Analgesia pp 421-435 | Cite as

Molecular Assays for Characterization of Alternatively Spliced Isoforms of the Mu Opioid Receptor (MOR)

  • Pavel Gris
  • Philip Cheng
  • John Pierson
  • William Maixner
  • Luda Diatchenko
Part of the Methods in Molecular Biology book series (MIMB, volume 617)


Mu-opioid receptor (MOR) belongs to a family of heptahelical G-protein-coupled receptors (GPCRs). Studies in humans and rodents demonstrated that the OPRM1 gene coding for MOR undergoes extensive alternative splicing afforded by the genetic complexity of OPRM1. Evidence from rodent studies also demonstrates an important role of these alternatively spliced forms in mediating opiate analgesia via their differential signaling properties. MOR signaling is predominantly Gia coupled. Release of the α subunit from G-protein complex results in the inhibition of adenylyl cyclase/cAMP pathway, whereas release of the βγ subunits activates G-protein-activated inwardly rectifying potassium channels and inhibits voltage-dependent calcium channels. These molecular events result in the suppression of cellular activities that diminish pain sensations. Recently, a new isoform of OPRM1, MOR3, has been identified that shows an increase in the production of nitric oxide (NO) upon stimulation with morphine. Hence, there is a need to describe molecular techniques that enable the functional characterization of MOR isoforms. In this review, we describe the methodologies used to assay key mediators of MOR activation including cellular assays for cAMP, free Ca2+, and NO, all of which have been implicated in the pharmacological effects of MOR agonists.

Key words

Alternative splicing OPRM1 Opioid Calcium cAMP Nitric oxide Fluo-4 Fluo-3 GPCR FSK Capsaicin 


