-like, aggressive and parental care behaviors. We eliminated expression of the oxytocin receptor in raphe neurons by crossing mice expressing Cre recombinase under control of the serotonin transporter promoter (Slc6a4) with our conditional oxytocin receptor knockout line. The knockout mice generated by this cross are normal across a range of behavioral measures: there are no effects for either sex on locomotion in an open-field, olfactory habituation/dishabituation or, surprisingly, anxiety-like behaviors in the elevated O and plus mazes. There was a profound deficit in male aggression: only one of 11 raphe oxytocin receptor knockouts showed any aggressive behavior, compared to eight of 11 wildtypes. In contrast, female knockouts displayed no deficits in maternal behavior or aggression. Our results show that oxytocin, via its effects on raphe neurons, is a key regulator of resident-intruder aggression in males but not maternal aggression. Furthermore, this reduction in male aggression is quite different from the effects reported previously after forebrain or total elimination of oxytocin receptors. Finally, we conclude that when constitutively eliminated, oxytocin receptors expressed by serotonin cells do not contribute to baseline anxiety-like behaviors or maternal care. PMID:25677455

  19. Collecting duct-specific endothelin B receptor knockout increases ENaC activity

    PubMed Central

    Bugaj, Vladislav; Mironova, Elena; Kohan, Donald E.

    2012-01-01

    Collecting duct (CD)-derived endothelin-1 (ET-1) acting via endothelin B (ETB) receptors promotes Na+ excretion. Compromise of ET-1 signaling or ETB receptors in the CD cause sodium retention and increase blood pressure. Activity of the epithelial Na+ channel (ENaC) is limiting for Na+ reabsorption in the CD. To test for ETB receptor regulation of ENaC, we combined patch-clamp electrophysiology with CD-specific knockout (KO) of endothelin receptors. We also tested how ET-1 signaling via specific endothelin receptors influences ENaC activity under differing dietary Na+ regimens. ET-1 significantly decreased ENaC open probability in CD isolated from wild-type (WT) and CD ETA KO mice but not CD ETB KO and CD ETA/B KO mice. ENaC activity in WT and CD ETA but not CD ETB and CD ETA/B KO mice was inversely related to dietary Na+ intake. ENaC activity in CD ETB and CD ETA/B KO mice tended to be elevated under all dietary Na+ regimens compared with WT and CD ETA KO mice, reaching significance with high (2%) Na+ feeding. These results show that the bulk of ET-1 inhibition of ENaC activity is mediated by the ETB receptor. In addition, they could explain the Na+ retention and elevated blood pressure observed in CD ET-1 KO, CD ETB KO, and CD ETA/B KO mice consistent with ENaC regulation by ET-1 via ETB receptors contributing to the antihypertensive and natriuretic effects of the local endothelin system in the mammalian CD. PMID:21918182

  20. Comparison of muscarinic receptor selectivity of solifenacin and oxybutynin in the bladder and submandibular gland of muscarinic receptor knockout mice.

    PubMed

    Ito, Yoshihiko; Oyunzul, Luvsandorj; Yoshida, Akira; Fujino, Tomomi; Noguchi, Yukiko; Yuyama, Hironori; Ohtake, Akiyoshi; Suzuki, Masanori; Sasamata, Masao; Matsui, Minoru; Yamada, Shizuo

    2009-08-01

    Solifenacin is a novel selective antagonist of M(3) muscarinic receptor developed for the treatment of overactive bladder. The current study was undertaken to characterize in vivo muscarinic receptor subtype selectivity of solifenacin in the bladder and submandibular gland by using muscarinic receptor subtype knockout (KO) mice. Muscarinic receptors in the bladder and submandibular gland of wild type, M(2)R KO and M(3)R KO mice under in vitro and after oral administration of solifenacin and oxybutynin were measured by radioligand binding assay using [N-methyl-(3)H]scopolamine ([(3)H]NMS). There was little difference between the bladder and submandibular gland of M(2)R KO mice in the receptor binding activities of oxybutynin and solifenacin in vitro, suggesting equal affinity for residual (predominantly M(3) subtype) muscarinic receptors in both tissues. In contrast, compared with oral oxybutynin, oral administration of solifenacin exerted a significantly greater activity to bind muscarinic receptors in the bladder of M(2)R KO mice, while exhibiting a significantly less activity to bind those in the submandibular gland. In the bladder and submandibular gland of M(3)R KO mice, the binding activity of solifenacin and oxybutynin showed no significant difference. Plasma concentrations of solifenacin and oxybutynin after oral administration differed little among wild type, M(2)R KO and M(3)R KO mice. The results indicate that oral solifenacin, unlike oral oxybutynin, may selectively bind to the muscarinic M(3) subtype in the bladder compared with such receptors in the submandibular gland in vivo. Oral solifenacin may be advantageous for the treatment of overactive bladder, in terms of high affinity for M(3) receptors in the bladder.

  1. Uptake and catabolism of modified LDL in scavenger-receptor class A type I/II knock-out mice.

    PubMed Central

    Van Berkel, T J; Van Velzen, A; Kruijt, J K; Suzuki, H; Kodama, T

    1998-01-01

    The liver is the major organ responsible for the uptake of modified low-density lipoprotein (LDL) from the blood circulation, with endothelial and Kupffer cells as major cellular uptake sites. Scavenger-receptors, which include various classes, are held responsible for this uptake. Mice deficient in scavenger-receptor class A types I and II were created and the fate of acetylated LDL (Ac-LDL) in vivo and its interaction with liver endothelial, Kupffer and peritoneal macrophages was characterized. Surprisingly, the decay in vivo (t12 < 2 min), tissue distribution and liver uptake (at 5 min it was 77.4 +/- 4.6% of the injected dose) of Ac-LDL in the knock-out mice were not significantly different from control mice (t12 < 2 min and liver uptake 79.1 +/- 4.6% of the injected dose). A separation of mice liver cells into parenchymal, endothelial and Kupffer cells 10 min after injection of Ac-LDL indicated that in both control and knock-out mice the liver endothelial cells were responsible for more than 70% of the liver uptake. Both in control and knock-out mice, preinjection of polyinosinic acid (poly I, 200 microg) completely blocked the liver uptake, indicating that both in control and knock-out mice the scavenger-receptors are sensitive to poly I. Preinjection of suboptimal poly I concentrations (20 and 50 microg) provided evidence that the serum decay and liver uptake of Ac-LDL is more readily inhibited in the knock-out mice as compared with the control mice, indicating less efficient removal of Ac-LDL in vivo in the knock-out mice under these conditions. Studies in vitro with isolated liver endothelial and Kupffer cells from knock-out mice indicate that the cell association of Ac-LDL during 2 h at 37 degrees C is 50 and 53% of the control, respectively, whereas the degradation reaches values of 58 and 63%. For peritoneal macrophages from knock-out mice the cell association of Ac-LDL was identical to the control mice whereas the Ac-LDL degradation in cells from the

  2. Body water balance and body temperature in vasopressin V1b receptor knockout mice.

    PubMed

    Daikoku, R; Kunitake, T; Kato, K; Tanoue, A; Tsujimoto, G; Kannan, H

    2007-10-30

    In an attempt to determine whether there is a specific vasopressin receptor (V(1b)) subtype involved in the regulation of body water balance and temperature, vasopressin V(1b) receptor knockout mice were used. Daily drinking behavior and renal excretory function were examined in V(1b)-deficient (V(1b)(-/-)) and control (V(1b)(+/+)) mice under the basal and stress-induced condition. In addition, body temperature and locomotor activity were measured with a biotelemetry system. The baseline daily water intake and urine volume were larger in V(1b)(-/-) mice than in V(1b)(+/+) mice. V(1b)(-/-) mice (V(1b)(-/-)) had significantly higher locomotor activity than wild-type, whereas the body temperature and oxygen consumption were lower in V(1b)(-/-) than in the V(1b)(+/+) mice. Next, the V(1b)(-/-) and V(1b)(+/+) mice were subjected to water deprivation for 48 hr. Under this condition, their body temperature decreased with the time course, which was significantly larger for V(1b)(-/-) than for V(1b)(+/+) mice. Central vasopressin has been reported to elicit drinking behavior and antipyretic action, and the V(1b) receptor has been reported to be located in the kidney. Thus, the findings suggest that the V(1b) receptor may be, at least in part, involved in body water balance and body temperature regulation.

  3. Acceleration of intestinal polyposis through prostaglandin receptor EP2 in Apc(Delta 716) knockout mice.

    PubMed

    Sonoshita, M; Takaku, K; Sasaki, N; Sugimoto, Y; Ushikubi, F; Narumiya, S; Oshima, M; Taketo, M M

    2001-09-01

    Arachidonic acid is metabolized to prostaglandin H(2) (PGH(2)) by cyclooxygenase (COX). COX-2, the inducible COX isozyme, has a key role in intestinal polyposis. Among the metabolites of PGH(2), PGE(2) is implicated in tumorigenesis because its level is markedly elevated in tissues of intestinal adenoma and colon cancer. Here we show that homozygous deletion of the gene encoding a cell-surface receptor of PGE(2), EP2, causes decreases in number and size of intestinal polyps in Apc(Delta 716) mice (a mouse model for human familial adenomatous polyposis). This effect is similar to that of COX-2 gene disruption. We also show that COX-2 expression is boosted by PGE(2) through the EP2 receptor via a positive feedback loop. Homozygous gene knockout for other PGE(2) receptors, EP1 or EP3, did not affect intestinal polyp formation in Apc(Delta 716) mice. We conclude that EP2 is the major receptor mediating the PGE2 signal generated by COX-2 upregulation in intestinal polyposis, and that increased cellular cAMP stimulates expression of more COX-2 and vascular endothelial growth factor in the polyp stroma.

  4. GABAA-receptor modification in taurine transporter knockout mice causes striatal disinhibition.

    PubMed

    Sergeeva, O A; Fleischer, W; Chepkova, A N; Warskulat, U; Häussinger, D; Siebler, M; Haas, H L

    2007-12-01

    The Striatum is involved in the regulation of movements and motor skills. We have shown previously, that the osmolyte and neuromodulator taurine plays a role in striatal plasticity. We demonstrate now that hereditary taurine deficiency in taurine-transporter knock-out (TAUT KO) mice results in disinhibition of striatal network activity, which can be corrected by taurine supplementation. Modification of GABAA but not glycine receptors (taurine is a ligand for both receptor types) underlies this disinhibition. Whole-cell recordings from acutely isolated as well as cultured striatal neurons revealed a decreased agonist sensitivity of the GABAA receptor in TAUT KO neurons in the absence of changes in the maximal GABA-evoked current amplitude. The striatal GABA level in TAUT KO mice was unchanged. The amplitude enhancement of spontaneous IPSCs by zolpidem was stronger in TAUT KO than in wild-type (WT) animals. Tonic inhibition was absent in striatal neurons under control conditions but was detected after incubation with the GABA-transaminase inhibitor vigabatrin: bicuculline induced a larger shift of baseline current in WT as compared to TAUT KO neurons. Lack of taurine leads to reduced sensitivity of synaptic and extrasynaptic GABAA receptors and consequently to disinhibition. These findings help in understanding neuropathologies accompanied by the loss of endogenous taurine, for instance in hepatic encephalopathy.

  5. Comparative effects of chlorpyrifos in wild type and cannabinoid Cb1 receptor knockout mice

    SciTech Connect

    Baireddy, Praveena; Liu, Jing; Hinsdale, Myron; Pope, Carey

    2011-11-15

    Endocannabinoids (eCBs) modulate neurotransmission by inhibiting the release of a variety of neurotransmitters. The cannabinoid receptor agonist WIN 55.212-2 (WIN) can modulate organophosphorus (OP) anticholinesterase toxicity in rats, presumably by inhibiting acetylcholine (ACh) release. Some OP anticholinesterases also inhibit eCB-degrading enzymes. We studied the effects of the OP insecticide chlorpyrifos (CPF) on cholinergic signs of toxicity, cholinesterase activity and ACh release in tissues from wild type (+/+) and cannabinoid CB1 receptor knockout (-/-) mice. Mice of both genotypes (n = 5-6/treatment group) were challenged with CPF (300 mg/kg, 2 ml/kg in peanut oil, sc) and evaluated for functional and neurochemical changes. Both genotypes exhibited similar cholinergic signs and cholinesterase inhibition (82-95% at 48 h after dosing) in cortex, cerebellum and heart. WIN reduced depolarization-induced ACh release in vitro in hippocampal slices from wild type mice, but had no effect in hippocampal slices from knockouts or in striatal slices from either genotype. Chlorpyrifos oxon (CPO, 100 {mu}M) reduced release in hippocampal slices from both genotypes in vitro, but with a greater reduction in tissues from wild types (21% vs 12%). CPO had no significant in vitro effect on ACh release in striatum. CPF reduced ACh release in hippocampus from both genotypes ex vivo, but reduction was again significantly greater in tissues from wild types (52% vs 36%). In striatum, CPF led to a similar reduction (20-23%) in tissues from both genotypes. Thus, while CB1 deletion in mice had little influence on the expression of acute toxicity following CPF, CPF- or CPO-induced changes in ACh release appeared sensitive to modulation by CB1-mediated eCB signaling in a brain-regional manner. -- Highlights: Black-Right-Pointing-Pointer C57Bl/6 mice showed dose-related cholinergic toxicity following subcutaneous chlorpyrifos exposure. Black-Right-Pointing-Pointer Wild type and

  6. Immune malfunction in the GPR39 zinc receptor of knockout mice: Its relationship to depressive disorder.

    PubMed

    Młyniec, Katarzyna; Trojan, Ewa; Ślusarczyk, Joanna; Głombik, Katarzyna; Basta-Kaim, Agnieszka; Budziszewska, Bogusława; Skrzeszewski, Jakub; Siwek, Agata; Holst, Birgitte; Nowak, Gabriel

    2016-02-15

    Depression is a serious psychiatric disorder affecting not only the monaminergic, glutamatergic, and GABAergic neurosystems, but also the immune system. Patients suffering from depression show disturbance in the immune parameters as well as increased susceptibility to infections. Zinc is well known as an anti-inflammatory agent, and its link with depression has been proved, zinc deficiency causing depression- and anxiety-like behavior with immune malfunction. It has been discovered that trace-element zinc acts as a neurotransmitter in the central nervous system via zinc receptor GPR39. In this study we investigated whether GPR39 knockout would cause depressive-like behavior as measured by the forced swim test, and whether these changes would coexist with immune malfunction. In GPR39 knockout mice versus a wild-type control we found: i) depressive-like behavior; ii) significantly reduced thymus weight; (iii) reduced cell viability of splenocytes; iv) reduced proliferative response of splenocytes; and v) increased IL-6 production of splenocytes after ConA stimulation and decreased IL-1b and IL-6 release after LPS stimulation. The results indicate depressive-like behavior in GPR39 KO animals with an immune response similar to that observed in depressive disorder. Here for the first time we show immunological changes under GPR39-deficient conditions. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Cholesterol lowering in low density lipoprotein receptor knockout mice overexpressing apolipoprotein E.

    PubMed Central

    Osuga, J; Yonemoto, M; Yamada, N; Shimano, H; Yagyu, H; Ohashi, K; Harada, K; Kamei, T; Yazaki, Y; Ishibashi, S

    1998-01-01

    Apo E is a key molecule in the lipoprotein metabolism; thus, genetic manipulation of apo E may prove useful in the treatment of hypercholesterolemia. To test the feasibility of this idea, we have generated low density lipoprotein receptor (LDLR) knockout mice that overexpress the rat apo E transgene (ETg+/+:LDLRKO), and compared their plasma lipoprotein profiles with those of nonexpressing LDLR knockout mice (ETg-/-:LDLRKO). On a normal chow diet, the mean plasma cholesterol level of ETg+/+:LDLRKO mice was significantly lower than that of ETg-/-:LDLRKO mice (189 versus 240 mg/dl, P < 0. 01). The LDL fraction was selectively reduced in the ETg+/+:LDLRKO mice. Despite the challenge with an atherogenic diet, cholesterol lowering was persistently observed and fatty streak lesions in the aortic sinus were significantly suppressed in the mice overexpressing apo E. These results imply that stimulation of hepatic production of apo E may be used as a promising adjunctive therapy for homozygous familial hypercholesterolemia. PMID:9664080

  8. Sex-dependence of anxiety-like behavior in cannabinoid receptor 1 (Cnr1) knockout mice

    PubMed Central

    Bowers, Mallory E.; Ressler, Kerry J.

