Advertisement

Upregulating G Protein-Coupled Receptors with Receptor Antagonists

  • Ellen M. UnterwaldEmail author
Protocol
Part of the Neuromethods book series (NM, volume 60)

Abstract

The phenomenon of antagonist-induced receptor upregulation is common to many G protein-coupled receptors (GPCRs) such as adrenergic, muscarinic, opioid, cannabinoid, histamine, GABA(B), serotonin, and dopamine receptors. This chapter reviews data that support antagonist-induced upregulation specifically of opioid receptors but many of the principles apply to other GPCRs as well. It is well documented that chronic exposure to opioid receptor antagonists reliably produces increases in binding to opioid receptors when the antagonists are administered in vivo or applied in vitro to cell culture systems. Antagonist exposure increases receptor number and is associated with functional supersensitivity to subsequent agonist administration. For example, the analgesic potency of morphine is increased following prior administration of opioid receptor antagonists. Likewise, coupling of opioid receptors to G proteins is increased following antagonist exposure, as is the ability of opioid agonists to regulate adenylyl cyclase activity. The most common approach used to measure receptor upregulation is radioligand receptor binding. This chapter includes methods to measure receptor number by radioligand binding and by immunohistochemical approaches. Also included are methods to assess alterations in receptor function following antagonist exposure. The methods can be applied to tissue or cell homogenates or to in situ preparations in order to increase the anatomical specificity of the resulting data.

Key words

Receptor upregulation Receptor antagonists Receptor binding Quantitative receptor autoradiography Opioid receptor regulation 

