Genetic dissociation of morphine analgesia from hyperalgesia in mice

Abstract

Rationale

Morphine is the prototypic mu opioid, producing its analgesic actions through traditional 7 transmembrane domain (7TM) G-protein-coupled receptors generated by the mu opioid receptor gene (Oprm1). However, the Oprm1 gene undergoes extensive alternative splicing to yield three structurally distinct sets of splice variants. In addition to the full-length 7TM receptors, it produces a set of truncated variants comprised of only 6 transmembrane domains (6TM).

Objectives

This study explored the relative contributions of 7TM and 6TM variants in a range of morphine actions.

Methods

Groups of male and mixed-gender wild-type and exon 11 Oprm1 knockout mice were examined in a series of behavioral assays measuring analgesia, hyperalgesia, respiration, and reward in conditioned place preference assays.

Results

Loss of the 6TM variants in an exon 11 knockout (E11 KO) mouse did not affect morphine analgesia, reward, or respiratory depression. However, E11 KO mice lacking 6TM variants failed to show morphine-induced hyperalgesia, developed tolerance more slowly than wild-type mice, and did not display hyperlocomotion.

Conclusions

Together, our findings confirm the established role of 7TM mu receptor variants in morphine analgesia, reward, and respiratory depression, but reveal an unexpected obligatory role for 6TM variants in morphine-induced hyperalgesia and a modulatory role in morphine tolerance and dependence.

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References

  1. Abbadie C, Pan Y-X, Pasternak GW (2004) Immunohistochemical study of the expression of exon11-containing mu opioid receptor variants in the mouse brain. Neuroscience i:419–430

  2. Bardo MT, Gehrke BJ, Shortridge BE, Rauhut AS (2003) Effects of beta-funaltrexamine and naloxonazine on single-trial morphine-conditioned place preference and locomotor activity. Pharmacol Biochem Behav 74:617–622

    CAS  Article  PubMed  Google Scholar 

  3. Becker A, Grecksch G, Brodemann R, Kraus J, Peters B, Schroeder H, Thiemann V, Loh HH, Hollt V (2000) Morphine self-administration in mu-opioid receptor-deficient mice. Naunyn Schmiedeberg's Arch Pharmacol 361:584–589

    CAS  Article  Google Scholar 

  4. Beckett AH (1959) Stereochemical factors in biological activity. Fortschr Arzneimittelforsch 1:455–530

    CAS  PubMed  Google Scholar 

  5. Cadet P, Mantione KJ, Stefano GB (2003) Molecular identification and functional expression of mu 3, a novel alternatively spliced variant of the human mu opiate receptor gene. JImmunol 170:5118–5123

    CAS  Article  Google Scholar 

  6. Charbogne P, Kieffer BL, Befort K (2014) 15 years of genetic approaches in vivo for addiction research: opioid receptor and peptide gene knockout in mouse models of drug abuse. Neuropharmacology 76 Pt B:204–217

    Article  PubMed  Google Scholar 

  7. Chen Y, Mestek A, Liu J, Hurley JA, Yu L (1993) Molecular cloning and functional expression of a μ-opioid receptor from rat brain. MolPharmacol 44:8–12

    CAS  Google Scholar 

  8. Corder G, Tawfik VL, Wang D, Sypek EI, Low SA, Dickinson JR, Sotoudeh C, Clark JD, Barres BA, Bohlen CJ, Scherrer G (2017) Loss of mu opioid receptor signaling in nociceptors, but not microglia, abrogates morphine tolerance without disrupting analgesia. Nat Med 23:164–173

    CAS  Article  PubMed  Google Scholar 

  9. Crain SM, Shen K-F (1990) Opioids can evoke direct receptor-mediated excitatory effects on sensory neurons. Trends Pharmacol Sci 11:77–81

    CAS  Article  PubMed  Google Scholar 

  10. D'Amour FE, Smith DL (1941) A method for determining loss of pain sensation. JPharmacolExpTher 72:74–79

    Google Scholar 

  11. Elhabazi K, Ayachi S, Ilien B, Simonin F (2014) Assessment of morphine-induced hyperalgesia and analgesic tolerance in mice using thermal and mechanical nociceptive modalities. J Vis Exp:e51264

  12. Gris P, Gauthier J, Cheng P, Gibson DG, Gris D, Laur O, Pierson J, Wentworth S, Nackley AG, Maixner W, Diatchenko L (2010) A novel alternatively spliced isoform of the mu-opioid receptor: functional antagonism. MolPain 6:33

    Google Scholar 

  13. Hahn EF, Carroll-Buatti M, Pasternak GW (1982) Irreversible opiate agonists and antagonists: the 14-hydroxydihydromorphinone azines. JNeurosci 2:572–576