    2015-01-01

    Epidemiological data suggest women are at increased risk for developing anxiety and depression, although the mechanisms for this sex/gender difference remain incompletely understood. Pre-clinical studies have begun to investigate sex-dependent emotional learning and behavior in rodents, particularly as it relates to psychopathology; however, information about how gonadal hormones interact with the central nervous system is limited. We observe greater anxiety-like behavior in male mice with global knockout of the cannabinoid 1 receptor (Cnr1) compared to male, wild-type controls as measured by percent open arm entries on an elevated plus maze test. A similar increase in anxiety-like behavior, however, is not observed when comparing female Cnr1 knockouts to female wild-type subjects. Although, ovariectomy in female mice did not reverse this effect, both male and female adult mice with normative development were sensitive to Cnr1 antagonist-mediated increases in anxiety-like behavior. Together, these data support an interaction between sex, potentially mediated by gonadal hormones, and the endocannabinoid system at an early stage of development that is critical for establishing adult anxiety-like behavior. PMID:26684509

  9. Receptor for advanced glycation end products (RAGE) knockout reduces fetal dysmorphogenesis in murine diabetic pregnancy.

    PubMed

    Ejdesjö, Andreas; Brings, Sebastian; Fleming, Thomas; Fred, Rikard G; Nawroth, Peter P; Eriksson, Ulf J

    2016-07-01

    The receptor for Advanced Glycation End products (RAGE) is implicated in the pathogenesis of diabetic complications, but its importance in diabetic embryopathy is unclear. We therefore investigated the role of RAGE in diabetic embryopathy using streptozotocin induced diabetes in female wild type (WT) C57Bl/6N and RAGE knockout C57Bl/6N (RAGE(-/-)) mice, mated with control males of the same genotype. Maternal diabetes induced more fetal resorption and malformation (facial skeleton, neural tube) in the WT than in the RAGE(-/-) fetuses. Maternal plasma glucose and methylgyoxal concentrations, as well as embryonic N(ε)-carboxymethyl-lysine (CML) levels were increased to the same extent in diabetic WT and RAGE(-/-) pregnancy. However, maternal diabetes induced increased fetal hepatic isoprostane 8-iso-PGF2α levels (oxidative stress marker) and embryonic activation of NFκB in WT only (not in RAGE(-/-) embryos). The association between RAGE knockout and diminished embryonic dysmorphogenesis in diabetic pregnancy suggests that embryonic RAGE activation is involved in diabetic embryopathy. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. Food intake, tumor growth, and weight loss in EP2 receptor subtype knockout mice bearing PGE2-producing tumors

    PubMed Central

    Iresjö, Britt-Marie; Wang, Wenhua; Nilsberth, Camilla; Andersson, Marianne; Lönnroth, Christina; Smedh, Ulrika

    2015-01-01

    Previous studies in our laboratory have demonstrated that prostaglandin (PG) E2 is involved in anorexia/cachexia development in MCG 101 tumor-bearing mice. In the present study, we investigate the role of PGE receptor subtype EP2 in the development of anorexia after MCG 101 implantation in wild-type (EP2+/+) or EP2-receptor knockout (EP2−/−) mice. Our results showed that host absence of EP2 receptors attenuated tumor growth and development of anorexia in tumor-bearing EP2 knockout mice compared to tumor-bearing wild-type animals. Microarray profiling of the hypothalamus revealed a relative twofold change in expression of around 35 genes including mRNA transcripts coding for Phospholipase A2 and Prostaglandin D2 synthase (Ptgds) in EP2 receptor knockout mice compared to wild-type mice. Prostaglandin D2 synthase levels were increased significantly in EP2 receptor knockouts, suggesting that improved food intake may depend on altered balance of prostaglandin production in hypothalamus since PGE2 and PGD2 display opposing effects in feeding control. PMID:26197930

  11. Effect of P2X7 Receptor Knockout on AQP-5 Expression of Type I Alveolar Epithelial Cells

    PubMed Central

    Ebeling, Georg; Bläsche, Robert; Hofmann, Falk; Augstein, Antje; Kasper, Michael; Barth, Kathrin

    2014-01-01

    P2X7 receptors, ATP-gated cation channels, are specifically expressed in alveolar epithelial cells. The pathophysiological function of this lung cell type, except a recently reported putative involvement in surfactant secretion, is unknown. In addition, P2X7 receptor-deficient mice show reduced inflammation and lung fibrosis after exposure with bleomycin. To elucidate the role of the P2X7 receptor in alveolar epithelial type I cells we characterized the pulmonary phenotype of P2X7 receptor knockout mice by using immunohistochemistry, western blot analysis and real-time RT PCR. No pathomorphological signs of fibrosis were found. Results revealed, however, a remarkable loss of aquaporin-5 protein and mRNA in young knockout animals. Additional in vitro experiments with bleomycin treated precision cut lung slices showed a greater sensitivity of the P2X7 receptor knockout mice in terms of aquaporin-5 reduction as wild type animals. Finally, P2X7 receptor function was examined by using the alveolar epithelial cell lines E10 and MLE-12 for stimulation experiments with bleomycin. The in vitro activation of P2X7 receptor was connected with an increase of aquaporin-5, whereas the inhibition of the receptor with oxidized ATP resulted in down regulation of aquaporin-5. The early loss of aquaporin-5 which can be found in different pulmonary fibrosis models does not implicate a specific pathogenetic role during fibrogenesis. PMID:24941004

  12. No further loss of dorsal root ganglion cells after axotomy in p75 neurotrophin receptor knockout mice.

    PubMed

    Sørensen, Bodil; Tandrup, Trine; Koltzenburg, Martin; Jakobsen, Johannes

    2003-05-05

    The role of the p75 neurotrophin receptor for neuronal survival after nerve crush was studied in L5 dorsal root ganglia (DRG) of knockout mice and controls with assumption-free stereological methods. Numbers of neuronal A- and B-cells were obtained using the optical fractionator and optical disector techniques. At birth, the total number of DRG neurons was 10,000 +/- 2,600 in control mice compared with 5,100 +/- 1,300 in p75 knockout mice. During postnatal development, 1,400 neuronal B-cell bodies were lost in p75 knockouts (2P < 0.05) and 1,100 in controls (NS), whereas the A-cell population remained stable. After a sciatic nerve crush, the total neuron loss in controls was 15.4% +/- 3.5% (2P < 0.05) and 22.7% +/- 5.1% (2P < 0.05) at days 14 and 42, respectively. In contrast, there was no loss in total number of neurons after crush in p75 knockout mice. Neuronal A-cell number was unchanged after the crush in p75 knockouts as well as in controls at both times. At 14 days, the population of B-cells was reduced by 24.8% +/- 3.6% in controls and by 6.1% +/- 3.5% in p75 knockouts, this difference being significant (2P < 0.001). At 42 days, the B-cell loss was 29.6% +/- 5.5% in controls and 4.2% +/- 6.4% in p75 knockouts (2P < 0.001). In conclusion, the lack of the p75 receptor results in neuronal DRG cells that are resistant to nerve injury, pointing to a role for the receptor in apoptosis. Copyright 2003 Wiley-Liss, Inc.

  13. Impairments in the Initiation of Maternal Behavior in Oxytocin Receptor Knockout Mice

    PubMed Central

    Rich, Megan E.; deCárdenas, Emily J.; Lee, Heon-Jin; Caldwell, Heather K.

    2014-01-01

    Oxytocin (Oxt) acting through its single receptor subtype, the Oxtr, is important for the coordination of physiology and behavior associated with parturition and maternal care. Knockout mouse models have been helpful in exploring the contributions of Oxt to maternal behavior, including total body Oxt knockout (Oxt −/−) mice, forebrain conditional Oxtr knockout (Oxtr FB/FB) mice, and total body Oxtr knockout (Oxtr −/−) mice. Since Oxtr −/− mice are unable to lactate, maternal behavior has only been examined in virgin females, or in dams within a few hours of parturition, and there have been no studies that have examined their anxiety-like and depression-like behavior following parturition. To improve our understanding of how the absence of Oxt signaling affects maternal behavior, mood and anxiety, we designed a study using Oxtr −/− mice that separated nursing behavior from other aspects of maternal care, such as licking and grooming by thelectomizing (i.e. removing the nipples) of Oxtr +/+ mice and sham-thelectomizing Oxtr −/− mice, and pairing both genotypes with a wet nurse. We then measured pup abandonment, maternal behavior, and postpartum anxiety-like and depression-like behaviors. We hypothesized that genetic disruption of the Oxtr would impact maternal care, mood and anxiety. Specifically, we predicted that Oxtr −/− dams would have impaired maternal care and increased anxiety-like and depression-like behaviors in the postpartum period. We found that Oxtr −/− dams had significantly higher levels of pup abandonment compared to controls, which is consistent with previous work in Oxtr FB/FB mice. Interestingly, Oxtr −/− dams that initiated maternal care did not differ from wildtype controls in measures of maternal behavior. We also did not find any evidence of altered anxiety-like or depressive-like behavior in the postpartum period of Oxtr −/− dams. Thus, our data suggest that Oxt lowers the threshold for the initiation of

  14. Trilostane exerts antidepressive effects among wild-type, but not estrogen receptor [beta] knockout mice.

    PubMed

    Koonce, Carolyn J; Walf, Alicia A; Frye, Cheryl A

    2009-08-05

    Women with estrogen receptor (ER) positive breast cancer, who are treated with the ER blocker, tamoxifen, have an increased risk of depression. Trilostane, a 3b-hydroxysteroid dehydrogenase inhibitor, is now being used to treat tamoxifen-insensitive breast cancer. In-vitro assays show that trilostane may have actions through ERb. Results of in-vivo research shows that actions at ERb may underline some antidepressant effects of estrogen. We hypothesized that trilostane may exert antidepressive effects in the forced swim in part due to actions through ERb. Trilostane (25 mg/kg, intraperitoneally), compared with vehicle, had significant antidepressant-like effects but only when administered to wild-type, not ERb knockout, mice. Thus, actions of trilostane through ERb may underlie some of its antidepressant-like effects.

  15. Trilostane exerts antidepressive effects among wild-type, but not estrogen receptor β knockout mice

    PubMed Central

    Koonce, Carolyn J.; Walf, Alicia A.; Frye, Cheryl A.

    2013-01-01

    Women with estrogen receptor (ER) positive breast cancer, who are treated with the ER blocker, tamoxifen, have an increased risk of depression. Trilostane, a 3β-hydroxysteroid dehydrogenase inhibitor, is now being used to treat tamoxifen-insensitive breast cancer. In-vitro assays show that trilostane may have actions through ERβ. Results of in-vivo research shows that actions at ERβ may underline some antidepressant effects of estrogen. We hypothesized that trilostane may exert antidepressive effects in the forced swim in part due to actions through ERβ. Trilostane (25 mg/kg, intraperitoneally), compared with vehicle, had significant antidepressant-like effects but only when administered to wild-type, not ERβ knockout, mice. Thus, actions of trilostane through ERβ may underlie some of its antidepressant-like effects. PMID:19593916

  16. Thromboelastography After Murine TBI and Implications of Beta-Adrenergic Receptor Knockout.

    PubMed

    Liou, Douglas Z; Ko, Ara; Volod, Oksana; Barmparas, Galinos; Harada, Megan Y; Martin, Matthew J; Salim, Ali; Dhillon, Navpreet; Thomsen, Gretchen M; Ley, Eric J

    2016-08-01

    The source of coagulopathy in traumatic brain injury (TBI) is multifactorial and may include adrenergic stimulation. The aim of this study was to assess coagulopathy after TBI using thromboelastography (TEG), and to investigate the implications of β-adrenergic receptor knockout. Adult male wild type c57/bl6 (WT) and β1/β2-adrenergic receptor knockout (BKO) mice were assigned to either TBI (WT-TBI, BKO-TBI) or sham injury (WT-sham, BKO-sham). Mice assigned to TBI were subject to controlled cortical impact (CCI). At 24 h post-injury, whole blood samples were obtained and taken immediately for TEG. At 24 h after injury, a trend toward increased fibrinolysis was seen in WT-TBI compared to WT-sham although this did not reach significance (EPL 8.1 vs. 0 %, p = 0.18). No differences were noted in fibrinolysis in BKO-TBI compared to BKO-sham (LY30 2.6 vs. 2.5 %, p = 0.61; EPL 3.4 vs. 2.9 %, p = 0.61). In addition BKO-TBI demonstrated increased clot strength compared to BKO-sham (MA 76.6 vs. 68.6, p = 0.03; G 18.2 vs. 11.3, p = 0.03). In a mouse TBI model, WT mice sustaining TBI demonstrated a trend toward increased fibrinolysis at 24 h after injury while BKO mice did not. These findings suggest β-blockade may attenuate the coagulopathy of TBI and minimize progression of intracranial hemorrhage by reducing fibrinolysis and increasing clot strength.

  17. Knockout of NMDA receptors in parvalbumin interneurons recreates autism-like phenotypes.

    PubMed

    Saunders, John A; Tatard-Leitman, Valerie M; Suh, Jimmy; Billingslea, Eddie N; Roberts, Timothy P; Siegel, Steven J

    2013-04-01

    Autism is a disabling neurodevelopmental disorder characterized by social deficits, language impairment, and repetitive behaviors with few effective treatments. New evidence suggests that autism has reliable electrophysiological endophenotypes and that these measures may be caused by n-methyl-d-aspartic acid receptor (NMDAR) disruption on parvalbumin (PV)-containing interneurons. These findings could be used to create new translational biomarkers. Recent developments have allowed for cell-type selective knockout of NMDARs in order to examine the perturbations caused by disrupting specific circuits. This study examines several electrophysiological and behavioral measures disrupted in autism using a PV-selective reduction in NMDA R1 subunit. Mouse electroencephalograph (EEG) was recorded in response to auditory stimuli. Event-related potential (ERP) component amplitude and latency analysis, social testing, and premating ultrasonic vocalizations (USVs) recordings were performed. Correlations were examined between the ERP latency and behavioral measures. The N1 ERP latency was delayed, sociability was reduced, and mating USVs were impaired in PV-selective NMDA Receptor 1 Knockout (NR1 KO) as compared with wild-type mice. There was a significant correlation between N1 latency and sociability but not between N1 latency and premating USV power or T-maze performance. The increases in N1 latency, impaired sociability, and reduced vocalizations in PV-selective NR1 KO mice mimic similar changes found in autism. Electrophysiological changes correlate to reduced sociability, indicating that the local circuit mechanisms controlling N1 latency may be utilized in social function. Therefore, we propose that behavioral and electrophysiological alterations in PV-selective NR1 KO mice may serve as a useful model for therapeutic development in autism. Autism Res 2013, 6: 69-77. © 2013 International Society for Autism Research, Wiley Periodicals, Inc. © 2013 International Society for

  18. Knockout of NMDA Receptors in Parvalbumin Interneurons Recreates Autism-Like Phenotypes

    PubMed Central

    Saunders, John A.; Tatard-Leitman, Valerie M.; Suh, Jimmy; Billingslea, Eddie N.; Roberts, Timothy P.; Siegel, Steven J.

    2014-01-01

    Autism is a disabling neurodevelopmental disorder characterized by social deficits, language impairment, and repetitive behaviors with few effective treatments. New evidence suggests that autism has reliable electrophysiological endophenotypes and that these measures may be caused by n-methyl-d-aspartic acid receptor (NMDAR) disruption on parvalbumin (PV)-containing interneurons. These findings could be used to create new translational biomarkers. Recent developments have allowed for cell-type selective knockout of NMDARs in order to examine the perturbations caused by disrupting specific circuits. This study examines several electrophysiological and behavioral measures disrupted in autism using a PV-selective reduction in NMDA R1 subunit. Mouse electroencephalograph (EEG) was recorded in response to auditory stimuli. Event-related potential (ERP) component amplitude and latency analysis, social testing, and premating ultrasonic vocalizations (USVs) recordings were performed. Correlations were examined between the ERP latency and behavioral measures. The N1 ERP latency was delayed, sociability was reduced, and mating USVs were impaired in PV-selective NMDA Receptor 1 Knockout (NR1 KO) as compared with wild-type mice. There was a significant correlation between N1 latency and sociability but not between N1 latency and premating USV power or T-maze performance. The increases in N1 latency, impaired sociability, and reduced vocalizations in PV-selective NR1 KO mice mimic similar changes found in autism. Electrophysiological changes correlate to reduced sociability, indicating that the local circuit mechanisms controlling N1 latency may be utilized in social function. Therefore, we propose that behavioral and electrophysiological alterations in PV-selective NR1 KO mice may serve as a useful model for therapeutic development in autism. PMID:23441094

  19. Conditional Knockout of NMDA Receptors in Dopamine Neurons Prevents Nicotine-Conditioned Place Preference

    PubMed Central

    Phillip Wang, Lei; Li, Fei; Shen, Xiaoming; Tsien, Joe Z.