References

  1. 1.
    Parashos SA, Barone P, Tucci I et al (1987) Attenuation of D-1 antagonist-induced D-1 receptor upregulation by concomitant D-2 receptor blockade. Life Sci 41:2279–2284PubMedCrossRefGoogle Scholar
  2. 2.
    Filtz TM, Guan W, Artymyshyn RP et al (1994) Mechanisms of up-regulation of D2L dopamine receptors by agonist and antagonists in transfected HEK-293 cells. J Pharmacol Exp Ther 271:1574–1582PubMedGoogle Scholar
  3. 3.
    Boundy VA, Pacheco MA, Guan W et al (1995) Agonists and antagonists differentially regulate the high affinity state of the D2L receptor in human embryonic kidney 293 cells. Mol Pharmacol 48:956–964PubMedGoogle Scholar
  4. 4.
    Fukamauchi F, Saunders PA, Hough C et al (1993) Agonist-induced down-regulation and antagonist-induced up-regulation of M2- and M3-muscarinic acetylcholine receptor mRNA and protein in cultured cerebellar granule cells. Mol Pharmacol 44:940–949PubMedGoogle Scholar
  5. 5.
    Brodde OE, Zerkowski HR, Doetsch N et al (1989) Subtype-selective up-regulation of human saphenous vein beta 2-adrenoceptors by chronic beta-adrenoceptor antagonist treatment. Naunyn Schmiedebergs Arch Pharmacol 339:479–482PubMedCrossRefGoogle Scholar
  6. 6.
    Nishi M, Azmitia EC (1999) Agonist- and antagonist-induced plasticity of rat 5-HT1A receptor in hippocampal cell culture. Synapse 31:186–195PubMedCrossRefGoogle Scholar
  7. 7.
    Osawa S, Kajimura M, Yamamoto S et al (2005) Alteration of intracellular histamine H2 receptor cycling precedes antagonist-induced upregulation. Am J Physiol Gastrointest Liver Physiol 289:G880-889PubMedCrossRefGoogle Scholar
  8. 8.
    Pibiri F, Carboni G, Carai MA et al (2005) Up-regulation of GABA(B) receptors by chronic administration of the GABA(B) receptor antagonist SCH 50,911. Eur J Pharmacol 515:94–98PubMedCrossRefGoogle Scholar
  9. 9.
    Unterwald EM, Howells RD (2008) Upregulation of opioid receptors. In: Dean R, Bilsky EJ, Negus SS (Eds) Opiate receptors and antagonists: From bench to clinic. Humana Press, New YorkGoogle Scholar
  10. 10.
    Hitzemann RJ, Hitzemann BA, Loh HH (1974) Binding of 3H-naloxone in the mouse brain: effect of ions and tolerance development. Life Sci 14:2393–2404PubMedCrossRefGoogle Scholar
  11. 11.
    Tang AH, Collins RJ (1978) Enhanced analgesic effects of morphine after chronic administration of naloxone in the rat. Eur. J Pharmacol 47:473–474PubMedCrossRefGoogle Scholar
  12. 12.
    Lahti RA, Collins RJ (1978) Chronic naloxone results in prolonged increases in opiate binding sites in brain. Eur J Pharmacol 51:185–186PubMedCrossRefGoogle Scholar
  13. 13.
    Schulz R, Wuster M, Herz A (1979) Supersensitivity to opioids following the chronic blockade of endorphin action by naloxone. Naunyn Schmiedebergs Arch Pharmacol 306:93–96PubMedCrossRefGoogle Scholar
  14. 14.
    Yoburn BC, Purohit V, Patel K et al (2004) Opioid agonist and antagonist treatment differentially regulates immunoreactive mu-opioid receptors and dynamin-2 in vivo. Eur J Pharmacol 498: 87–96PubMedCrossRefGoogle Scholar
  15. 15.
    Tempel A, Zukin RS, Gardner EL (1982) Supersensitivity of brain opiate receptor subtypes after chronic naltrexone treatment. Life Sci 31:1401–1404PubMedCrossRefGoogle Scholar
  16. 16.
    Zukin RS, Sugarman JR, Fitz-Syage ML et al (1982) Naltrexone-induced opiate receptor supersensitivity. Brain Res 245:285–292PubMedCrossRefGoogle Scholar
  17. 17.
    Yoburn BC, Nunes FA, Adler B et al (1986) Pharmacodynamic supersensitivity and opioid receptor upregulation in the mouse. J Pharmacol Exp Ther 239:132–135PubMedGoogle Scholar
  18. 18.
    Tempel A, Gardner EL, Zukin RS (1985). Neurochemical and functional correlates of naltrexone-induced opiate receptor up-regulation. J Pharmacol Exp Ther 232:439–444PubMedGoogle Scholar
  19. 19.
    Attali B, Vogel Z (1990) Characterization of kappa opiate receptors in rat spinal cord-dorsal root ganglion co-cultures and their regulation by chronic opiate treatment. Brain Res 517:182–188PubMedCrossRefGoogle Scholar
  20. 20.
    Bhargava HN, Matwyshyn GA, Reddy PL et al (1993). Effects of naltrexone on the binding of [3H]D-Ala2, MePhe4, Gly-ol5-enkephalin to brain regions and spinal cord and pharmacological responses to morphine in the rat. Gen Pharmacol 24:1351–1357PubMedCrossRefGoogle Scholar
  21. 21.
    Yoburn BC, Shah S, Chan K et al (1995) Supersensitivity to opioid analgesics following chronic opioid antagonist treatment: relationship to receptor selectivity. Pharmacol Biochem Behav 51:535–539PubMedCrossRefGoogle Scholar