    CAS  Google Scholar 

  14. Heyman JS, Williams CL, Burks TF, Mosberg HI, Porreca F (1988) Dissociation of opioid antinociception and central gastrointestinal propulsion in the mouse: studies with naloxonazine. JPharmacolExpTher 245:238–243

    CAS  Google Scholar 

  15. Juni A, Klein G, Pintar JE, Kest B (2007) Nociception increases during opioid infusion in opioid receptor triple knock-out mice. Neuroscience 147:439–444

    CAS  Article  PubMed  Google Scholar 

  16. Kitanaka N, Sora I, Kinsey S, Zeng ZZ, Uhl GR (1998) No heroin or morphine 6β-glucuronide analgesia in μ-opioid receptor knockout mice. Eur J Pharmacol 355:R1–R3

    CAS  Article  PubMed  Google Scholar 

  17. Ling GSF, MacLeod JM, Lee S, Lockhart SH, Pasternak GW (1984) Separation of morphine analgesia from physical dependence. Science 226:462–464

    CAS  Article  PubMed  Google Scholar 

  18. Ling GSF, Spiegel K, Lockhart SH, Pasternak GW (1985) Separation of opioid analgesia from respiratory depression: evidence for different receptor mechanisms. JPharmacolExpTher 232:149–155

    CAS  Google Scholar 

  19. Ling GSF, Spiegel K, Nishimura S, Pasternak GW (1983) Dissociation of morphine’s analgesic and respiratory depressant actions. EurJPharmacol 86:487–488

    CAS  Google Scholar 

  20. Loh HH, Liu HC, Cavalli A, Yang WL, Chen YF, Wei LN (1998) Μυ opioid receptor knockout in mice: effects on ligand-induced analgesia and morphine lethality. Mol Brain Res 54:321–326

    CAS  Article  PubMed  Google Scholar 

  21. Lu Z, Xu J, Rossi GC, Majumdar S, Pasternak GW, Pan YX (2015) Mediation of opioid analgesia by a truncated 6-transmembrane GPCR. J Clin Invest 125:2626–2630

    Article  PubMed  PubMed Central  Google Scholar 

  22. Majumdar S, Grinnell S, Le RV, Burgman M, Polikar L, Ansonoff M, Pintar J, Pan YX, Pasternak GW (2011) Truncated G protein-coupled mu opioid receptor MOR-1 splice variants are targets for highly potent opioid analgesics lacking side effects. ProcNatlAcadSciUSA 108:19776–19783

    Google Scholar 

  23. Marrone GF, Grinnell SG, Lu Z, Rossi GC, Le Rouzic V, Xu J, Majumdar S, Pan YX, Pasternak GW (2016) Truncated mu opioid GPCR variant involvement in opioid-dependent and opioid-independent pain modulatory systems within the CNS. Proc Natl Acad Sci U S A 113:3663–3668

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  24. Matthes HW, Maldonado R, Simonin F, Valverde O, Slowe S, Kitchen I, Befort K, Dierich A, Le Meur M, Dollé P, Tzavara E, Hanoune J, Roques BP, Kieffer BL (1996) Loss of morphine-induced analgesia, reward effect and withdrawal symptoms in mice lacking the μ-opioid-receptor gene. Nature 383:819–823

    CAS  Article  PubMed  Google Scholar 

  25. Oladosu FA, Conrad MS, O'Buckley SC, Rashid NU, Slade GD, Nackley AG (2015a) Mu opioid splice variant MOR-1K contributes to the development of opioid-induced hyperalgesia. PLoS One 10:e0135711

    Article  PubMed  PubMed Central  Google Scholar 

  26. Oladosu FA, Maixner W, Nackley AG (2015b) Alternative splicing of G protein-coupled receptors: relevance to pain management. Mayo Clin Proc 90:1135–1151

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  27. Pan YX, Xu J, Xu M, Rossi GC, Matulonis JE, Pasternak GW (2009) Involvement of exon 11-associated variants of the mu opioid receptor MOR-1 in heroin, but not morphine, actions. ProcNatlAcadSciUSA 106:4917–4922

    CAS  Article  Google Scholar 

  28. Pasternak GW (2001) Incomplete cross tolerance and multiple mu opioid peptide receptors. Trends PharmacolSci 22:67–70

    CAS  Article  Google Scholar 

  29. Pasternak GW, Childers SR, Snyder SH (1980a) Naloxazone, a long-acting opiate antagonist: effects on analgesia in intact animals and on opiate receptor binding in vitro. JPharmacolExpTher 214:455–462

    CAS  Google Scholar 

  30. Pasternak GW, Childers SR, Snyder SH (1980b) Opiate analgesia: evidence for mediation by a subpopulation of opiate receptors. Science 208:514–516