    2010-01-01

    Nicotine from smoking tobacco produces one of the most common forms of addictive behavior and has major societal and health consequences. It is known that nicotine triggers tobacco addiction by activating nicotine acetylcholine receptors (nAChRs) in the midbrain dopaminergic reward system, primarily via the ventral tegmental area. Heterogeneity of cell populations in the region has made it difficult for pharmacology-based analyses to precisely assess the functional significance of glutamatergic inputs to dopamine neurons in nicotine addiction. By generating dopamine neuron-specific NR1 knockout mice using cre/loxP-mediated method, we demonstrate that genetic inactivation of the NMDA receptors in ventral tegmental area dopamine neurons selectively prevents nicotine-conditioned place preference. Interestingly, the mutant mice exhibit normal performances in the conditioned place aversion induced by aversive air puffs. Therefore, this selective effect on addictive drug-induced reinforcement behavior suggests that NMDA receptors in the dopamine neurons are critical for the development of nicotine addiction. PMID:20062537

  20. Conditional knockout of NMDA receptors in dopamine neurons prevents nicotine-conditioned place preference.

    PubMed

    Wang, Lei Phillip; Li, Fei; Shen, Xiaoming; Tsien, Joe Z

    2010-01-07

    Nicotine from smoking tobacco produces one of the most common forms of addictive behavior and has major societal and health consequences. It is known that nicotine triggers tobacco addiction by activating nicotine acetylcholine receptors (nAChRs) in the midbrain dopaminergic reward system, primarily via the ventral tegmental area. Heterogeneity of cell populations in the region has made it difficult for pharmacology-based analyses to precisely assess the functional significance of glutamatergic inputs to dopamine neurons in nicotine addiction. By generating dopamine neuron-specific NR1 knockout mice using cre/loxP-mediated method, we demonstrate that genetic inactivation of the NMDA receptors in ventral tegmental area dopamine neurons selectively prevents nicotine-conditioned place preference. Interestingly, the mutant mice exhibit normal performances in the conditioned place aversion induced by aversive air puffs. Therefore, this selective effect on addictive drug-induced reinforcement behavior suggests that NMDA receptors in the dopamine neurons are critical for the development of nicotine addiction.

  1. Pregnane X Receptor Knockout Mice Display Aging-Dependent Wearing of Articular Cartilage

    PubMed Central

    Azuma, Kotaro; Casey, Stephanie C.; Urano, Tomohiko; Horie-Inoue, Kuniko; Ouchi, Yasuyoshi; Blumberg, Bruce; Inoue, Satoshi

    2015-01-01

    Steroid and xenobiotic receptor (SXR) and its murine ortholog, pregnane X receptor (PXR), are nuclear receptors that are expressed at high levels in the liver and the intestine where they function as xenobiotic sensors that induce expression of genes involved in detoxification and drug excretion. Recent evidence showed that SXR and PXR are also expressed in bone tissue where they mediate bone metabolism. Here we report that systemic deletion of PXR results in aging-dependent wearing of articular cartilage of knee joints. Histomorphometrical analysis showed remarkable reduction of width and an enlarged gap between femoral and tibial articular cartilage in PXR knockout mice. We hypothesized that genes induced by SXR in chondrocytes have a protective effect on articular cartilage and identified Fam20a (family with sequence similarity 20a) as an SXR-dependent gene induced by the known SXR ligands, rifampicin and vitamin K2. Lastly, we demonstrated the biological significance of Fam20a expression in chondrocytes by evaluating osteoarthritis-related gene expression of primary articular chondrocytes. Consistent with epidemiological findings, our results indicate that SXR/PXR protects against aging-dependent wearing of articular cartilage and that ligands for SXR/PXR have potential role in preventing osteoarthritis caused by aging. PMID:25749104

  2. Improved androgen specificity of AR-EcoScreen by CRISPR based glucocorticoid receptor knockout.

    PubMed

    Zwart, Nick; Andringa, Dave; de Leeuw, Willem-Jan; Kojima, Hiroyuki; Iida, Mitsuru; Houtman, Corine J; de Boer, Jacob; Kool, Jeroen; Lamoree, Marja H; Hamers, Timo

    2017-08-11

    The AR-EcoScreen is a widely used reporter assay for the detection of androgens and anti-androgens. Endogenous expression of glucocorticoid receptors and their affinity for the androgen responsive element that drives reporter expression, however, makes the reporter cells sensitive to interference by glucocorticoids and less specific for (anti-)androgens. To create a glucocorticoid insensitive derivative of the AR-EcoScreen, CRISPR/Cas9 genome editing was used to develop glucocorticoid receptor knockout mutants by targeting various sites in the glucocorticoid gene. Two mutant cell lines were further characterized and validated against the unmodified AR-EcoScreen with a set of 19 environmentally relevant chemicals and a series of environmental passive sampler extracts with (anti-)androgenic activity. Sequencing of the targeted sites revealed premature stop codons following frame-shift mutations, leading to an absence of functional glucocorticoid receptor expression. The introduced mutations rendered cell lines insensitive to glucocorticoid activation and caused no significant difference in the responsiveness towards (anti-)androgens, compared to the unmodified AR-EcoScreen cells, allowing the selective, GR-independent, determination of (anti-)androgenicity in environmental passive sampler extracts. The increase in selectivity for (anti-)androgens improves reliability of the AR-EcoScreen and will provide higher accuracy in determining (anti-)androgenic potential when applied in toxicity screening and environmental monitoring of both single compounds and mixtures. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. [Upregulation of P2X3 receptors in dorsal root ganglion of TRPV1 knockout female mice].

    PubMed

    Fang, Xiao; Shi, Xiao-Han; Huang, Li-Bin; Rong, Wei-Fang; Ma, Bei

    2014-08-25

    The study was aimed to investigate the changes in mechanical pain threshold in the condition of chronic inflammatory pain after transient receptor potential vanilloid 1 (TRPV1) gene was knockout. Hind-paw intraplantar injection of complete freund's adjuvant (CFA, 20 μL) produced peripheral inflammation in wild-type and TRPV1 knockout female mice. The mechanical pain thresholds were measured during the 8 days after injection and pre-injection by using Von-Frey hair. Nine days after injection, mice were killed and the differences of expression of c-Fos and P2X3 receptor in the dorsal root ganglia (DRG) and spinal cord dorsal horn were examined by Western blotting between the two groups. Compared with that in wild-type mice, the mechanical pain threshold was increased significantly in TRPV1 knockout mice (P < 0.05); 3 days after CFA injection, the baseline mechanical pain threshold in the TRPV1 knockout mice group was significantly higher than that in the wild-type mice group (P < 0.05); The result of Western blotting showed that the expression of c-Fos protein both in DRG and spinal cord dorsal horn of TRPV1 knockout mice group was decreased significantly compared with that in wild-type mice group (P < 0.01, P < 0.05), while the expression of P2X3 receptor in DRG of TRPV1 knockout mice group was increased significantly compared with that in wild-type mice group (P < 0.05). Our findings indicate that TRPV1 may influence the peripheral mechanical pain threshold by mediating the expression of c-Fos protein both in DRG and spinal cord dorsal horn and changing the expression of P2X3 receptor in DRG.

  4. Chronic allergic pulmonary inflammation is aggravated in angiotensin-(1-7) Mas receptor knockout mice.

    PubMed

    Magalhães, Giselle S; Rodrigues-Machado, Maria Glória; Motta-Santos, Daisy; Alenina, Natalia; Bader, Michael; Santos, Robson A; Barcelos, Lucíola S; Campagnole-Santos, Maria José

    2016-12-01

    The angiotensin-(1-7) [ANG-(1-7)]/Mas receptor pathway is currently recognized as a counterbalancing mechanism of the renin-angiotensin system in different pathophysiological conditions. We have previously described that treatment with ANG-(1-7) attenuates lung inflammation and remodeling in an experimental model of asthma. In the present study, we investigated whether lack of the Mas receptor could alter the inflammatory response in a model of chronic allergic lung inflammation induced by ovalbumin (OVA). Mas receptor wild-type (MasWT) and knockout (MasKO) mice were subjected to four doses of OVA (20 μg/mice ip) with a 14-day interval. At the 21st day, nebulization with OVA (1%) was started, three times per week until the 46th day. Control groups received saline (0.9% ip) and were nebulized with saline (0.9%). MasWT-OVA developed a modest inflammatory response and minor pulmonary remodeling to OVA challenge. Strikingly, MasKO-OVA presented a significant increase in inflammatory cell infiltrate, increase in extracellular matrix deposition, increase in thickening of the alveolar parenchyma, increase in thickening of the smooth muscle layer of the pulmonary arterioles, increase in proinflammatory cytokine and chemokine levels in the lungs, characteristic of chronic asthma. Additionally, MasKO-OVA presented an increase in ERK1/2 phosphorylation compared with MasWT-OVA. Furthermore, MasKO-OVA showed a worse performance in a test of maximum physical exercise compared with MasWT-OVA. Our study shows that effects triggered by the Mas receptor are important to attenuate the inflammatory and remodeling processes in a model of allergic lung inflammation in mice. Our data indicate that impairment of the ANG-(1-7)/Mas receptor pathway may lead to worsening of the pathophysiological changes of asthma. Copyright © 2016 the American Physiological Society.

  5. Brain Region-Specific Effects of cGMP-Dependent Kinase II Knockout on AMPA Receptor Trafficking and Animal Behavior

    ERIC Educational Resources Information Center

    Kim, Seonil; Pick, Joseph E.; Abera, Sinedu; Khatri, Latika; Ferreira, Danielle D. P.; Sathler, Matheus F.; Morison, Sage L.; Hofmann, Franz; Ziff, Edward B.

    2016-01-01

    Phosphorylation of GluA1, a subunit of AMPA receptors (AMPARs), is critical for AMPAR synaptic trafficking and control of synaptic transmission. cGMP-dependent protein kinase II (cGKII) mediates this phosphorylation, and cGKII knockout (KO) affects GluA1 phosphorylation and alters animal behavior. Notably, GluA1 phosphorylation in the KO…

  6. Brain Region-Specific Effects of cGMP-Dependent Kinase II Knockout on AMPA Receptor Trafficking and Animal Behavior

    ERIC Educational Resources Information Center

    Kim, Seonil; Pick, Joseph E.; Abera, Sinedu; Khatri, Latika; Ferreira, Danielle D. P.; Sathler, Matheus F.; Morison, Sage L.; Hofmann, Franz; Ziff, Edward B.

    2016-01-01

    Phosphorylation of GluA1, a subunit of AMPA receptors (AMPARs), is critical for AMPAR synaptic trafficking and control of synaptic transmission. cGMP-dependent protein kinase II (cGKII) mediates this phosphorylation, and cGKII knockout (KO) affects GluA1 phosphorylation and alters animal behavior. Notably, GluA1 phosphorylation in the KO…

  7. CB1 receptor knockout mice display reduced ethanol-induced conditioned place preference and increased striatal dopamine D2 receptors.

    PubMed

    Houchi, Hakim; Babovic, Daniela; Pierrefiche, Olivier; Ledent, Catherine; Daoust, Martine; Naassila, Mickaël

    2005-02-01

    Cannabinoids and ethanol activate the same reward pathways, and recent advances in the understanding of the neurobiological basis of alcoholism suggest that the CB1 receptor system may play a key role in the reinforcing effects of ethanol and in modulating ethanol intake. In the present study, male CB1 receptors knockout mice generated on a CD1 background displayed decreased ethanol-induced conditioned place preference (CPP) compared to wild-type (CB1(+/+)) mice. Ethanol (0.5, 1.0, 1.5, and 2.0 g/kg) induced significant CPP in CB1(+/+) mice at all doses tested, whereas it induced significant CPP only at the highest dose of ethanol (2.0 g/kg) in CB1(-/-) mice. However, there was no genotypic difference in cocaine (20 mg/kg)-induced CPP. There was also no genotypic difference, neither in cocaine (10-50 mg/kg) nor in D-amphetamine (1.2-5 mg/kg)-induced locomotor effects. In addition, mutant and wild-type mice did not differ in sensitivity to the anxiolytic effects of ethanol (1.5 g/kg) when tested using the elevated plus maze. Interestingly, this decrease in ethanol efficacy to induce CPP in CB1(-/-) mice was correlated with an increase in D2/D3 receptors, as determined by [3H]raclopride binding, whereas there was no difference in D1-like receptors, as determined by [3H]SCH23390 binding, measured in the striatum from drug-naive mice. This increase in D2/D3 binding sites observed in CB1 knockout mice was associated with an altered locomotor response to the D2/D3 agonist quinpirole (low doses 0.02-0.1 mg/kg) but not to an alteration of quinpirole (0.1-1.0 mg/kg)-induced CPP compared to wild-type mice. Altogether, the present results indicate that lifelong deletion of CB1 receptors reduced ethanol-induced CPP and that these reduced rewarding effects of ethanol are correlated to an overexpression of striatal dopamine D2 receptors.

  8. Differences in triglyceride and cholesterol metabolism and resistance to obesity in male and female vitamin D receptor knockout mice.

    PubMed

    Weber, K; Erben, R G

    2013-08-01

    A lean phenotype has been detected in vitamin D receptor (VDR) knockout mice; however, the gender differences in fat metabolism between male and female mice both with age and in response to a high-fat diet have not been studied before. The objective of our study was to assess changes in body and fat tissue weight, food intake and serum cholesterol and triglyceride in VDR knockout mice from weaning to adulthood and after a challenge of adult animals with a high-fat diet. Although VDR knockout mice of both sexes consumed more food than wild-type and heterozygous littermates, their body weight and the weight of fat depots was lower after 6 months on a diet with 5% crude fat content. When adult animals were challenged with a high-fat diet containing 21% crude fat content for 8 weeks, VDR knockout mice of both sexes had a significantly higher food intake but gained less weight than their wild-type littermates. Cholesterol levels were higher after 2 days on the high-fat diet in both sexes, but in the VDR knockout mice, less cholesterol was detected in the serum after 8 weeks. Wild-type male mice showed signs of fatty liver disease at the end of the experiment, which was not detected in the other groups. In conclusion, lack of the VDR receptor results in reduced fat accumulation with age and when adult mice are fed a high-fat diet, despite a higher food intake of VDR knockout mice relative to their wild-type littermates. These effects can be detected in both sexes. Wild-type male mice react with the highest weight gain and cholesterol levels of all groups and develop fatty liver disease after 8 weeks on a high-fat diet, while male VDR knockout mice appear to be protected.

  9. Regulation of dopamine presynaptic markers and receptors in the striatum of DJ-1 and Pink1 knockout rats

    PubMed Central

    Sun, Jianjun; Kouranova, Evguenia; Cui, Xiaoxia; Mach, Robert H.; Xu, Jinbin

    2014-01-01

    Pathogenic autosomal recessive mutations in the DJ-1 (Park7) or the PTEN-induced putative kinase 1 (Pink1 or PARK6) genes are associated with familial Parkinson’s disease (PD). It is not well known regarding the pathological mechanisms involving the DJ-1 and Pink1 mutations. Here we characterized DJ-1 and Pink1 knockout rats both through expression profiling and using quantitative autoradiography to measure the densities of the dopamine D1, D2, D3 receptors, vesicular monoamine transporter type-2 (VMAT2) and dopamine transporter (DAT) in the striatum of transgenic rats and wild type controls. Expression profiling with a commercially available array of 84 genes known to be involved in PD indicated that only the target gene was significantly downregulated in each transgenic rat model. D1 receptor, VMAT2, and DAT were measured using [3H]SCH23390, [3H]dihydrotetrabenazine, and [3H]WIN35428, respectively. No significant changes were observed in the density of DAT in either model. Although the densities of VMAT2 and D1 receptor were unchanged in Pink1 knockout, but both were increased in DJ-1 knockout rats. The densities of D2 and D3 receptors, determined by mathematical analysis of binding of radioligands [3H]WC-10 and [3H]raclopride, were significantly increased in both knockout models. These distinctive changes in the expression of dopamine presynaptic markers and receptors in the striatum may reflect different compensatory regulation of dopamine system in DJ-1 versus Pink1 knockout rat models of familial PD. PMID:24157858

  10. Homeostatic regulation of synaptic excitability: tonic GABAA receptor currents replace Ih in cortical pyramidal neurons of HCN1 knockout mice

    PubMed Central

    Chen, Xiangdong; Shu, Shaofang; Schwartz, Lauren C.; Sun, Chengsan; Kapur, Jaideep; Bayliss, Douglas A.