  22. 22.
    Giordano AL, Nock B, Cicero TJ (1990) Antagonist-induced up-regulation of the putative epsilon opioid receptor in rat brain: comparison with kappa, mu and delta opioid receptors. J Pharmacol Exp Ther 255:536–540PubMedGoogle Scholar
  23. 23.
    Morris BJ, Millan MJ, Herz A (1988) Antagonist-induced opioid receptor up-regulation. II. Regionally specific modulation of mu, delta and kappa binding sites in rat brain revealed by quantitative autoradiography. J Pharmacol Exp Ther 247:729–736PubMedGoogle Scholar
  24. 24.
    Yoburn BC, Luke MC, Pasternak GW et al (1988) Upregulation of opioid receptor subtypes correlates with potency changes of morphine and DADLE. Life Sci 43:1319–1324PubMedCrossRefGoogle Scholar
  25. 25.
    Lesscher HMB, Bailey A, Burbach JPH et al (2003) Receptor-selective changes in mu-, delta-, and kappa-opioid receptors after chronic naltrexone treatment in mice. Eur J Neurosci 17:1006–1012PubMedCrossRefGoogle Scholar
  26. 26.
    Hummel M, Ansonoff MA, Pintar JE et al (2004) Genetic and pharmacological manipulation of mu opioid receptors in mice reveals a differential effect on behavioral sensitization to cocaine. Neuroscience 125:211–220PubMedCrossRefGoogle Scholar
  27. 27.
    Raynor K, Kong H, Chen Y et al (1994) Pharmacological characterization of the cloned kappa-, delta-, and mu-opioid receptors. Mol Pharmacol 45:330–334.PubMedGoogle Scholar
  28. 28.
    Unterwald EM, Anton B, To T et al (1998) Quantitative immuno-localization of mu opioid receptors: regulation by naltrexone. Neuroscience 85:897–905PubMedCrossRefGoogle Scholar
  29. 29.
    Tempel A, Gardner EL, Zukin RS (1984) Visualization of opiate receptor upregulation by light microscopy autoradiography. Proc Natl Acad Sci USA 81:3893–3897PubMedCrossRefGoogle Scholar
  30. 30.
    Zaki PA, Keith DE, Brine GA et al (2000) Ligand-induced changes in surface mu-opioid receptor number: relationship to G protein Activation. J Pharmacol Exp Ther 292:1127–1134PubMedGoogle Scholar
  31. 31.
    Zadina JE, Chang SL, Ge LJ et al (1993) Mu opiate receptor down-regulation by morphine and up-regulation by naloxone in SHSY5Y human neuroblastoma cells. J Pharmacol Exp Ther 265:254–262PubMedGoogle Scholar
  32. 32.
    Yoburn BC, Kreuscher SP, Inturrisi CE et al (1989) Opioid receptor upregulation and supersensitivity in mice: effect on morphine sensitivity. Pharmacol Biochem Behav 32:727–731PubMedCrossRefGoogle Scholar
  33. 33.
    Volterra BN, DiGiulio AM, Cuomo V et al (1984) Modulation of opioid system in C57 mice after repeated treatment with morphine and naloxone: biochemical and behavioral correlates. Life Sci 34:1669–1678PubMedCrossRefGoogle Scholar
  34. 34.
    Bardo MT, Bhatnagar RK, Gebhart GF (1983) Chronic naltrexone increases binding in brain and produces supersensitivity to morphine in the locus coeruleus of the rat. Brain Res 289:223–234PubMedCrossRefGoogle Scholar
  35. 35.
    Suzuki T, Fukagawa Y, Misawa M (1990) Enhancement of morphine withdrawal signs in the rat after chronic treatment with naloxone. Eur J Pharmacol 178:239–242PubMedCrossRefGoogle Scholar
  36. 36.
    Yoburn BC, Nunes FA, Adler B et al (1986) Pharmacodynamic supersensitivity and opioid receptor upregulation in the mouse. J Pharmacol Exp Ther 239:132–135PubMedGoogle Scholar
  37. 37.
    Narita M, Mizoguchi H, Nagase H et al (2001) Up-regulation of spinal mu-opioid receptor function to activate G-protein by chronic naloxone treatment. Brain Res 913:170–173PubMedCrossRefGoogle Scholar
  38. 38.
    Cote TE, Izenwasser S, Weems HB (1993) Naltexone-induced upregulation of mu opioid receptors on 7315c cell and brain membranes: enhancement of opioid efficacy in inhibiting adenylyl cyclase. J Pharmacol Exp Ther 267:238–244PubMedGoogle Scholar
  39. 39.
    Unterwald EM, Rubenfeld JM, Imai Y et al (1995) Chronic opioid antagonist administration upregulates mu opioid receptor binding without altering mu opioid receptor mRNA levels. Mol Brain Res 33:351–355PubMedCrossRefGoogle Scholar
  40. 40.
    Castelli MP, Melis M, Mameli M et al (1997) Chronic morphine and naltrexone fail to modify mu-opioid receptor mRNA levels in the rat brain. Mol Brain Res 45:149–153PubMedCrossRefGoogle Scholar
  41. 41.
    Jenab S, Kest B, Inturrisi CE (1995) Assessment of delta opioid antinociception and receptor mRNA levels in mouse after chronic naltrexone treatment. Brain Res 691:69–75PubMedCrossRefGoogle Scholar