    CAS  Article  PubMed  Google Scholar 

  31. Pasternak GW, Pan Y-X (2013) Mu opioids and their receptors: evolution of a concept. PharmacolRev 65:1257–1317

    CAS  Google Scholar 

  32. Pasternak GW, Snyder SH (1975) Identification of a novel high affinity opiate receptor binding in rat brain. Nature 253:563–565

    CAS  Article  PubMed  Google Scholar 

  33. Patrick GA, Dewey WL, Spaulding TC, Harris LS (1975) Relationship of brain morphine levels to analgesic activity in acutely treated mice and rats and in pellet implanted mice. J Pharmacol Exp Ther 193:876–883

    CAS  PubMed  Google Scholar 

  34. Paul D, Pasternak GW (1988) Differential blockade by naloxonazine of two μ opiate actions: analgesia and inhibition of gastrointestinal transit. EurJPharmacol 149:403–404

    CAS  Google Scholar 

  35. Pert CB, Snyder SH (1973) Opiate receptor: demonstration in nervous tissue. Science 179:1011–1014

    CAS  Article  PubMed  Google Scholar 

  36. Piepponen TP, Kivastik T, Katajamäki J, Zharkovsky A, Ahtee L (1997) Involvement of opioid μ1 receptors in morphine-induced conditioned place preference in rats. PharmacolBiochemBehav 58:275–279

    CAS  Google Scholar 

  37. Portoghese PS (1966) Stereochemical factors and receptor interactions associated with narcotic analgesics. JPharmacSciences 55:865–887

    CAS  Google Scholar 

  38. Roeckel LA, Le Coz GM, Gaveriaux-Ruff C, Simonin F (2016) Opioid-induced hyperalgesia: cellular and molecular mechanisms. Neuroscience.

  39. Rossi GC, Leventhal L, Pan YX, Cole J, Su W, Bodnar RJ, Pasternak GW (1997) Antisense mapping of MOR-1 in rats: distinguishing between morphine and morphine-6beta-glucuronide antinociception. JPharmacolExpTher 281:109–114

    CAS  Google Scholar 

  40. Rossi GC, Pan Y-X, Cheng J, Pasternak GW (1994) Blockade of morphine analgesia by an antisense oligodeoxynucleotide against the mu receptor. Life Sci 54:L375–L379

    Article  Google Scholar 

  41. Samoshkin A, Convertino M, Viet CT, Wieskopf JS, Kambur O, Marcovitz J, Patel P, Stone LS, Kalso E, Mogil JS, Schmidt BL, Maixner W, Dokholyan NV, Diatchenko L (2015) Structural and functional interactions between six-transmembrane mu-opioid receptors and beta2-adrenoreceptors modulate opioid signaling. Sci Rep 5:18198

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  42. Schuller AG, King MA, Zhang J, Bolan E, Pan YX, Morgan DJ, Chang A, Czick ME, Unterwald EM, Pasternak GW, Pintar JE (1999) Retention of heroin and morphine-6 beta-glucuronide analgesia in a new line of mice lacking exon 1 of MOR-1. NatNeurosci 2:151–156

    CAS  Google Scholar 

  43. Shabalina SA, Zaykin DV, Gris P, Ogurtsov AY, Gauthier J, Shibata K, Tchivileva IE, Belfer I, Mishra B, Kiselycznyk C, Wallace MR, Staud R, Spiridonov NA, Max MB, Goldman D, Fillingim RB, Maixner W, Diatchenko L (2009) Expansion of the human mu-opioid receptor gene architecture: novel functional variants. HumMolGenet 18:1037–1051

    CAS  Google Scholar 

  44. Simon EJ, Hiller JM, Edelman I (1973) Stereospecific binding of the potent narcotic analgesic [3H]etorphine to rat-brain homogenate. ProcNatlAcadSciUSA 70:1947–1949

    CAS  Article  Google Scholar 

  45. Sora I, Elmer G, Funada M, Pieper J, Li XF, Hall FS, Uhl GR (2001) Mu opiate receptor gene dose effects on different morphine actions: evidence for differential in vivo mu receptor reserve. Neuropsychopharmacology 25:41–54

    CAS  Article  PubMed  Google Scholar 

  46. Sora I, Takahashi N, Funada M, Ujike H, Revay RS, Donovan DM, Miner LL, Uhl GR (1997) Opiate receptor knockout mice define μ receptor roles in endogenous nociceptive responses and morphine-induced analgesia. ProcNatlAcadSciUSA 94:1544–1549

    CAS  Article  Google Scholar 

  47. Spiegel K, Kourides I, Pasternak GW (1982) Prolactin and growth hormone release by morphine in the rat: different receptor mechanisms. Science 217:745–747