    2010-01-01

    Homeostatic control of synaptic efficacy is often mediated by dynamic regulation of excitatory synaptic receptors. Here, we report a novel form of homeostatic synaptic plasticity based on regulation of shunt currents that control dendritosomatic information transfer. In cortical pyramidal neurons from wild type mice, HCN1 channels underlie a dendritic hyperpolarization-activated cationic current (Ih) that serves to limit temporal summation of synaptic inputs. In HCN1 knockout mice, as expected, Ih is reduced in pyramidal neurons and its effects on synaptic summation are strongly diminished. Unexpectedly, we found a markedly enhanced bicuculline- and L-655,708-sensitive background GABAA current in these cells that could be attributed to selective up-regulation of GABAA α5 subunit expression in the cortex of HCN1 knockout mice. Strikingly, despite diminished Ih, baseline sub-linear summation of evoked EPSPs was unchanged in pyramidal neurons from HCN1 knockout mice; however, blocking tonic GABAA currents with bicuculline enhanced synaptic summation more strongly in pyramidal cells from HCN1 knockout mice than in those cells from wild type mice. Increasing tonic GABAA receptor conductance in the context of reduced Ih, using computational or pharmacological approaches, restored normal baseline synaptic summation, as observed in neurons from HCN1 knockout mice. These data indicate that up-regulation of α5 subunit-mediated GABAA receptor tonic current compensates quantitatively for loss of dendritic Ih in cortical pyramidal neurons from HCN1 knockout mice to maintain normal synaptic summation; they further imply that dendritosomatic synaptic efficacy is a controlled variable for homeostatic regulation of cortical neuron excitability in vivo. PMID:20164346

  11. Dopamine D3 receptor knockout mice exhibit abnormal nociception in a sex-different manner.

    PubMed

    Liu, Peng; Xing, Bo; Chu, Zheng; Liu, Fei; Lei, Gang; Zhu, Li; Gao, Ya; Chen, Teng; Dang, Yong-Hui

    2016-09-26

    Pain is a complex and subjective experience. Previous studies have shown that mice lacking the dopamine D3 receptor (D3RKO) exhibit hypoalgesia, indicating a role of the D3 receptor in modulation of nociception. Given that there are sex differences in pain perception, there may be differences in responses to nociceptive stimuli between male and female D3RKO mice. In the current study, we examined the role of the D3 receptor in modulating nociception in male and female D3RKO mice. Acute thermal pain was modeled by hot-plate test. This test was performed at different temperatures including 52°C, 55°C, and 58°C. The von Frey hair test was applied to evaluate mechanical pain. And persistent pain produced by peripheral tissue injury and inflammation was modeled by formalin test. In the hot-plate test, compared with wild-type (WT) mice, D3RKO mice generally exhibited longer latencies at each of the three temperatures. Specially, male D3RKO mice showed hypoalgesia compared with male WT mice when the temperature was 55°C, while for the female mice, there was a statistical difference between genotypes when the test condition was 52°C. In the von Frey hair test, both male and female D3RKO mice exhibited hypoalgesia. In the formalin test, the male D3RKO mice displayed a similar nociceptive behavior as their sex-matched WT littermates, whereas significantly depressed late-phase formalin-induced nociceptive behaviors were observed in the female mutants. These findings indicated that the D3 receptor affects nociceptive behaviors in a sex-specific manner and that its absence induces more analgesic behavior in the female knockout mice. © 2016 Wiley Periodicals, Inc.

  12. Selective enhancement of fentanyl-induced antinociception by the delta agonist SNC162 but not by ketamine in rhesus monkeys: Further evidence supportive of delta agonists as candidate adjuncts to mu opioid analgesics.

    PubMed

    Banks, Matthew L; Folk, John E; Rice, Kenner C; Negus, S Stevens

    2010-12-01

    Mu-opioid receptor agonists such as fentanyl are effective analgesics, but their clinical use is limited by untoward effects. Adjunct medications may improve the effectiveness and/or safety of opioid analgesics. This study compared interactions between fentanyl and either the noncompetitive N-methyl-D-aspartate (NMDA) glutamate receptor antagonist ketamine or the delta-opioid receptor agonist SNC162 [(+)-4-[(alphaR)-alpha-[(2S,5R)-2,5-dimethyl-4-(2-propenyl)-1-piperazinyl]-(3-phenyl)methyl]-N,N-diethylbenzamide] in two behavioral assays in rhesus monkeys. An assay of thermal nociception evaluated tail-withdrawal latencies from water heated to 50 and 54°C. An assay of schedule-controlled responding evaluated response rates maintained under a fixed-ratio 30 schedule of food presentation. Effects of each drug alone and of three mixtures of ketamine+fentanyl (22:1, 65:1, 195:1 ketamine/fentanyl) or SNC162+fentanyl (59:1, 176:1, 528:1 SNC162/fentanyl) were evaluated in each assay. All drugs and mixtures dose-dependently decreased rates of food-maintained responding, and drug proportions in the mixtures were based on relative potencies in this assay. Ketamine and SNC162 were inactive in the assay of thermal antinociception, but fentanyl and all mixtures produced dose-dependent antinociception. Drug interactions were evaluated using dose-addition and dose-ratio analysis. Dose-addition analysis revealed that interactions for all ketamine/fentanyl mixtures were additive in both assays. SNC162/fentanyl interactions were usually additive, but one mixture (176:1) produced synergistic antinociception at 50°C. Dose-ratio analysis indicated that ketamine failed to improve the relative potency of fentanyl to produce antinociception vs. rate suppression, whereas two SNC162/fentanyl mixtures (59:1 and 176:1) increased the relative potency of fentanyl to produce antinociception. These results suggest that delta agonists may produce more selective enhancement than ketamine of mu

  13. Further Evaluation of Delta Opioid Agonists as Candidate Adjuncts to Mu Opioid Analgesics: A Comparison of Interactions between Fentanyl and either Ketamine or the Delta Agonist SNC162 in Rhesus Monkeys

    PubMed Central

    Banks, Matthew L.; Folk, John E.; Rice, Kenner C.; Negus, S. Stevens

    2010-01-01

    Mu-opioid receptor agonists such as fentanyl are effective analgesics, but their clinical use is limited by untoward effects. Adjunct medications may improve the effectiveness and/or safety of opioid analgesics. This study compared interactions between fentanyl and either the noncompetitive N-methyl-D-aspartate (NMDA) glutamate receptor antagonist ketamine or the delta-opioid receptor agonist SNC162 [(+)-4-[(alphaR)-alpha-[(2S,5R)-2,5-dimethyl-4-(2-propenyl)-1-piperazinyl]-(3-phenyl)methyl]-N,N-diethylbenzamide] in two behavioral assays in rhesus monkeys. An assay of thermal nociception evaluated tail-withdrawal latencies from water heated to 50 and 54°C. An assay of schedule-controlled responding evaluated response rates maintained under a fixed-ratio 30 schedule of food presentation. Effects of each drug alone and of three mixtures of ketamine +fentanyl (22:1, 65:1, 195:1 ketamine/fentanyl) or SNC162+fentanyl (59:1, 176:1, 528:1 SNC162/fentanyl) were evaluated in each assay. All drugs and mixtures dose-dependently decreased rates of food-maintained responding, and drug proportions in the mixtures were based on relative potencies in this assay. Ketamine and SNC162 were inactive in the assay of thermal antinociception, but fentanyl and all mixtures produced dose-dependent antinociception. Drug interactions were evaluated using dose-addition and dose-ratio analysis. Dose-addition analysis revealed that interactions for all ketamine/fentanyl mixtures were additive in both assays. SNC162/fentanyl interactions were usually additive, but one mixture (176:1) produced synergistic antinociception at 50°C. Dose-ratio analysis indicated that ketamine failed to improve the relative potency of fentanyl to produce antinociception vs. rate suppression, whereas two SNC162/fentanyl mixtures (59:1 and 176:1) increased the relative potency of fentanyl to produce antinociception. These results suggest that delta agonists may produce more selective enhancement than ketamine of mu

  14. alpha7 Nicotinic acetylcholine receptor knockout selectively enhances ethanol-, but not beta-amyloid-induced neurotoxicity.

    PubMed

    de Fiebre, Nancyellen C; de Fiebre, Christopher M

    2005-01-03

    The alpha7 subtype of nicotinic acetylcholine receptor (nAChR) has been implicated as a potential site of action for two neurotoxins, ethanol and the Alzheimer's disease related peptide, beta-amyloid. Here, we utilized primary neuronal cultures of cerebral cortex from alpha7 nAChR null mutant mice to examine the role of this receptor in modulating the neurotoxic properties of subchronic, "binge" ethanol and beta-amyloid. Knockout of the alpha7 nAChR gene selectively enhanced ethanol-induced neurotoxicity in a gene dosage-related fashion. Susceptibility of cultures to beta-amyloid induced toxicity, however, was unaffected by alpha7 nAChR gene null mutation. Further, beta-amyloid did not inhibit the binding of the highly alpha7-selective radioligand, [(125)I]alpha-bungarotoxin. On the other hand, in studies in Xenopus oocytes ethanol efficaciously inhibited alpha7 nAChR function. These data suggest that alpha7 nAChRs modulate the neurotoxic effects of binge ethanol, but not the neurotoxicity produced by beta-amyloid. It is hypothesized that inhibition of alpha7 nAChRs by ethanol provides partial protection against the neurotoxic properties of subchronic ethanol.

  15. Hypersomnolence and reduced activity in pan-leptin receptor knockout mice.

    PubMed

    Wang, Yuping; He, Junyun; Kastin, Abba J; Hsuchou, Hung; Pan, Weihong

    2013-11-01

    Excessive obesity correlates with hypersomnolence and impaired cognitive function, presumably induced by metabolic factors and cytokines. Production of the adipokine leptin correlates with the amount of adiposity, and leptin has been shown to promote sleep. To determine whether leptin plays a major role in the hypersomnolence of obesity, we measured sleep architecture in pan-leptin receptor knockout (POKO) mice that do not respond to leptin because of the production of a mutant, non-signaling receptor. The obese POKO mice had more non-rapid eye movement (NREM) sleep and less waking time than their littermate controls. This was mainly seen during the light span, although increased bouts of rapid eye movement sleep were also seen in the dark span. The increase of NREM sleep correlated with the extent of obesity. The POKO mice also had decreased locomotor activity and more immobility in the open field test, but there was no increase of forced immobility nor reduction of sucrose intake as would be seen in depression. The increased NREM sleep and reduced locomotor activity in the POKO mice suggest that it was obesity, rather than leptin signaling, that played a predominant role in altering sleep architecture and activity.

  16. Dysregulation of Uterine Signaling Pathways in Progesterone Receptor-Cre Knockout of Dicer

    PubMed Central

    Andreu-Vieyra, Claudia V.; Kim, Tae Hoon; Jeong, Jae-Wook; Hodgson, Myles C.; Chen, Ruihong; Creighton, Chad J.; Lydon, John P.; Gunaratne, Preethi H.; DeMayo, Francesco J.; Matzuk, Martin M.

    2012-01-01

    Epithelial-stromal interactions in the uterus are required for normal uterine functions such as pregnancy, and multiple signaling pathways are essential for this process. Although Dicer and microRNA (miRNA) have been implicated in several reproductive processes, the specific roles of Dicer and miRNA in uterine development are not known. To address the roles of miRNA in the regulation of key uterine pathways, we generated a conditional knockout of Dicer in the postnatal uterine epithelium and stroma using progesterone receptor-Cre. These Dicer conditional knockout females are sterile with small uteri, which demonstrate significant defects, including absence of glandular epithelium and enhanced stromal apoptosis, beginning at approximately postnatal d 15, with coincident expression of Cre and deletion of Dicer. Specific miRNA (miR-181c, −200b, −101, let-7d) were down-regulated and corresponding predicted proapoptotic target genes (Bcl2l11, Aldh1a3) were up-regulated, reflecting the apoptotic phenomenon. Although these mice had normal serum hormone levels, critical uterine signaling pathways, including progesterone-responsive genes, Indian hedgehog signaling, and the Wnt/β-catenin canonical pathway, were dysregulated at the mRNA level. Importantly, uterine stromal cell proliferation in response to progesterone was absent, whereas uterine epithelial cell proliferation in response to estradiol was maintained in adult uteri. These data implicate Dicer and appropriate miRNA expression as essential players in the regulation of multiple uterine signaling pathways required for uterine development and appropriate function. PMID:22798293

  17. Hematopoietic G-protein-coupled receptor kinase 2 deficiency decreases atherosclerotic lesion formation in LDL receptor-knockout mice

    PubMed Central

    Otten, Jeroen J. T.; de Jager, Saskia C. A.; Kavelaars, Annemieke; Seijkens, Tom; Bot, Ilze; Wijnands, Erwin; Beckers, Linda; Westra, Marijke M.; Bot, Martine; Busch, Matthias; Bermudez, Beatriz; van Berkel, Theo J. C.; Heijnen, Cobi J.; Biessen, Erik A. L.

    2013-01-01

    Leukocyte chemotaxis is deemed instrumental in initiation and progression of atherosclerosis. It is mediated by G-protein-coupled receptors (e.g., CCR2 and CCR5), the activity of which is controlled by G-protein-coupled receptor kinases (GRKs). In this study, we analyzed the effect of hematopoietic deficiency of a potent regulator kinase of chemotaxis (GRK2) on atherogenesis. LDL receptor-deficient (LDLr−/−) mice with heterozygous hematopoietic GRK2 deficiency, generated by bone marrow transplantation (n=15), displayed a dramatic attenuation of plaque development, with 79% reduction in necrotic core and increased macrophage content. Circulating monocytes decreased and granulocytes increased in GRK2+/− chimeras, which could be attributed to diminished granulocyte colony-forming units in bone marrow. Collectively, these data pointed to myeloid cells as major mediators of the impaired atherogenic response in GRK2+/− chimeras. LDLr−/− mice with macrophage/granulocyte-specific GRK2 deficiency (LysM-Cre GRK2flox/flox; n=8) failed to mimic the aforementioned phenotype, acquitting these cells as major responsible subsets for GRK2 deficiency-associated atheroprotection. To conclude, even partial hematopoietic GRK2 deficiency prevents atherosclerotic lesion progression beyond the fatty streak stage, identifying hematopoietic GRK2 as a potential target for intervention in atherosclerosis.—Otten, J. J. T., de Jager, S. C. A., Kavelaars, A., Seijkens, T., Bot, I., Wijnands, E., Beckers, L., Westra, M. M., Bot, M., Busch, M., Bermudez, B., van Berkel, T. J. C., Heijnen, C. J., Biessen, E. A. L. Hematopoietic G-protein-coupled receptor kinase 2 deficiency decreases atherosclerotic lesion formation in LDL receptor-knockout mice. PMID:23047899

  18. Pyrrolo- and pyridomorphinans: non-selective opioid antagonists and delta opioid agonists/mu opioid partial agonists.

    PubMed

    Kumar, V; Clark, M J; Traynor, J R; Lewis, J W; Husbands, S M

    2014-08-01

    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.