  42. 42.
    Wannemacher K, Yadav P, Howells RD (2007) A select set of opioid ligands induce up-regulation by promoting the maturation and stability of the rat kappa opioid receptor in human embryonic kidney 293 cells. J Pharmacol Exp Ther 323:614625PubMedCrossRefGoogle Scholar
  43. 43.
    Jenab S, Inturrisi CE (1994) Ethanol and naloxone differentially upregulate delta opioid receptor gene expression in neuroblastoma hybrid (NG108-15) cells. Mol Brain Res 27:95–102PubMedCrossRefGoogle Scholar
  44. 44.
    Morello J-P, Salahpour A, Laperriere A et al (2000) Pharmacological chaperones rescue cell-surface expression and function of misfolded V2 vasopressin receptor mutants. J Clin Invest 105:887–895PubMedCrossRefGoogle Scholar
  45. 45.
    Petaja-Repo UE, Hogue M, Bhalla S et al (2002) Ligands act as pharmacological chaperones and increase the efficiency of delta opioid receptor maturation. EMBO J 21:1628–1637PubMedCrossRefGoogle Scholar
  46. 46.
    Chaipatikul V, Erickson-Herbrandson LJ, Loh HH et al (2003) Rescuing the traffic-deficient mutants of rat mu-opioid receptors with hydrophobic ligands. Mol Pharmacol 64:32–41PubMedCrossRefGoogle Scholar
  47. 47.
    Chen Y, Chen C, Wang Y et al (2006) Ligands regulate cell surface level of the human kappa opioid receptor (hKOR) by activation-induced down-regulation and pharmacological chaperone-mediated enhancement: differential effects of non-peptide and peptide agonists. J Pharmacol Exp Ther 319:765–775PubMedCrossRefGoogle Scholar
  48. 48.
    Li J, Chen C, Huang P et al (2001) Inverse agonist up-regulates the constitutively active D3.49(164)Q mutant of the rat mu-opioid receptor by stabilizing the structure and blocking constitutive internalization and down-regulation. Mol Pharmacol 60:1064–1075PubMedGoogle Scholar
  49. 49.
    Li J, Huang P, Chen C et al (2001) Constitutive activation of the mu opioid receptor by mutation of D3.49(164), but not D3.32(147): D3.49(164) is critical for stabilization of the inactive form of the receptor and for its expression. Biochemistry 40:12039–12050PubMedCrossRefGoogle Scholar
  50. 50.
    Yamamura HI, Enna SJ, Kuhar MJ (1985) Neurotransmitter receptor binding. Raven Press, New York.Google Scholar
  51. 51.
    Unterwald EM (2008) Naltrexone in the treatment of alcohol dependence. J Addict Med 2:121–127PubMedCrossRefGoogle Scholar
  52. 52.
    Unterwald EM, Rubenfeld JM, Kreek MJ (1994) Repeated cocaine administration upregulates kappa and mu, but not delta, opioid receptors. NeuroReport 5:1613–1616PubMedCrossRefGoogle Scholar
  53. 53.
    Sim LJ, Selley DE, Childers SR (1995) In vitro autoradiography of receptor-activated G proteins in rat brain by agonist-stimulated guanylyl 5’-[gamma-[35S]thio]-triphosphate binding. Proc Natl Acad Sci USA 92:7242–7246PubMedCrossRefGoogle Scholar
  54. 54.
    Milligan G (2003) Principles: extending the utility of [35S]GTP gammaS binding assays. TIPS 24:87–90PubMedGoogle Scholar
  55. 55.
    Harrison C and Traynor JR (2003) The [35S]GTP gammaS binding assay: approaches and applications in pharmacology. Life Sci 74:489–508PubMedCrossRefGoogle Scholar
  56. 56.
    Sim-Selley LJ and Childers SR (2002) Neuroanatomical localization of receptor-activated G proteins in brain. Methods Enzymol 344:42–58PubMedCrossRefGoogle Scholar
  57. 57.
    Garcia-Jimenez A, Cowbuurn RF, Winblad B et al (1997) Autoradiographic characterization of [3535]GTPgammaS binding sites in rat brain. Neurochem Res 22:1055–1063PubMedCrossRefGoogle Scholar
  58. 58.
    Schroeder JA, Niculescu M, Unterwald EM (2003) Cocaine alters mu but not delta or kappa opioid receptor-stimulated in situ [35S]GTPgammaS binding in rat brain. Synapse 47:26–32PubMedCrossRefGoogle Scholar
  59. 59.
    Zhang X, de Araujo LG, Elde R et al (1999) Effect of morphine on cholecystokinin and mu-opioid receptor-like immunoreactivities in rat spinal dorsal horn neurons after peripheral axotomy and inflammation. Neuroscience 95:197–207CrossRefGoogle Scholar
  60. 60.
    Millan MJ, Morris BJ, Herz A (1988). Antagonist-induced opioid receptor up-regulation. I. Characterization of supersensitivity to selective mu and kappa agonists. J Pharmacol Exp Ther 247:721–728Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Department of Pharmacology and Center for Substance Abuse ResearchTemple University School of MedicinePhiladelphiaUSA

Personalised recommendations