    CAS  Article  PubMed  Google Scholar 

  48. Stefano GB, Hartman A, Bilfinger TV, Magazine HI, Liu Y, Casares F, Goligorsky MS (1995) Presence of the μ3 opiate receptor in endothelial cells—coupling to nitric oxide production and vasodilation. JBiolChem 270:30290–30293

    CAS  Google Scholar 

  49. Terenius L (1973) Characteristics of the "receptor" for narcotic analgesics in synaptic plasma membrane from rat brain. Acta Pharmacolet toxicol 33:377–384

    CAS  Article  Google Scholar 

  50. Thompson RC, Mansour A, Akil H, Watson SJ (1993) Cloning and pharmacological characterization of a rat μ opioid receptor. Neuron 11:903–913

    CAS  Article  PubMed  Google Scholar 

  51. Tian M, Broxmeyer HE, Fan Y, Lai Z, Zhang S, Aronica S, Cooper S, Bigsby RM, Steinmetz R, Engle SJ, Mestek A, Pollock JD, Lehman MN, Jansen HT, Ying M, Stambrook PJ, Tischfield JA, Yu L (1997) Altered hematopoiesis, behavior, and sexual function in μ opioid receptor-deficient mice. JExpMed 185:1517–1522

    CAS  Article  Google Scholar 

  52. Uhl GR, Childers S, Pasternak GW (1994) An opiate-receptor gene family reunion. Trends Neurosci 17:89–93

    CAS  Article  PubMed  Google Scholar 

  53. Wang JB, Imai Y, Eppler CM, Gregor P, Spivak CE, Uhl GR (1993) μ opiate receptor: cDNA cloning and expression. ProcNatlAcadSciUSA 90:10230–10234

    CAS  Article  Google Scholar 

  54. Wolozin BL, Pasternak GW (1981) Classification of multiple morphine and enkephalin binding sites in the central nervous system. ProcNatlAcadSciUSA 78:6181–6185

    CAS  Article  Google Scholar 

  55. Xu J, Faskowitz AJ, Rossi GC, Xu M, Lu Z, Pan YX, Pasternak GW (2015) Stabilization of morphine tolerance with long-term dosing: association with selective upregulation of mu-opioid receptor splice variant mRNAs. Proc Natl Acad Sci U S A 112:279–284

    CAS  Article  PubMed  Google Scholar 

  56. Xu J, Lu Z, Xu M, Rossi GC, Kest B, Waxman AR, Pasternak GW, Pan YX (2014) Differential expressions of the alternatively spliced variant mRNAs of the mu opioid receptor gene, OPRM1, in brain regions of four inbred mouse strains. PLoS One 9:e111267

    Article  PubMed  PubMed Central  Google Scholar 

  57. Xu J, Xu M, Brown T, Rossi GC, Hurd YL, Inturrisi CE, Pasternak GW, Pan YX (2013) Stabilization of the mu opioid receptor by truncated single transmembrane splice variants through a chaperone-like action. JBioChem 288:21211–21227

    CAS  Google Scholar 

  58. Xu J, Xu M, Hurd YL, Pasternak GW, Pan YX (2009) Isolation and characterization of new exon 11-associated N-terminal splice variants of the human mu opioid receptor gene. JNeurochem 108:962–972

    CAS  Article  Google Scholar 

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Acknowledgements

This work was supported in part by grants from the Peter F. McManus Charitable Trust, Mr. William H. Goodwin and Mrs. Alice Goodwin and the Commonwealth Foundation for Cancer Research, The Experimental Therapeutics Center of Memorial Sloan Kettering Cancer Center, and the National Institutes on Drug Abuse of the National Institutes of Health (DA06241 and DA07242) to GWP and DA0291122 to AMR; a core grant from the National Cancer Institute of the National Institutes of Health (CA08748) to MSKCC; and a National Science Foundation Graduate Research Fellowship Grant (DGE-1257284) to GFM. There are no competing financial interests.

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Correspondence to Gavril W. Pasternak.

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All animal studies were approved by the Institutional Animal Care and Use Committee of Memorial Sloan Kettering Cancer Center and performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals in an AAALAC-accredited facility.

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Marrone, G.F., Le Rouzic, V., Varadi, A. et al. Genetic dissociation of morphine analgesia from hyperalgesia in mice. Psychopharmacology 234, 1891–1900 (2017). https://doi.org/10.1007/s00213-017-4600-2

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Keywords

  • Mu opioid
  • Mu opioid receptor
  • KNOCKOUT
  • Alternative splicing
  • MOR-1
  • GPCR
  • Truncated
  • 6TM