  19. Ovarian wedge resection restores fertility in estrogen receptor beta knockout (ERbeta-/-) mice.

    PubMed

    Inzunza, José; Morani, Andrea; Cheng, Guojun; Warner, Margaret; Hreinsson, Julius; Gustafsson, Jan-Ake; Hovatta, Outi

    2007-01-09

    Ovulation rarely occurs in mice in which the estrogen receptor beta (ERbeta) gene has been inactivated (ERbeta-/- mice). Here, we investigated whether this subfertility is due to a defect in the ovary itself or to more general endocrine changes in ERbeta-/- mice. We transplanted ERbeta-/- ovaries into WT mice and WT ovaries into ERbeta-/- mice. Upon mating with ERbeta-/- males, fertility increased from 20% in control intact ERbeta-/- group to 40% in the WT recipients with ERbeta-/- ovaries. The transplantation procedure was not efficient, and when WT ovaries were transplanted into WT mice, fertility was only 36%. Surgical ovarian wedge resection, a procedure which induces ovulation in anovulatory women with polycystic ovarian syndrome, resulted in 100% fertility of ERbeta-/- mice. In ERbeta-/- mice, as the follicles enlarged, the thecal layer remained very compact (revealed by H&E and collagen staining), and there was no increase in vascularization (measured as smooth muscle actin). In addition, there was an increase in PDGF receptor alpha (PDGFRalpha) and a decrease in PDGFbeta expression in the granulosa cells, similar to what has been found in follitropin receptor knockout mice. After wedge resection, expression of both smooth muscle actin and PDGFRs was normalized. During normal follicular development, increased vascularization of the thecal layer is a prerequisite for further follicular growth. We suggest that the defect in ERbeta-/- mouse ovaries is a failure of communication between the granulosa and thecal layers. The follicles do not mature because of insufficient blood supply. This problem is overcome by stimulating neovascularization by simple wedge resection of the ovaries.

  20. Ovarian wedge resection restores fertility in estrogen receptor β knockout (ERβ−/−) mice

    PubMed Central

    Inzunza, José; Morani, Andrea; Cheng, Guojun; Warner, Margaret; Hreinsson, Julius; Gustafsson, Jan-Åke; Hovatta, Outi

    2007-01-01

    Ovulation rarely occurs in mice in which the estrogen receptor β (ERβ) gene has been inactivated (ERβ−/− mice). Here, we investigated whether this subfertility is due to a defect in the ovary itself or to more general endocrine changes in ERβ−/− mice. We transplanted ERβ−/− ovaries into WT mice and WT ovaries into ERβ−/− mice. Upon mating with ERβ−/− males, fertility increased from 20% in control intact ERβ−/− group to 40% in the WT recipients with ERβ−/− ovaries. The transplantation procedure was not efficient, and when WT ovaries were transplanted into WT mice, fertility was only 36%. Surgical ovarian wedge resection, a procedure which induces ovulation in anovulatory women with polycystic ovarian syndrome, resulted in 100% fertility of ERβ−/− mice. In ERβ−/− mice, as the follicles enlarged, the thecal layer remained very compact (revealed by H&E and collagen staining), and there was no increase in vascularization (measured as smooth muscle actin). In addition, there was an increase in PDGF receptor α (PDGFRα) and a decrease in PDGFβ expression in the granulosa cells, similar to what has been found in follitropin receptor knockout mice. After wedge resection, expression of both smooth muscle actin and PDGFRs was normalized. During normal follicular development, increased vascularization of the thecal layer is a prerequisite for further follicular growth. We suggest that the defect in ERβ−/− mouse ovaries is a failure of communication between the granulosa and thecal layers. The follicles do not mature because of insufficient blood supply. This problem is overcome by stimulating neovascularization by simple wedge resection of the ovaries. PMID:17197418

  1. Sensorimotor gating and spatial learning in α7-nicotinic receptor knockout mice.

    PubMed

    Azzopardi, E; Typlt, M; Jenkins, B; Schmid, S

    2013-06-01

    The role of acetylcholine and specific nicotinic receptors in sensorimotor gating and higher cognitive function has been controversial. Here, we used a commercially available mouse with a null mutation in the Chrna7(tm1Bay) gene [α7-nicotinic acetylcholine receptor (nAChR) knockout (KO) mouse] in order to assess the role of the α7-nAChR in sensorimotor gating and spatial learning. We examined prepulse inhibition (PPI) of startle and nicotine-induced enhancement of PPI. We also tested short- and long-term habituation of the startle response as well as of locomotor behaviour in order to differentiate the role of this receptor in the habituation of evoked behaviour (startle) vs. motivated behaviour (locomotion). To address higher cognition, mice were also tested in a spatial learning task. Our results showed a mild but consistent PPI deficit in α7-nAChR KO mice. Furthermore, they did not show nicotine-induced enhancement of startle or PPI. Short- and long-term habituation was normal in KO mice for both types of behaviours, evoked or motivated, and they also showed normal learning and memory in the Barnes maze. Thorough analysis of the behavioural data indicated a slightly higher degree of anxiety in α7-nAChR KO mice; however, this could only be partially confirmed in an elevated plus maze test. In summary, our data suggest that α7-nAChRs play a minor role in PPI, but seem to mediate nicotine-induced PPI enhancement. We found no evidence to suggest that they are important for habituation or spatial learning. © 2013 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

  2. CXC receptor knockout mice: characterization of skeletal features and membranous bone healing in the adult mouse.

    PubMed

    Bischoff, David S; Sakamoto, Taylor; Ishida, Kenji; Makhijani, Nalini S; Gruber, Helen E; Yamaguchi, Dean T

    2011-02-01

    The potential role of CXC chemokines bearing the glu-leu-arg (ELR) motif in bone repair was studied using a cranial defect (CD) model in mice lacking the CXC receptor (mCXCR(-/-) knockout mice), which is homologous to knockout of the human CXC receptor 2 (CXCR2) gene. During the inflammatory stage of bone repair, ELR CXC chemokines are released by inflammatory cells and serve as chemotactic and angiogenic factors. mCXCR(-/-) mice were smaller in weight and length from base of tail to nose tip, compared to WT littermates. DEXA analysis indicated that bone mineral density (BMD), bone mineral content (BMC), total area (TA), bone area (BA), and total tissue mass (TTM) were decreased in the mCXCR(-/-) mice at 6, 12, and 18 weeks of age. Trabecular bone characteristics in mCXCR(-/-) (% bone, connectivity, number, and thickness) were reduced, and trabecular spacing was increased as evidenced by μCT. There was no difference in bone formation or resorption indices measured by bone histomorphometry. Trabecular BMD was not altered. Cortical bone volume, BMD, and thickness were reduced; whereas, bone marrow volume was increased in mCXCR(-/-). Decreased polar moment of inertia (J) in the tibias/femurs suggested that the mCXCR(-/-) long bones are weaker. This was confirmed by three-point bending testing of the femurs. CDs created in 6-week-old male mCXCR(-/-) and WT littermates were not completely healed at 12 weeks; WT animals, however, had significantly more bone in-growth than mCXCR(-/-). New bone sites were identified using polarized light and assessed for numbers of osteocyte (OCy) lacunae and blood vessels (BlV) around the original CD. In new bone, the number of BlV in WT was >2× that seen in mCXCR(-/-). Bone histomorphometry parameters in the cranial defect did not show any difference in bone formation or resorption markers. In summary, studies showed that mCXCR(-/-) mice have (1) reduced weight and size; (2) decreased BMD and BMC; (3) decreased amounts of trabecular

  3. Effects of dopamine D1-like and D2-like antagonists on cocaine discrimination in muscarinic receptor knockout mice.

    PubMed

    Thomsen, Morgane; Caine, Simon Barak

    2016-04-05

    Muscarinic and dopamine brain systems interact intimately, and muscarinic receptor ligands, like dopamine ligands, can modulate the reinforcing and discriminative stimulus (S(D)) effects of cocaine. To enlighten the dopamine/muscarinic interactions as they pertain to the S(D) effects of cocaine, we evaluated whether muscarinic M1, M2 or M4 receptors are necessary for dopamine D1 and/or D2 antagonist mediated modulation of the S(D) effects of cocaine. Knockout mice lacking M1, M2, or M4 receptors, as well as control wild-type mice and outbred Swiss-Webster mice, were trained to discriminate 10mg/kg cocaine from saline in a food-reinforced drug discrimination procedure. Effects of pretreatments with the dopamine D1 antagonist SCH 23390 and the dopamine D2 antagonist eticlopride were evaluated. In intact mice, both SCH 23390 and eticlopride attenuated the cocaine discriminative stimulus effect, as expected. SCH 23390 similarly attenuated the cocaine discriminative stimulus effect in M1 knockout mice, but not in mice lacking M2 or M4 receptors. The effects of eticlopride were comparable in each knockout strain. These findings demonstrate differences in the way that D1 and D2 antagonists modulate the S(D) effects of cocaine, D1 modulation being at least partially dependent upon activity at the inhibitory M2/M4 muscarinic subtypes, while D2 modulation appeared independent of these systems. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Soy milk versus simvastatin for preventing atherosclerosis and left ventricle remodeling in LDL receptor knockout mice.

    PubMed

    Santos, L; Davel, A P; Almeida, T I R; Almeida, M R; Soares, E A; Fernandes, G J M; Magalhães, S F; Barauna, V G; Garcia, J A D

    2017-02-20

    Functional food intake has been highlighted as a strategy for the prevention of cardiovascular diseases by reducing risk factors. In this study, we compared the effects of oral treatment with soy milk and simvastatin on dyslipidemia, left ventricle remodeling and atherosclerotic lesion of LDL receptor knockout mice (LDLr-/-) fed a hyperlipidic diet. Forty 3-month old male LDLr-/- mice were distributed into four groups: control group (C), in which animals received standard diet; HL group, in which animals were fed a hyperlipidic diet; HL+SM or HL+S groups, in which animals were submitted to a hyperlipidic diet plus soy milk or simvastatin, respectively. After 60 days, both soy milk and simvastatin treatment prevented dyslipidemia, atherosclerotic lesion progression and left ventricle hypertrophy in LDLr-/- mice. These beneficial effects of soy milk and simvastatin were associated with reduced oxidative stress and inflammatory state in the heart and aorta caused by the hyperlipidic diet. Treatment with soy milk was more effective in preventing HDLc reduction and triacylglycerol and VLDLc increase. On the other hand, simvastatin was more effective in preventing an increase in total cholesterol, LDLc and superoxide production in aorta, as well as CD40L both in aorta and left ventricle of LDLr-/-. In conclusion, our results suggest a cardioprotective effect of soy milk in LDLr-/- mice comparable to the well-known effects of simvastatin.

  5. Deficit in acoustic signal-in-noise detection in glycine receptor α3 subunit knockout mice.

    PubMed

    Tziridis, Konstantin; Buerbank, Stefanie; Eulenburg, Volker; Dlugaiczyk, Julia; Schulze, Holger

    2017-02-01

    Hearing is an essential sense for communication in animals and humans. Normal function of the cochlea of higher vertebrates relies on a fine-tuned interplay of afferent and efferent innervation of both inner and outer hair cells. Efferent inhibition is controlled via olivocochlear feedback loops, mediated mainly by acetylcholine, γ-aminobutyric acid (GABA) and glycine, and is one of the first sites affected by synapto- and neuropathy in the development of hearing loss. While the functions of acetylcholine, GABA and other inhibitory transmitters within these feedback loops are at least partially understood, especially the function of glycine still remains elusive. To address this question, we investigated hearing in glycine receptor (GlyR) α3 knockout (KO) and wildtype (WT) mice. We found no differences in pure tone hearing thresholds at 11.3 and 16 kHz between the two groups as assessed by auditory brainstem response (ABR) measurements. Detailed analysis of the ABR waves at 11.3 kHz, however, revealed a latency decrease of wave III and an amplitude increase of wave IV in KO compared to WT animals. GlyRα3 KO animals showed significantly impaired prepulse inhibition of the auditory startle response in a noisy environment, indicating that GlyRα3-mediated glycinergic inhibition is important for signal-in-noise detection.

  6. Glucose-dependent insulinotropic polypeptide receptor knockout mice have altered bone turnover.

    PubMed

    Xie, Ding; Cheng, Hua; Hamrick, Mark; Zhong, Qing; Ding, Ke-Hong; Correa, Daniel; Williams, Sandra; Mulloy, Anthony; Bollag, Wendy; Bollag, Roni J; Runner, Royce R; McPherson, James C; Insogna, Karl; Isales, Carlos M

    2005-12-01

    Glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone, which is secreted from endocrine cells in the small intestine after meal ingestion. GIP has been shown to affect osteoblastic function in vitro; however, the in vivo effects of GIP on bone remodeling remain unclear. In the present study, we investigated the role of GIP in modulating bone turnover, by evaluating serum markers of bone turnover, bone density, bone morphology, and changes in biomechanical bone strength over time (one to five months) in GIP receptor knockout mice (GIPR-/- mice). The GIPR-/- mice showed a decreased bone size, lower bone mass, altered bone microarchitecture and biomechanical properties, and altered parameters for bone turnover, especially in bone formation. Moreover, the effects of GIP on bone mass were site-specific and compensatory mechanism developed over time and ameliorated the impact of the loss of GIP signaling on bone mass. Further, GIPR-/- mice had earlier age-related changes than wild-type mice in body composition, including bone mass, lean body mass, and fat percentage. In summary, our results indicate that GIP has an anabolic effect on bone mass and bone quality and suggests that GIP may be a hormonal link between nutrient ingestion and utilization.

  7. Soy milk versus simvastatin for preventing atherosclerosis and left ventricle remodeling in LDL receptor knockout mice

    PubMed Central

    Santos, L.; Davel, A.P.; Almeida, T.I.R.; Almeida, M.R.; Soares, E.A.; Fernandes, G.J.M.; Magalhães, S.F.; Barauna, V.G.; Garcia, J.A.D.

    2017-01-01

    Functional food intake has been highlighted as a strategy for the prevention of cardiovascular diseases by reducing risk factors. In this study, we compared the effects of oral treatment with soy milk and simvastatin on dyslipidemia, left ventricle remodeling and atherosclerotic lesion of LDL receptor knockout mice (LDLr-/-) fed a hyperlipidic diet. Forty 3-month old male LDLr-/- mice were distributed into four groups: control group (C), in which animals received standard diet; HL group, in which animals were fed a hyperlipidic diet; HL+SM or HL+S groups, in which animals were submitted to a hyperlipidic diet plus soy milk or simvastatin, respectively. After 60 days, both soy milk and simvastatin treatment prevented dyslipidemia, atherosclerotic lesion progression and left ventricle hypertrophy in LDLr-/- mice. These beneficial effects of soy milk and simvastatin were associated with reduced oxidative stress and inflammatory state in the heart and aorta caused by the hyperlipidic diet. Treatment with soy milk was more effective in preventing HDLc reduction and triacylglycerol and VLDLc increase. On the other hand, simvastatin was more effective in preventing an increase in total cholesterol, LDLc and superoxide production in aorta, as well as CD40L both in aorta and left ventricle of LDLr-/-. In conclusion, our results suggest a cardioprotective effect of soy milk in LDLr-/- mice comparable to the well-known effects of simvastatin. PMID:28225891

  8. Investigation of the cardiomyocyte dysfunction in bradykinin type 2 receptor knockout mice.

    PubMed

    Roman-Campos, Danilo; Duarte, Hugo Leonardo; Gomes, Enéas Ricardo; Castro, Carlos Henrique; Guatimosim, Silvia; Natali, Antonio José; Almeida, Alvair Pinto; Pesquero, João Bosco; Pesquero, Jorge Luiz; Cruz, Jader Santos

    2010-12-18

    Bradykinin type 2 receptor (B(2)R) is the key component to trigger the intracellular signaling pathway in response to bradykinin under physiological conditions. The present study sought to investigate whether the B(2)R gene deletion will have an impact on myocardial function. Isolated cell shortening, patch-clamp technique, Western blot and confocal microscopy. Isolated cell shortening measurements showed significant reduction in B(2)R knockout (B(2)R(-/-)) left ventricular cardiac myocytes' shortening. Whole-cell recordings were used to study the electrophysiological aspects of the left ventricular B(2)R(-/-) cardiomyocytes. Results showed: 1) action potential lengthening; 2) unchanged inwardly rectifying K(+) current; 3) reduced transient outward K(+) (I(to)) and L-type Ca(2+) current densities; 5) changes in kinetic properties related to I(to) and I(Ca,L). In addition, transient sarcoplasmic reticulum (SR) Ca(2+) release was found to be smaller in B(2)R(-/-) cardiomyocytes. Importantly, evidence is provided that NO constitutive production is, at least in part, responsible for the reported electrophysiological modifications observed in cardiomyocytes from B(2)R(-/-) mice. Surprisingly, NO is not involved in the SR Ca(2+) release reduction as demonstrated in the present study. Taken together, our findings indicate that B(2)R plays a fundamental role in the regulation of cardiac function and Ca(2+) homeostasis, probably through a NO dependent pathway. These results may contribute to our understanding of the kinins participation in the control of cardiac function. Copyright © 2010 Elsevier Inc. All rights reserved.