  1. 1.
    Pasternak GW (2004) Multiple opiate receptors: deja vu all over again. Neuropharmacology 47(Suppl. 1):312–323PubMedCrossRefGoogle Scholar
  2. 2.
    Pan YX et al (2003) Identification and characterization of two new human mu opioid receptor splice variants, hMOR-1O and hMOR-1X. Biochem Biophys Res Commun 301(4):1057–1061PubMedCrossRefGoogle Scholar
  3. 3.
    Pasternak DA et al (2004) Identification of three new alternatively spliced variants of the rat mu opioid receptor gene: dissociation of affinity and efficacy. J Neurochem 91(4):881–890PubMedCrossRefGoogle Scholar
  4. 4.
    Pan YX et al (2005) Identification of four novel exon 5 splice variants of the mouse mu-opioid receptor gene: functional consequences of C-terminal splicing. Mol Pharmacol 68(3):866–875PubMedGoogle Scholar
  5. 5.
    Pan L et al (2005) Identification and characterization of six new alternatively spliced variants of the human mu opioid receptor gene, Oprm. Neuroscience 133(1):209–220PubMedCrossRefGoogle Scholar
  6. 6.
    Shabalina SA et al (2008) Expansion of the human {micro}-opioid receptor gene architecture: novel functional variants. Hum Mol Genet. 2009 Mar 15;18(6):1037–51PubMedCrossRefGoogle Scholar
  7. 7.
    Gris P, Cheng P, Pierson J, Gauthier J, Shabalina S, Spiridonov N, Maixner W, Diatchenko L (2008) Functional characterization of the novel alternatively spliced form of mu-opioid receptor OPRM1. In: 12th World congress on pain. Glasgow, UKGoogle Scholar
  8. 8.
    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. J Immunol 170(10):5118–5123PubMedGoogle Scholar
  9. 9.
    Rubovitch V, Gafni M, Sarne Y (2003) The mu opioid agonist DAMGO stimulates cAMP production in SK-N-SH cells through a PLC-PKC-Ca++ pathway. Brain Res Mol Brain Res 110(2):261–266PubMedCrossRefGoogle Scholar
  10. 10.
    Galeotti N et al (2006) Signaling pathway of morphine induced acute thermal hyperalgesia in mice. Pain 123(3):294–305PubMedCrossRefGoogle Scholar
  11. 11.
    Costigan M, Woolf CJ (2000) Pain: molecular mechanisms. J Pain 1(3 Suppl):35–44PubMedGoogle Scholar
  12. 12.
    Dolan S, Nolan AM (2001) Biphasic modulation of nociceptive processing by the cyclic AMP-protein kinase A signalling pathway in sheep spinal cord. Neurosci Lett 309(3):157–160PubMedCrossRefGoogle Scholar
  13. 13.
    Vetter I et al (2006) The mu opioid agonist morphine modulates potentiation of capsaicin-evoked TRPV1 responses through a cyclic AMP-dependent protein kinase A pathway. Mol Pain 2:22PubMedCrossRefGoogle Scholar
  14. 14.
    North RA et al (1987) Mu and delta receptors belong to a family of receptors that are coupled to potassium channels. Proc Natl Acad Sci U S A 84(15):5487–5491PubMedCrossRefGoogle Scholar
  15. 15.
    Chieng BC et al (2008) Functional coupling of mu-receptor-Galphai-tethered proteins in AtT20 cells. Neuroreport 19(18):1793–1796PubMedCrossRefGoogle Scholar
  16. 16.
    Ikeda K et al (2000) Involvement of G-protein-activated inwardly rectifying K (GIRK) channels in opioid-induced analgesia. Neurosci Res 38(1):113–116PubMedCrossRefGoogle Scholar
  17. 17.
    Saegusa H et al (2000) Altered pain responses in mice lacking alpha 1E subunit of the voltage-dependent Ca2+ channel. Proc Natl Acad Sci U S A 97(11):6132–6137PubMedCrossRefGoogle Scholar
  18. 18.
    Wang L, Gintzler AR (1997) Altered mu-opiate receptor-G protein signal transduction following chronic morphine exposure. J Neurochem 68(1):248–254PubMedCrossRefGoogle Scholar
  19. 19.
    Ito A et al (2000) Mechanisms for ovariectomy-induced hyperalgesia and its relief by calcitonin: participation of 5-HT1A-like receptor on C-afferent terminals in substantia gelatinosa of the rat spinal cord. J Neurosci 20(16):6302–6308PubMedGoogle Scholar
  20. 20.
    Fields A, Sarne Y (1997) The stimulatory effect of opioids on cyclic AMP production in SK-N-SH cells is mediated by calcium ions. Life Sci 61(6):595–602PubMedCrossRefGoogle Scholar
  21. 21.
    Sarne Y et al (1998) Dissociation between the inhibitory and stimulatory effects of opioid peptides on cAMP formation in SK-N-SH neuroblastoma cells. Biochem Biophys Res Commun 246(1):128–131PubMedCrossRefGoogle Scholar