  9. Novel insight into glucagon receptor action: lessons from knockout and transgenic mouse models

    PubMed Central

    Vuguin, P. M.; Charron, M. J.

    2014-01-01

    Using knockout and transgenic technology, genetically modified animal models allowed us to understand the role of glucagon signalling in metabolism. Mice with a global deletion of the glucagon receptor gene (Gcgr) were designed using gene targeting. The phenotype of Gcgr−/− mouse provided important clues about the role of Gcgr in foetal growth, pancreatic development and glucose and lipid homeostasis. The lack of Gcgr activation was associated with: (i) hypoglycaemic pregnancies, poor foetal growth and increased foetal–neonatal demise; (ii) altered cytoarchitecture of pancreatic islets; (iii) altered glucose, lipid and hormonal milieu; (iv) reduced gastric emptying; (v) altered body composition and protection from diet-induced obesity; (vi) altered energy state; (vii) impaired hepatocyte survival; (viii) altered metabolic response to prolonged fasting and exercise and (ix) prevented development of diabetes in insulin-deficient mice. In contrast, mice overexpressing the Gcgr on pancreatic β-cells displayed an increase insulin secretion, pancreatic insulin content and β-cell mass, and partially protected against hyperglycaemia and impaired glucose tolerance when fed a high-fat diet. These findings suggest that glucagon signalling plays a significant role in the regulation of glucose and lipid homeostasis. Treatment options designed to block Gcgr activation may have negative implications in the treatment of diabetes. PMID:21824268

  10. P2X7 receptor knockout prevents streptozotocin-induced type 1 diabetes in mice.

    PubMed

    Vieira, Flávia Sarmento; Nanini, Hayandra Ferreira; Takiya, Christina Maeda; Coutinho-Silva, Robson

    2016-01-05

    Type 1 diabetes (T1D) is caused by autoimmune destruction of islet of Langerhans β-cells. P2X7 receptors (P2X7R) modulate proinflammatory immune responses by binding extracellular ATP, a classic 'danger signal'. Here, we evaluated whether the P2X7R has a role in T1D development. P2X7(-/-) mice are resistant to TD1 induction by streptozotocin (STZ) treatment, with no increase in blood glucose, decrease in insulin-positive cells, and pancreatic islet reduction, compared to WT (C57BL/6) mice. Also, the levels of proinflammatory mediators (IL-1β, IFN-γ and NO) did not increase after STZ treatment in P2X7(-/-) animals, with reduced infiltration of CD4(+), CD8(+), B220(+), CD11b(+) and CD11c(+) cells in the pancreatic lymph nodes. Treatment with a P2X7 antagonist mimicked the effect of P2X7 knockout, preventing STZ-induced diabetes. Our results show that the absence of the P2X7R provides resistance in the induction of diabetes in this model, and suggest that therapy targeting the P2X7R may be useful against clinical T1D.

  11. Schmallenberg virus infection of adult type I interferon receptor knock-out mice.

    PubMed

    Wernike, Kerstin; Breithaupt, Angele; Keller, Markus; Hoffmann, Bernd; Beer, Martin; Eschbaumer, Michael

    2012-01-01

    Schmallenberg virus (SBV), a novel orthobunyavirus, was discovered in Europe in late 2011. It causes mild and transient disease in adult ruminants, but fetal infection can lead to abortion or severe malformations. There is considerable demand for SBV research, but in vivo studies in large animals are complicated by their long gestation periods and the cost of high containment housing. The goal of this study was to investigate whether type I interferon receptor knock-out (IFNAR(-/-)) mice are a suitable small animal model for SBV. Twenty IFNAR(-/-) mice were inoculated with SBV, four were kept as controls. After inoculation, all were observed and weighed daily; two mice per day were sacrificed and blood, brain, lungs, liver, spleen, and intestine were harvested. All but one inoculated mouse lost weight, and two mice died spontaneously at the end of the first week, while another two had to be euthanized. Real-time RT-PCR detected large amounts of SBV RNA in all dead or sick mice; the controls were healthy and PCR-negative. IFNAR(-/-) mice are susceptible to SBV infection and can develop fatal disease, making them a handy and versatile tool for SBV vaccine research.

  12. Dietary corn fractions reduce atherogenesis in low-density lipoprotein receptor knockout mice.

    PubMed

    Masisi, Kabo; Le, Khuong; Ghazzawi, Nora; Moghadasian, Mohammed H; Beta, Trust

    2017-01-01

    Accumulating evidence has suggested that intake of whole grains is a protective factor against pathogenesis of coronary artery disease. The exact mechanisms, however, are still not clearly understood. In this study, we hypothesized that adequate intake of corn fractions (aleurone, endosperm and germ) can modify lipid profiles in relation to atherosclerotic lesion development in low-density lipoprotein receptor knockout (LDLr-KO) mice. The purpose of the present study was to investigate the potential cardiovascular benefits of corn fractions in LDLr-KO mice through a number of biomarkers including lipid profile, and morphologic and morphometrical analysis of atherosclerotic lesions in aortic root. Four groups of male LDLr-KO mice were fed with the experimental diets supplemented with (3 treated) or without (control) 5% (wt/wt) of each of corn fractions for 10 weeks. All diets were supplemented with 0.06% (wt/wt) cholesterol. Compared with mice in the control group, atherosclerotic lesions in the aortic roots were significantly reduced (P=.003) in the mice that were fed diet supplemented with aleurone and germ fractions. This effect was associated with significant reductions in plasma total (P=.02) and LDL (P=.03) cholesterol levels, and an increase in fecal cholesterol excretion (P=.04). Furthermore, abdominal fat mass was significantly reduced by consumption of aleurone (P=.03). In summary, the consumption of aleurone and germ may help attenuate atherosclerosis by reducing plasma total and LDL cholesterol levels.

  13. Principal function of mineralocorticoid signaling suggested by constitutive knockout of the mineralocorticoid receptor in medaka fish

    PubMed Central

    Sakamoto, Tatsuya; Yoshiki, Madoka; Takahashi, Hideya; Yoshida, Masayuki; Ogino, Yukiko; Ikeuchi, Toshitaka; Nakamachi, Tomoya; Konno, Norifumi; Matsuda, Kouhei; Sakamoto, Hirotaka

    2016-01-01

    As in osmoregulation, mineralocorticoid signaling is implicated in the control of brain-behavior actions. Nevertheless, the understanding of this role is limited, partly due to the mortality of mineralocorticoid receptor (MR)-knockout (KO) mice due to impaired Na+ reabsorption. In teleost fish, a distinct mineralocorticoid system has only been identified recently. Here, we generated a constitutive MR-KO medaka as the first adult-viable MR-KO animal, since MR expression is modest in osmoregulatory organs but high in the brain of adult medaka as for most teleosts. Hyper- and hypo-osmoregulation were normal in MR-KO medaka. When we studied the behavioral phenotypes based on the central MR localization, however, MR-KO medaka failed to track moving dots despite having an increase in acceleration of swimming. These findings reinforce previous results showing a minor role for mineralocorticoid signaling in fish osmoregulation, and provide the first convincing evidence that MR is required for normal locomotor activity in response to visual motion stimuli, but not for the recognition of these stimuli per se. We suggest that MR potentially integrates brain-behavioral and visual responses, which might be a conserved function of mineralocorticoid signaling through vertebrates. Importantly, this fish model allows for the possible identification of novel aspects of mineralocorticoid signaling. PMID:27897263

  14. Atypical chemokine receptor 1 deficiency reduces atherogenesis in ApoE-knockout mice.

    PubMed

    Wan, Wuzhou; Liu, Qian; Lionakis, Michail S; Marino, Ana Paula M P; Anderson, Stasia A; Swamydas, Muthulekha; Murphy, Philip M

    2015-06-01

    Atypical chemokine receptor 1 (Ackr1; previously known as the Duffy antigen receptor for chemokines or Darc) is thought to regulate acute inflammatory responses in part by scavenging inflammatory CC and CXC chemokines; however, evidence for a role in chronic inflammation has been lacking. Here we investigated the role of Ackr1 in chronic inflammation, in particular in the setting of atherogenesis, using the apolipoprotein E-deficient (ApoE(-/-)) mouse model. Ackr1(-/-)ApoE(-/-) and Ackr1(+/+)ApoE(-/-) littermates were obtained by crossing ApoE(-/-) mice and Ackr1(-/-) mice on a C57BL/6J background. Ackr1 (+/+)ApoE(-/-)mice fed a Western diet up-regulated Ackr1 expression in the aorta and had markedly increased atherosclerotic lesion size compared with Ackr1(-/-)ApoE(-/-) mice. This difference was observed in both the whole aorta and the aortic root in both early and late stages of the model. Ackr1 deficiency did not affect serum cholesterol levels or macrophage, collagen or smooth muscle cell content in atherosclerotic plaques, but significantly reduced the expression of Ccl2 and Cxcl1 in the whole aorta of ApoE(-/-) mice. In addition, Ackr1 deficiency resulted in a modest decrease in T cell subset frequency and inflammatory mononuclear phagocyte content in aorta and blood in the model. Ackr1 deficiency appears to be protective in the ApoE knockout model of atherogenesis, but it is associated with only modest changes in cytokine and chemokine expression as well as T-cell subset frequency and inflammatory macrophage content. Published by Oxford University Press on behalf of the European Society of Cardiology 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.

  15. Oxytocin receptor knockout mice display deficits in the expression of autism-related behaviors.

    PubMed

    Pobbe, Roger L H; Pearson, Brandon L; Defensor, Erwin B; Bolivar, Valerie J; Young, W Scott; Lee, Heon-Jin; Blanchard, D Caroline; Blanchard, Robert J

    2012-03-01

    A wealth of studies has implicated oxytocin (Oxt) and its receptors (Oxtr) in the mediation of social behaviors and social memory in rodents. It has been suggested that failures in this system contribute to deficits in social interaction that characterize autism spectrum disorders (ASD). In the current analyses, we investigated the expression of autism-related behaviors in mice that lack the ability to synthesize the oxytocin receptor itself, Oxtr knockout (KO) mice, as compared to their wild-type (WT) littermates. In the visible burrow system, Oxtr KO mice showed robust reductions in frontal approach, huddling, allo-grooming, and flight, with more time spent alone, and in self-grooming, as compared to WT. These results were corroborated in the three-chambered test: unlike WT, Oxtr KO mice failed to spend more time in the side of the test box containing an unfamiliar CD-1 mouse. In the social proximity test, Oxtr KO mice showed clear reductions in nose to nose and anogenital sniff behaviors oriented to an unfamiliar C57BL/6J (B6) mouse. In addition, our study revealed no differences between Oxtr WT and KO genotypes in the occurrence of motor and cognitive stereotyped behaviors. A significant genotype effect was found in the scent marking analysis, with Oxtr KO mice showing a decreased number of scent marks, as compared to WT. Overall, the present data indicate that the profile for Oxtr KO mice, including consistent social deficits, and reduced levels of communication, models multiple components of the ASD phenotype. This article is part of a Special Issue entitled Oxytocin, Vasopressin, and Social Behavior.

  16. Attenuation of lithium-induced natriuresis and kaliuresis in P2Y₂ receptor knockout mice.

    PubMed

    Zhang, Yue; Li, Lijun; Kohan, Donald E; Ecelbarger, Carolyn M; Kishore, Bellamkonda K

    2013-08-01

    Whole body knockout (KO) of the P2Y₂ receptor (P2Y₂R) results in enhanced vasopressin V2 receptor activity and increased renal Na⁺ conservation. We hypothesized that P2Y₂R KO mice would be less sensitive to lithium-induced natriuresis and kaliuresis due to attenuated downregulation of one or more of the major renal Na⁺ or K⁺ transporter/channel proteins. KO and wild-type (WT) mice were fed a control or lithium-added diet (40 mmol/kg food) for 14 days. Lithium-induced natriuresis and kaliuresis were significantly (~25%) attenuated in KO mice. The subunits of the epithelial Na⁺ channel (ENaC) were variably affected by lithium and genotype, but, overall, medullary levels were decreased substantially by lithium (15-60%) in both genotypes. In contrast, cortical, β-, and γ-ENaC were increased by lithium (~50%), but only in WT mice. Moreover, an assessment of ENaC activity by benzamil sensitivity suggested that lithium increased ENaC activity in WT mice but in not KO mice. In contrast, medullary levels of Na⁺-K⁺-2Cl⁻ cotransporter 2 and cortical levels of the renal outer medullary K⁺ channel were not downregulated by lithium and were significantly (15-76%) higher in KO mice under both dietary conditions. In addition, under control conditions, tissue osmolality of the inner medulla as well as furosemide sensitivity were significantly higher in KO mice versus WT mice. Therefore, we suggest that increased expression of these proteins, particularly in the control state, reduces Na⁺ delivery to the distal nephron and provides a buffer to attenuate collecting duct-mediated natriuresis and kaliuresis. Additional studies are warranted to explore the potential therapeutic benefits of purinergic antagonism.

  17. Role of the TNF-α receptor type 1 on prostate carcinogenesis in knockout mice.

    PubMed

    Galheigo, Maria Raquel Unterkircher; Cruz, Amanda Rodrigues; Cabral, Ágata Silva; Faria, Paulo Rogério; Cordeiro, Renato Simões; Silva, Marcelo José Barbosa; Tomiosso, Tatiana Carla; Gonçalves, Bianca Fachim; Pinto-Fochi, Maria Etelvina; Taboga, Sebastião Roberto; Góes, Rejane Maira; Ribeiro, Daniele Lisboa

    2016-07-01

    TNF-α is a key cytokine involved in prostate carcinogenesis and is mediated by the TNF-α receptor type 1 (TNFR-1). This receptor triggers two opposite pathways: cell death or cell survival and presents a protective or stimulator role in cancer. Thus, the purpose of this study was to evaluate the role of TNF signaling in chemically induced prostate carcinogenesis in mice. C57bl/6 wild type (WT) and p55 TNFR-1 knockout mice (KO) were treated with mineral oil (control) or N-methyl N-nitrosurea (MNU) in association with testosterone (MNU+T, single injection of 40 mg/kg and weekly injection 2 mg/kg, respectively) over the course of 6 months. After this induction period, prostate samples were processed for histological and biochemical analysis. MNU+T treatment led to the development of prostate intraepithelial neoplasia (PIN) and adenocarcinoma (PCa) in both WT and KO animals; however, the incidence of PCa was lower in KO group than in WT. Cell proliferation analysis showed that PCNA levels were significantly lower in the KO group, even after carcinogenesis induction. Furthermore, the prostate of KO animals had lower levels of p65 and p-mTOR after treatment with MNU+T than WT. There was also a decrease in prostate androgen receptor levels after induction of carcinogenesis in both KO and WT mice. Regarding the extracellular matrix in the prostate, KO mice had higher levels of fibronectin and lower levels of matrix metalloproteinase 2 (MMP2) after carcinogenesis. Finally, there was a similar increase in apoptosis in both groups after carcinogenesis, indicating that the TNAFr1 pathway in prostate carcinogenesis presented proliferative, and not apoptotic, stimuli. TNF-α, through its receptor TNFR-1, promoted cell proliferation and cell survival in prostate by activation of the AKT/mTOR and NFKB pathway, which stimulated prostate carcinogenesis in chemically induced mice. Prostate 76: 917-926, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  18. Small changes in bone structure of female α7 nicotinic acetylcholine receptor knockout mice.

    PubMed

    Lips, Katrin S; Yanko, Özcan; Kneffel, Mathias; Panzer, Imke; Kauschke, Vivien; Madzharova, Maria; Henss, Anja; Schmitz, Peter; Rohnke, Marcus; Bäuerle, Tobias; Liu, Yifei; Kampschulte, Marian; Langheinrich, Alexander C; Dürselen, Lutz; Ignatius, Anita; Heiss, Christian; Schnettler, Reinhard; Kilian, Olaf

    2015-01-31

    Recently, analysis of bone from knockout mice identified muscarinic acetylcholine receptor subtype M3 (mAChR M3) and nicotinic acetylcholine receptor (nAChR) subunit α2 as positive regulator of bone mass accrual whereas of male mice deficient for α7-nAChR (α7KO) did not reveal impact in regulation of bone remodeling. Since female sex hormones are involved in fair coordination of osteoblast bone formation and osteoclast bone degradation we assigned the current study to analyze bone strength, composition and microarchitecture of female α7KO compared to their corresponding wild-type mice (α7WT). Vertebrae and long bones of female 16-week-old α7KO (n = 10) and α7WT (n = 8) were extracted and analyzed by means of histological, radiological, biomechanical, cell- and molecular methods as well as time of flight secondary ion mass spectrometry (ToF-SIMS) and transmission electron microscopy (TEM). Bone of female α7KO revealed a significant increase in bending stiffness (p < 0.05) and cortical thickness (p < 0.05) compared to α7WT, whereas gene expression of osteoclast marker cathepsin K was declined. ToF-SIMS analysis detected a decrease in trabecular calcium content and an increase in C4H6N(+) (p < 0.05) and C4H8N(+) (p < 0.001) collagen fragments whereas a loss of osteoid was found by means of TEM. Our results on female α7KO bone identified differences in bone strength and composition. In addition, we could demonstrate that α7-nAChRs are involved in regulation of bone remodelling. In contrast to mAChR M3 and nAChR subunit α2 the α7-nAChR favours reduction of bone strength thereby showing similar effects as α7β2-nAChR in male mice. nAChR are able to form heteropentameric receptors containing α- and β-subunits as well as the subunits α7 can be arranged as homopentameric cation channel. The different effects of homopentameric and heteropentameric α7-nAChR on bone need to be analysed in future studies as well as gender effects of cholinergic receptors on

  19. Sympathetic neural activation evoked by mu-receptor blockade in patients addicted to opioids is abolished by intravenous clonidine.

    PubMed

    Kienbaum, Peter; Heuter, Thorsten; Michel, Martin C; Scherbaum, Norbert; Gastpar, Markus; Peters, Jürgen

    2002-02-01

    Mu-opioid receptor blockade by naloxone administered for acute detoxification in patients addicted to opioids markedly increases catecholamine plasma concentrations, muscle sympathetic activity (MSA), and is associated with cardiovascular stimulation despite general anesthesia. The current authors tested the hypothesis that the alpha2-adrenoceptor agonist clonidine (1) attenuates increased MSA during mu-opioid receptor blockade for detoxification, and (2) prevents cardiovascular activation when given before detoxification. Fourteen mono-opioid addicted patients received naloxone during propofol anesthesia. Clonidine (10 microg x kg(-1) administered over 5 min + 5 microg x kg(-1) x h(-1) intravenous) was infused either before (n = 6) or after (n = 6) naloxone administration. Two patients without immediate clonidine administration occurring after naloxone administration served as time controls. Muscle sympathetic activity (n = 8) in the peroneal nerve, catecholamine plasma concentrations (n = 14), arterial blood pressure, and heart rate were assessed in awake patients, during propofol anesthesia before and after mu-opioid receptor blockade, and after clonidine administration. Mu-receptor blockade markedly increased MSA from a low activity (burst frequency: from 2 burst/min +/- 1 to 24 +/- 8, means +/- SD). Similarly, norepinephrine (41 pg/ml +/- 37 to 321 +/- 134) and epinephrine plasma concentration (13 pg/ml +/- 6 to 627 +/- 146) significantly increased, and were associated with, increased arterial blood pressure and heart rate. Clonidine immediately abolished both increased MSA (P < 0.001) and catecholamine plasma concentrations (P < 0.001). When clonidine was given before mu-opioid receptor blockade, catecholamine plasma concentrations and hemodynamic variables did not change. Administration of the alpha2-adrenoceptor agonist clonidine decreases both increased MSA and catecholamine plasma concentrations observed after mu-opioid receptor blockade for

  20. Nicotinic alpha7- or beta2-containing receptor knockout: effects on radial-arm maze learning and long-term nicotine consumption in mice.

    PubMed

    Levin, Edward D; Petro, Ann; Rezvani, Amir H; Pollard, Ninitia; Christopher, N Channelle; Strauss, Mariel; Avery, Jessica; Nicholson, Jessica; Rose, Jed E

    2009-01-23

    Classically, it has been thought that high-affinity nicotinic receptors-containing beta2 subunits are the most important receptor subtypes for nicotinic involvement in cognitive function and nicotine self-administration, while low affinity alpha7-containing nicotinic receptors have not been thought to be important. In the current study, we found that knockout of either beta2 or alpha7 subunits caused significant deficits in spatial discrimination in mice. The character of the impairment in the two knockouts was different. The beta2 knockout preferentially impaired cognition in males while the alpha7 caused impairment regardless of sex. Both beta2- and alpha7-containing nicotinic receptors also are important for nicotine self-administration, also in different ways. Most animal model studies of nicotine self-administration are relatively short-term whereas the problem of tobacco addiction is considerably longer-term. To better model the impact of nicotinic receptor subtypes on nicotine self-administration over the long-term, we studied the impact of genetic knockout of low affinity alpha7 receptors vs. high-affinity beta2-containing nicotinic receptors. Mice with knockouts of either of these receptors and their wildtype counter parts were given free access to a choice of nicotine-containing and nicotine-free solution in their home cages on a continuous basis over a period of 5 months. During the first few weeks, the beta2-containing nicotinic receptor knockout mice showed a significant decrease in nicotine consumption relative to wildtype mice, whereas the alpha7 knockout mice did not significantly differ from wildtype controls at the beginning of their access to nicotine. Interestingly, in the longer-term after the first few weeks of nicotine access, the beta2 knockout mice returned to wildtype mouse levels of nicotine consumption, whereas the alpha7 knockout mice developed an emergent decrease in nicotine consumption. The alpha7 receptor knockout-induced decrease

  1. [Neural stem cell-specific peroxisome proliferator-activated receptor γ knockout mice: breeding and genetic identification].

    PubMed

    Wu, Qiaoqi; Zhang, Hongyan; Wang, Zhen; Lin, Lifang; Chen, Lu; Wang, Xuemin

    2014-12-01

    To breed neual stem cell-specific peroxisome proliferator-activated receptor γ (PPARγ) knockout mice. Two transgenic mouse models, namely B6.PPARγloxp/loxp and B6.Nestin-Cre were interbred, and the first- generation offsprings were backcrossed with B6.PPARγloxp/loxp to obtain the second-generation mice. Genomic DNA was extracted from the second-generation mice for PCR to amplify the loxp and Cre gene fragments followed by agarose gel electrophoresis to verify their sizes. The mice with the PPARγloxp/loxp.Nestin-Cre (KO) genotype were selected as the neural stem cell-specific knockout PPARγ mice, with B6.PPARγloxp/loxp (loxp) mice as the control. Tissue samples were collected from specific regions of the mouse brain and peripheral tissue for detecting the expression of PPARγ mRNA using RT-PCR and real-time quantitative PCR. Genotyping results showed PPARγloxp and Cre bands in the knockout mice, which showed obviously decreased mRNA expression of PPARγ, suggesting successful establishment of neural stem cell-specific PPARγ knockout mice. The two transgenic mice we used were fertile, and their breeding pattern followed the laws of Mendelian inheritance.

  2. Aberrant expression of PDGF ligands and receptors in the tumor prone ovary of follitropin receptor knockout (FORKO) mouse.

    PubMed

    Chen, Xinlei; Aravindakshan, Jayaprakash; Yang, Yinzhi; Tiwari-Pandey, Rashmi; Sairam, M Ram

    2006-05-01

    Although PDGF family members play a vital role in cell proliferation, motility and chemotaxis via activation of structurally similar alpha- and beta-receptors, little is known of their function in ovarian regulation and induction of tumorigenesis. Microarray analyses of ovaries from young follitropin receptor knockout (FORKO) mice that are prone to late ovarian tumors upon aging have revealed significant imbalances in PDGF ligands and receptors. We hypothesized that FSH/FSH-R signaling may exert effects partly by regulation of PDGF the family. To further understand their implications for ovarian tumorigenesis, we studied FORKO ovaries and hormonal regulation of the PDGF family members in normal mice, by using RT-PCR, Q-PCR, immunohistochemistry and western blotting. While PDGF-C and PDGFR-alpha increased, PDGFR-beta mRNA and protein decreased significantly in absence of FSH-R signaling. In the normal ovary, PDGFR-alpha was not affected by gonadotropin (eCG) stimulation but PDGF-C and PDGFR-beta decreased. Administration of estradiol decreased PDGF and their receptors. To further probe the differential regulation of PDGF family members by eCG and estradiol, we co-administered eCG with estrogen antagonist, ICI 182780. Increase in PDGFR-alpha in the absence of estradiol suggests direct effects of FSH signaling. During the estrous cycle in mice PDGF-C, PDGF-D and PDGFR-alpha mRNA levels were higher at the proestrous. By IHC, we report for the first time the localization of PDGF-C, PDGFR-alpha and PDGFR-beta protein in mouse ovarian compartments including the surface epithelium that is also altered in mutants. Immunostaining of PDGFRs increased as the follicle developed to preantral stage and declined thereafter. Thus, FSH modulates PDGF family members, partly via E2, suggesting that loss of FSH-R signaling causes an imbalance of PDGF family members predisposing the abnormal ovarian follicular environment for inducing tumorigenesis in aging FORKO mice.

  3. Impaired coordination of nutrient intake and substrate oxidation in melanocortin-4 receptor knockout mice.

    PubMed

    Albarado, Diana C; McClaine, Jennifer; Stephens, Jacqueline M; Mynatt, Randall L; Ye, Jianping; Bannon, Anthony W; Richards, William G; Butler, Andrew A

    2004-01-01

    Mutations in the melanocortin-4 receptor (MC4R) are associated with obesity. The obesity syndrome observed in humans with MC4R haploinsufficiency is similar to that observed in MC4R knockout mice, including increased longitudinal growth, hyperphagia, and fasting hyperinsulinemia. For comparison with other commonly investigated models of obesity and insulin resistance, we have backcrossed Mc4r-/- mice into the C57BL/6J (B6) background. Female obese Mc4r-/- mice exhibit reduced energy expenditure and an attenuated increase in fatty acid (FA) oxidation after exposure to high-fat diets compared with obese Lepob/Lepob mice. The reduced energy expenditure and FA oxidation correlates with changes in hepatic gene expression. The expression of genes involved in FA oxidation increased in obese Lepob/Lepob mice compared with wild-type and obese Mc4r-/- mice. In contrast, a key lipogenic enzyme, FA synthase (FAS), is increased in obese Mc4r-/- mice compared with obese Lepob/Lepob mice. Hyperinsulinemia, increased FAS mRNA expression and hepatic steatosis appear to be secondary to obesity in B6 Mc4r-/- mice. However, Mc4r-/- mice in a mixed genetic background develop severe hepatic steatosis at an early age. This might suggest an important role of the MC4R in regulating liver FA metabolism that is masked on the B6 background. Interestingly, the 10- to 20-fold increase in liver triglyceride in the outbred strain of Mc4r-/- mice is not always associated with fasting hyperinsulinemia or increased FAS mRNA expression. This observation suggests that changes in liver secondary to triglyceride accumulation lead to hyperinsulinemia and increased hepatic FAS expression in Mc4r-/- mice.

  4. Behavioural phenotype of histamine H4 receptor knockout mice: Focus on central neuronal functions.

    PubMed

    Sanna, Maria Domenica; Ghelardini, Carla; Thurmond, Robin L; Masini, Emanuela; Galeotti, Nicoletta

    2017-03-01

    The functional expression of H4 receptors (H4R) within neurons of the central nervous system has been recently reported, but their role is poorly understood. The present study aims to elucidate the role of neuronal H4R by providing the first description of the behavioural phenotype of H4R-deficient (H4R knockout, H4R-KO) mice. Mice lacking H4R underwent behavioural studies to evaluate locomotor activity, pain perception, anxiety, depression, memory and feeding behaviour. H4R-KO mice showed a significant increase in ambulation in an open field as well as in exploratory activity in the absence of any modification of motor coordination. The sensitivity of mutant mice to a thermal or a mechanical stimulus was identical to that of the wild type mice, but H4R-KO showed sensory hypersensitivity toward a condition of neuropathic pain. The lack of H4R is associated with the promotion of anxiety in the light-dark box test. H4R-KO mice showed an increased immobility time in the tail suspension test, experimental procedure used to evaluate the response of H4R deficient mice to a behavioural despair paradigm. Cognitive function parameters of H4R deficient mice, examined using the passive avoidance and the novel object recognition tests, were unaltered showing the lack of influence of H4R on working and recognition memory. Finally, H4R-deficient mice showed an orectic phenotype. These results illustrate that H4R modulates various neurophysiological functions such as locomotor activity, anxiety, nociception and feeding behaviour, confirming the importance of the integrity and functionality of neuronal H4R in the histaminergic regulation of neuronal functions. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Knockout of the neurokinin-1 receptor reduces cholangiocyte proliferation in bile duct-ligated mice

    PubMed Central

    Glaser, Shannon; Gaudio, Eugenio; Renzi, Anastasia; Mancinelli, Romina; Ueno, Yoshiyuki; Venter, Julie; White, Mellanie; Kopriva, Shelley; Chiasson, Valorie; DeMorrow, Sharon; Francis, Heather; Meng, Fanyin; Marzioni, Marco; Franchitto, Antonio; Alvaro, Domenico; Supowit, Scott; DiPette, Donald J.; Onori, Paolo

    2011-01-01

    In bile duct-ligated (BDL) rats, cholangiocyte proliferation is regulated by neuroendocrine factors such as α-calcitonin gene-related peptide (α-CGRP). There is no evidence that the sensory neuropeptide substance P (SP) regulates cholangiocyte hyperplasia. Wild-type (WT, +/+) and NK-1 receptor (NK-1R) knockout (NK-1R−/−) mice underwent sham or BDL for 1 wk. Then we evaluated 1) NK-1R expression, transaminases, and bilirubin serum levels; 2) necrosis, hepatocyte apoptosis and steatosis, and the number of cholangiocytes positive by CK-19 and terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling in liver sections; 3) mRNA expression for collagen 1α and α-smooth muscle (α-SMA) actin in total liver samples; and 4) PCNA expression and PKA phosphorylation in cholangiocytes. In cholangiocyte lines, we determined the effects of SP on cAMP and d-myo-inositol 1,4,5-trisphosphate levels, proliferation, and PKA phosphorylation. Cholangiocytes express NK-1R with expression being upregulated following BDL. In normal NK-1R−/− mice, there was higher hepatocyte apoptosis and scattered hepatocyte steatosis compared with controls. In NK-1R −/− BDL mice, there was a decrease in serum transaminases and bilirubin levels and the number of CK-19-positive cholangiocytes and enhanced biliary apoptosis compared with controls. In total liver samples, the expression of collagen 1α and α-SMA increased in BDL compared with normal mice and decreased in BDL NK-1R−/− compared with BDL mice. In cholangiocytes from BDL NK-1R −/− mice there was decreased PCNA expression and PKA phosphorylation. In vitro, SP increased cAMP levels, proliferation, and PKA phosphorylation of cholangiocytes. Targeting of NK-1R may be important in the inhibition of biliary hyperplasia in cholangiopathies. PMID:21596993

  6. Uric acid stones in the urinary bladder of aryl hydrocarbon receptor (AhR) knockout mice

    PubMed Central

    Butler, Ryan; Inzunza, Jose; Suzuki, Hitoshi; Fujii-Kuriyama, Yoshiaki; Warner, Margaret; Gustafsson, Jan-Åke

    2012-01-01

    The aryl hydrocarbon receptor (AhR) knockout mice raised in the laboratory of Fujii-Kuriyama have been under investigation for several years because of the presence in their urinary bladder of large, yellowish stones. The stones are composed of uric acid and become apparent in the bladders as tiny stones when mice are 10 wk of age. By the time the mice are 6 mo of age, there are usually two or three stones with diameters of 3–4 mm. The urate concentration in the serum was normal but in the urine the concentration was 40–50 mg/dL, which is 10 times higher than that in the WT littermates. There were no apparent histological pathologies in the kidney or joints and the levels of enzymes involved in elimination of purines were normal. The source of the uric acid was therefore judged to be from degradation of nucleic acids due to a high turnover of cells in the bladder itself. The bladder was fibrotic and the luminal side of the bladder epithelium was filled with eosinophilic granules. There was loss of E-cadherin between some epithelial cells, with an enlarged submucosal area filled with immune cells and sometimes invading epithelial cells. We hypothesize that in the absence of AhR there is loss of detoxifying enzymes, which leads to accumulation of unconjugated cytotoxins and carcinogens in the bladder. The presence of bladder toxins may have led to the increased apoptosis and inflammation as well as invasion of epithelial cells in the bladders of older mice. PMID:22232670

  7. Uric acid stones in the urinary bladder of aryl hydrocarbon receptor (AhR) knockout mice.

    PubMed

    Butler, Ryan; Inzunza, Jose; Suzuki, Hitoshi; Fujii-Kuriyama, Yoshiaki; Warner, Margaret; Gustafsson, Jan-Åke

    2012-01-24

    The aryl hydrocarbon receptor (AhR) knockout mice raised in the laboratory of Fujii-Kuriyama have been under investigation for several years because of the presence in their urinary bladder of large, yellowish stones. The stones are composed of uric acid and become apparent in the bladders as tiny stones when mice are 10 wk of age. By the time the mice are 6 mo of age, there are usually two or three stones with diameters of 3-4 mm. The urate concentration in the serum was normal but in the urine the concentration was 40-50 mg/dL, which is 10 times higher than that in the WT littermates. There were no apparent histological pathologies in the kidney or joints and the levels of enzymes involved in elimination of purines were normal. The source of the uric acid was therefore judged to be from degradation of nucleic acids due to a high turnover of cells in the bladder itself. The bladder was fibrotic and the luminal side of the bladder epithelium was filled with eosinophilic granules. There was loss of E-cadherin between some epithelial cells, with an enlarged submucosal area filled with immune cells and sometimes invading epithelial cells. We hypothesize that in the absence of AhR there is loss of detoxifying enzymes, which leads to accumulation of unconjugated cytotoxins and carcinogens in the bladder. The presence of bladder toxins may have led to the increased apoptosis and inflammation as well as invasion of epithelial cells in the bladders of older mice.