  22. 22.
    Crain SM, Shen KF (2000) Antagonists of excitatory opioid receptor functions enhance morphine’s analgesic potency and attenuate opioid tolerance/dependence liability. Pain 84(2-3):121–131PubMedCrossRefGoogle Scholar
  23. 23.
    Olmstead MC, Burns LH (2005) Ultra-low-dose naltrexone suppresses rewarding effects of opiates and aversive effects of opiate withdrawal in rats. Psychopharmacology (Berl) 181(3):576–581CrossRefGoogle Scholar
  24. 24.
    Crain SM, Shen KF (2001) Acute thermal hyperalgesia elicited by low-dose morphine in normal mice is blocked by ultra-low-dose naltrexone, unmasking potent opioid analgesia. Brain Res 888(1):75–82PubMedCrossRefGoogle Scholar
  25. 25.
    Martin NP et al (2004) PKA-mediated phosphorylation of the beta1-adrenergic receptor promotes Gs/Gi switching. Cell Signal 16(12):1397–1403PubMedCrossRefGoogle Scholar
  26. 26.
    Hill SJ, Baker JG (2003) The ups and downs of Gs- to Gi-protein switching. Br J Pharmacol 138(7):1188–1189PubMedCrossRefGoogle Scholar
  27. 27.
    Malmberg AB et al (1997) Diminished inflammation and nociceptive pain with preservation of neuropathic pain in mice with a targeted mutation of the type I regulatory subunit of cAMP-dependent protein kinase. J Neurosci 17(19):7462–7470PubMedGoogle Scholar
  28. 28.
    Chen GD et al (2008) Calcium/calmodulin-dependent kinase II mediates NO-elicited PKG activation to participate in spinal reflex potentiation in anesthetized rats. Am J Physiol Regul Integr Comp Physiol 294(2):R487-R493PubMedCrossRefGoogle Scholar
  29. 29.
    Selbie LA, Hill SJ (1998) G protein-coupled-receptor cross-talk: the fine-tuning of multiple receptor-signalling pathways. Trends Pharmacol Sci 19(3):87–93PubMedCrossRefGoogle Scholar
  30. 30.
    Tell GP, Pasternak GW, Cuatrecasas P (1975) Brain and caudate nucleus adenylate cyclase: effects of dopamine, GTP, E prostaglandins and morphine. FEBS Lett 51(1):242–245PubMedCrossRefGoogle Scholar
  31. 31.
    Connor M, Christie MD (1999) Opioid receptor signalling mechanisms. Clin Exp Pharmacol Physiol 26(7):493–499PubMedCrossRefGoogle Scholar
  32. 32.
    Aronson JK (2007) Concentration-effect and dose-response relations in clinical pharmacology. Br J Clin Pharmacol 63(3):255–257PubMedCrossRefGoogle Scholar
  33. 33.
    Minta A, Kao JP, Tsien RY (1989) Fluorescent indicators for cytosolic calcium based on rhodamine and fluorescein chromophores. J Biol Chem 264(14):8171–8178PubMedGoogle Scholar
  34. 34.
    Vetter I et al (2008) Mechanisms involved in potentiation of transient receptor potential vanilloid 1 responses by ethanol. Eur J Pain 12(4):441–454PubMedCrossRefGoogle Scholar
  35. 35.
    Vetter I et al (2008) Rapid, opioid-sensitive mechanisms involved in transient receptor potential vanilloid 1 sensitization. J Biol Chem 283(28):19540–19550PubMedCrossRefGoogle Scholar
  36. 36.
    Vasko MR, Campbell WB, Waite KJ (1994) Prostaglandin E2 enhances bradykinin-stimulated release of neuropeptides from rat sensory neurons in culture. J Neurosci 14(8):4987–4997PubMedGoogle Scholar
  37. 37.
    Berg KA et al (2007) Integrins regulate opioid receptor signaling in trigeminal ganglion neurons. Neuroscience 144(3):889–897PubMedCrossRefGoogle Scholar
  38. 38.
    Berg KA et al (1994) Signal transduction differences between 5-hydroxytryptamine type 2A and type 2C receptor systems. Mol Pharmacol 46(3):477–484PubMedGoogle Scholar
  39. 39.
    Cadet P et al (2007) A functionally coupled mu3-like opiate receptor/nitric oxide regulatory pathway in human multi-lineage progenitor cells. J Immunol 179(9):5839–5844PubMedGoogle Scholar
  40. 40.
    Takahashi A et al (1999) Measurement of intracellular calcium. Physiol Rev 79(4):1089–1125PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Pavel Gris
    • 1
  • Philip Cheng
    • 1
  • John Pierson
    • 1
  • William Maixner
    • 2
  • Luda Diatchenko
    • 2
  1. 1.Center for Neurosensory Disorders, University of North CarolinaChapel HillUSA
  2. 2.Center for Neurosensory Disorders, Carolina Center for Genome Sciences, University of North Carolina at Chapel HillChapel HillUSA

Personalised recommendations