  8. Derivation of rat embryonic stem cells and generation of protease-activated receptor-2 knockout rats.

    PubMed

    Yamamoto, Satoshi; Nakata, Mitsugu; Sasada, Reiko; Ooshima, Yuki; Yano, Takashi; Shinozawa, Tadahiro; Tsukimi, Yasuhiro; Takeyama, Michiyasu; Matsumoto, Yoshio; Hashimoto, Tadatoshi

    2012-08-01

    One of the remarkable achievements in knockout (KO) rat production reported during the period 2008-2010 is the derivation of authentic embryonic stem (ES) cells from rat blastocysts using a novel culture medium containing glycogen synthase kinase 3 and mitogen-activated protein kinase kinase inhibitors (2i medium). Here, we report gene-targeting technology via homologous recombination in rat ES cells, demonstrating its use through production of a protease-activated receptor-2 gene (Par-2) KO rat. We began by generating germline-competent ES cells from Dark Agouti rats using 2i medium. These ES cells, which differentiate into cardiomyocytes in vitro, can produce chimeras with high ES cell contribution when injected into blastocysts. We then introduced a targeting vector with a neomycin-resistant gene driven by the CAG promoter to disrupt Par-2. After a 7-day drug selection, 489 neomycin-resistant colonies were obtained. Following screening by polymerase chain reaction (PCR) genotyping and quantitative PCR analysis, we confirmed three homologous recombinant clones, resulting in chimeras that transmitted the Par-2 targeted allele to offspring. Par-2 KO rats showed a loss of Par-2 messenger RNA expression in their stomach cells and a lack of PAR-2 mediated smooth muscle relaxation in the aorta as indicated by pharmacological testing. Compared with mice, rats offer many advantages in biomedical research, including a larger body size; consequently, they are widely used in scientific investigation. Thus, the establishment of a gene-targeting technology using rat ES cells will be a valuable tool in human disease model production and drug discovery.

  9. M4 muscarinic receptor knockout mice display abnormal social behavior and decreased prepulse inhibition

    PubMed Central

    2012-01-01

    Background In the central nervous system (CNS), the muscarinic system plays key roles in learning and memory, as well as in the regulation of many sensory, motor, and autonomic processes, and is thought to be involved in the pathophysiology of several major diseases of the CNS, such as Alzheimer's disease, depression, and schizophrenia. Previous studies reveal that M4 muscarinic receptor knockout (M4R KO) mice displayed an increase in basal locomotor activity, an increase in sensitivity to the prepulse inhibition (PPI)-disrupting effect of psychotomimetics, and normal basal PPI. However, other behaviorally significant roles of M4R remain unclear. Results In this study, to further investigate precise functional roles of M4R in the CNS, M4R KO mice were subjected to a battery of behavioral tests. M4R KO mice showed no significant impairments in nociception, neuromuscular strength, or motor coordination/learning. In open field, light/dark transition, and social interaction tests, consistent with previous studies, M4R KO mice displayed enhanced locomotor activity compared to their wild-type littermates. In the open field test, M4R KO mice exhibited novelty-induced locomotor hyperactivity. In the social interaction test, contacts between pairs of M4R KO mice lasted shorter than those of wild-type mice. In the sensorimotor gating test, M4R KO mice showed a decrease in PPI, whereas in the startle response test, in contrast to a previous study, M4R KO mice demonstrated normal startle response. M4R KO mice also displayed normal performance in the Morris water maze test. Conclusions These findings indicate that M4R is involved in regulation of locomotor activity, social behavior, and sensorimotor gating in mice. Together with decreased PPI, abnormal social behavior, which was newly identified in the present study, may represent a behavioral abnormality related to psychiatric disorders including schizophrenia. PMID:22463818

  10. Enhanced induction of Mucin Depleted Foci in Estrogen Receptor β Knockout Mice

    PubMed Central

    Saleiro, D.; Murillo, G.; Lubahn, D. B.; Kopelovich, L.; Korach, K. S.; Mehta, R. G.

    2010-01-01

    The role of the estrogen receptor β (ERβ) in the colon has received considerable interest, yet in vivo models are needed to better define its protective actions. In the present study, wild-type (WT), ERα and ERβ knockout (αERKO and βERKO) mice were injected with azoxymethane (AOM), a colon chemical carcinogen. Fourteen weeks after AOM exposure the incidence of aberrant crypt foci (ACF) was assessed by methylene blue staining. βERKO mice showed significantly higher incidence (P < 0.001) of ACF (15.0 ± 2.5) as compared to αERKO (3.4 ± 1.0) and WT (4.6 ± 1.0) mice. The colons in several βERKO mice had increased thickness and loss of normal morphology. It has been reported that ERβ plays a role in maintenance of the colonic crypt architecture; this may explain the loss of crypt organization in the colonic epithelium of βERKO mice. The presence of mucin depleted foci (MDF) has been shown, both in humans and in rodents, as an early event in colon cancer. Therefore, in order to surpass the limitations with ACF scoring, we performed alcian blue-neutral red staining to assess the presence of MDF. This assay allowed the assessment of precancerous lesions on all the βERKO mice colons (38.3 ± 4.0; P < 0.001), comparing to WT and αERKO mice (6.6 ± 1.5, and 10.0 ± 1.9, respectively), and served to confirm the ACF results. Together these data support the use of MDF staining as a biomarker for precancerous lesions and the protective role of ERβ in colon carcinogenesis. PMID:20716634

  11. Opioid Receptors Mediate Direct Predictive Fear Learning: Evidence from One-Trial Blocking

    ERIC Educational Resources Information Center

    Cole, Sindy; McNally, Gavan P.

    2007-01-01

    Pavlovian fear learning depends on predictive error, so that fear learning occurs when the actual outcome of a conditioning trial exceeds the expected outcome. Previous research has shown that opioid receptors, including [mu]-opioid receptors in the ventrolateral quadrant of the midbrain periaqueductal gray (vlPAG), mediate such predictive fear…

  12. Opioid Receptors Mediate Direct Predictive Fear Learning: Evidence from One-Trial Blocking

    ERIC Educational Resources Information Center

    Cole, Sindy; McNally, Gavan P.

    2007-01-01

    Pavlovian fear learning depends on predictive error, so that fear learning occurs when the actual outcome of a conditioning trial exceeds the expected outcome. Previous research has shown that opioid receptors, including [mu]-opioid receptors in the ventrolateral quadrant of the midbrain periaqueductal gray (vlPAG), mediate such predictive fear…

  13. A satellite cell-specific knockout of the androgen receptor reveals myostatin as a direct androgen target in skeletal muscle.

    PubMed

    Dubois, Vanessa; Laurent, Michaël R; Sinnesael, Mieke; Cielen, Nele; Helsen, Christine; Clinckemalie, Liesbeth; Spans, Lien; Gayan-Ramirez, Ghislaine; Deldicque, Louise; Hespel, Peter; Carmeliet, Geert; Vanderschueren, Dirk; Claessens, Frank

    2014-07-01

    Androgens have well-established anabolic actions on skeletal muscle, although the direct effects of the androgen receptor (AR) in muscle remain unclear. We generated satellite cell-specific AR-knockout (satARKO) mice in which the AR is selectively ablated in satellite cells, the muscle precursor cells. Total-limb maximal grip strength is decreased by 7% in satARKO mice, with soleus muscles containing ∼10% more type I fibers and 10% less type IIa fibers than the corresponding control littermates. The weight of the perineal levator ani muscle is markedly reduced (-52%). Thus, muscle AR is involved in fiber-type distribution and force production of the limb muscles, while it is a major determinant of the perineal muscle mass. Surprisingly, myostatin (Mstn), a strong inhibitor of skeletal muscle growth, is one of the most androgen-responsive genes (6-fold reduction in satARKO) through direct transcription activation by the AR. Consequently, muscle hypertrophy in response to androgens is augmented in Mstn-knockout mice. Our finding that androgens induce Mstn signaling to restrain their own anabolic actions has implications for the treatment of muscle wasting disorders.-Dubois, V., Laurent, M. R., Sinnesael, M., Cielen, N., Helsen, C., Clinckemalie, L., Spans, L., Gayan-Ramirez, G., Deldicque, L., Hespel, P., Carmeliet, G., Vanderschueren, D., and Claessens, F. A satellite cell-specific knockout of the androgen receptor reveals myostatin as a direct androgen target in skeletal muscle.

  14. Loss of Myocardial Ischemic Postconditioning in Adenosine A1 and Bradykinin B2 Receptors Gene Knockout Mice

    PubMed Central

    Xi, Lei; Das, Anindita; Zhao, Zhi-Qing; Merino, Vanessa F.; Bader, Michael; Kukreja, Rakesh C.

    2011-01-01

    Background Ischemic postconditioning (PostC) is a recently described cardioprotective modality against reperfusion injury, through series of brief re-flow interruptions applied at the very onset of reperfusion. It is proposed that PostC can activate a complex cellular signaling cascade, in which cell membrane receptors could serve as the upstream triggers of PostC. However, the exact subtypes of such receptors remain controversial or uninvestigated. To this context, the purpose of present study was to determine the definitive role of adenosine A1, bradykinin B1 and B2 receptors in PostC. Methods and Results The hearts isolated from adult male C57BL/6J wild-type mice or the mice lacking adenosine A1, or bradykinin B1 or B2 receptors subjected to zero-flow global ischemia and reperfusion in a Langendorff model. PostC, consisting of 6 cycles of 10 sec of reperfusion and 10 sec of ischemia, demonstrated significantly reduced myocardial infarct size (22.8±3.1%, Mean±SEM) as compared with the non-PostC wild-type controls (35.1±2.8%, P<0.05). The infarct-limiting protection of PostC was absent in adenosine A1 receptor knockout mice (34.9±2.7%) or bradykinin B2 receptor knockout mice (33.3±1.7%) and was partially attenuated in bradykinin B1 receptor deficient mice (25.6±2.9%; P>0.05). On the other hand, PostC did not significantly alter post-ischemic cardiac contractile function and coronary flow. Conclusions With the use of three distinctive strains of gene knockout mice, the current study has provided the first conclusive evidence showing PostC-induced infarct-limiting cardioprotection could be triggered by activation of multiple types of cell membrane receptors, which include adenosine A1 and bradykinin B2 receptors. PMID:18824766

  15. Distinct Mu, Delta, and Kappa Opioid Receptor Mechanisms Underlie Low Sociability and Depressive-Like Behaviors During Heroin Abstinence

    PubMed Central

    Lutz, Pierre-Eric; Ayranci, Gulebru; Chu-Sin-Chung, Paul; Matifas, Audrey; Koebel, Pascale; Filliol, Dominique; Befort, Katia; Ouagazzal, Abdel-Mouttalib; Kieffer, Brigitte L

    2014-01-01

    Addiction is a chronic disorder involving recurring intoxication, withdrawal, and craving episodes. Escaping this vicious cycle requires maintenance of abstinence for extended periods of time and is a true challenge for addicted individuals. The emergence of depressive symptoms, including social withdrawal, is considered a main cause for relapse, but underlying mechanisms are poorly understood. Here we establish a mouse model of protracted abstinence to heroin, a major abused opiate, where both emotional and working memory deficits unfold. We show that delta and kappa opioid receptor (DOR and KOR, respectively) knockout mice develop either stronger or reduced emotional disruption during heroin abstinence, establishing DOR and KOR activities as protective and vulnerability factors, respectively, that regulate the severity of abstinence. Further, we found that chronic treatment with the antidepressant drug fluoxetine prevents emergence of low sociability, with no impact on the working memory deficit, implicating serotonergic mechanisms predominantly in emotional aspects of abstinence symptoms. Finally, targeting the main serotonergic brain structure, we show that gene knockout of mu opioid receptors (MORs) in the dorsal raphe nucleus (DRN) before heroin exposure abolishes the development of social withdrawal. This is the first result demonstrating that intermittent chronic MOR activation at the level of DRN represents an essential mechanism contributing to low sociability during protracted heroin abstinence. Altogether, our findings reveal crucial and distinct roles for all three opioid receptors in the development of emotional alterations that follow a history of heroin exposure and open the way towards understanding opioid system-mediated serotonin homeostasis in heroin abuse. PMID:24874714

  16. Enhanced response to mouse thyroid-stimulating hormone (TSH) receptor immunization in TSH receptor-knockout mice.

    PubMed

    Nakahara, Mami; Mitsutake, Norisato; Sakamoto, Hikaru; Chen, Chun-Rong; Rapoport, Basil; McLachlan, Sandra M; Nagayama, Yuji

    2010-08-01

    Graves-like hyperthyroidism is induced in BALB/c mice by immunization with adenovirus expressing the human TSH receptor (TSHR) A-subunit (amino acids 1-289). However, because of nonidentity between the human and mouse TSHR ( approximately 87% amino acid homology), we compared the responses of mice immunized with adenoviruses expressing either the mouse or the human TSHR A-subunit. Wild-type (wt) BALB/c mice immunized with the mouse A-subunit developed neither TSHR antibodies (measured by flow cytometry) nor thyroid lymphocytic infiltration. However, wt C57BL/6 mice developed sparse intrathyroidal lymphocyte infiltration without antibody production. Depletion of naturally occurring regulatory CD4(+)CD25(+) T cells had little effect. These results indicate the inability to break tolerance to the mouse TSHR in wt mice. In contrast, TSHR knockout (KO) BALB/c mice generated mouse TSHR antibodies in response to mouse A-subunit immunization and augmented human TSHR antibody response to human A-subunit immunization. Thyroid-stimulating antibody titers measured in a functional bioassay were comparable in human A-subunit immunized wt mice and in TSHR KO mice immunized with either the mouse or human A-subunit. In conclusion, immune response to the mouse TSHR is readily induced in TSHR KO but not in wt mice. Only in the former does immunization with adenovirus expressing the mouse A-subunit generate antibodies capable of activating the mouse TSHR. TSHR KO mice are, therefore, of value for future studies dissecting the autoimmune response to the mouse TSHR.

  17. Enhanced dihydropyridine receptor calcium channel activity restores muscle strength in JP45/CASQ1 double knockout mice

    PubMed Central

    Mosca, Barbara; Delbono, Osvaldo; Messi, Maria Laura; Bergamelli, Leda; Wang, Zhong-Min; Vukcevic, Mirko; Lopez, Ruben; Treves, Susan; Nishi, Miyuki; Takeshima, Hiroshi; Paolini, Cecilia; Martini, Marta; Rispoli, Giorgio; Protasi, Feliciano; Zorzato, Francesco

    2016-01-01

    Muscle strength declines with age in part due to a decline of Ca2+ release from sarcoplasmic reticulum calcium stores. Skeletal muscle dihydropyridine receptors (Cav1.1) initiate muscle contraction by activating ryanodine receptors in the sarcoplasmic reticulum. Cav1.1 channel activity is enhanced by a retrograde stimulatory signal delivered by the