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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
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 41:2279–2284
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 271:1574–1582
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 48:956–964
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 44:940–949
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 339:479–482
Nishi M, Azmitia EC (1999) Agonist- and antagonist-induced plasticity of rat 5-HT1A receptor in hippocampal cell culture. Synapse 31:186–195
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:G880-889
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 515:94–98
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 York
Hitzemann RJ, Hitzemann BA, Loh HH (1974) Binding of 3H-naloxone in the mouse brain: effect of ions and tolerance development. Life Sci 14:2393–2404
Tang AH, Collins RJ (1978) Enhanced analgesic effects of morphine after chronic administration of naloxone in the rat. Eur. J Pharmacol 47:473–474
Lahti RA, Collins RJ (1978) Chronic naloxone results in prolonged increases in opiate binding sites in brain. Eur J Pharmacol 51:185–186
Schulz R, Wuster M, Herz A (1979) Supersensitivity to opioids following the chronic blockade of endorphin action by naloxone. Naunyn Schmiedebergs Arch Pharmacol 306:93–96
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: 87–96
Tempel A, Zukin RS, Gardner EL (1982) Supersensitivity of brain opiate receptor subtypes after chronic naltrexone treatment. Life Sci 31:1401–1404
Zukin RS, Sugarman JR, Fitz-Syage ML et al (1982) Naltrexone-induced opiate receptor supersensitivity. Brain Res 245:285–292
Yoburn BC, Nunes FA, Adler B et al (1986) Pharmacodynamic supersensitivity and opioid receptor upregulation in the mouse. J Pharmacol Exp Ther 239:132–135
Tempel A, Gardner EL, Zukin RS (1985). Neurochemical and functional correlates of naltrexone-induced opiate receptor up-regulation. J Pharmacol Exp Ther 232:439–444
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 517:182–188
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 24:1351–1357
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 51:535–539
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 255:536–540
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 247:729–736
Yoburn BC, Luke MC, Pasternak GW et al (1988) Upregulation of opioid receptor subtypes correlates with potency changes of morphine and DADLE. Life Sci 43:1319–1324
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 17:1006–1012
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 125:211–220
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.
Unterwald EM, Anton B, To T et al (1998) Quantitative immuno-localization of mu opioid receptors: regulation by naltrexone. Neuroscience 85:897–905
Tempel A, Gardner EL, Zukin RS (1984) Visualization of opiate receptor upregulation by light microscopy autoradiography. Proc Natl Acad Sci USA 81:3893–3897
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 292:1127–1134
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 265:254–262
Yoburn BC, Kreuscher SP, Inturrisi CE et al (1989) Opioid receptor upregulation and supersensitivity in mice: effect on morphine sensitivity. Pharmacol Biochem Behav 32:727–731
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 34:1669–1678
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 289:223–234
Suzuki T, Fukagawa Y, Misawa M (1990) Enhancement of morphine withdrawal signs in the rat after chronic treatment with naloxone. Eur J Pharmacol 178:239–242
Yoburn BC, Nunes FA, Adler B et al (1986) Pharmacodynamic supersensitivity and opioid receptor upregulation in the mouse. J Pharmacol Exp Ther 239:132–135
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 913:170–173
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 267:238–244
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 33:351–355
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 45:149–153
Jenab S, Kest B, Inturrisi CE (1995) Assessment of delta opioid antinociception and receptor mRNA levels in mouse after chronic naltrexone treatment. Brain Res 691:69–75
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 323:614–625
Jenab S, Inturrisi CE (1994) Ethanol and naloxone differentially upregulate delta opioid receptor gene expression in neuroblastoma hybrid (NG108-15) cells. Mol Brain Res 27:95–102
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 105:887–895
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 21:1628–1637
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 64:32–41
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 319:765–775
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 60:1064–1075
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 40:12039–12050
Yamamura HI, Enna SJ, Kuhar MJ (1985) Neurotransmitter receptor binding. Raven Press, New York.
Unterwald EM (2008) Naltrexone in the treatment of alcohol dependence. J Addict Med 2:121–127
Unterwald EM, Rubenfeld JM, Kreek MJ (1994) Repeated cocaine administration upregulates kappa and mu, but not delta, opioid receptors. NeuroReport 5:1613–1616
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 92:7242–7246
Milligan G (2003) Principles: extending the utility of [35S]GTP gammaS binding assays. TIPS 24:87–90
Harrison C and Traynor JR (2003) The [35S]GTP gammaS binding assay: approaches and applications in pharmacology. Life Sci 74:489–508
Sim-Selley LJ and Childers SR (2002) Neuroanatomical localization of receptor-activated G proteins in brain. Methods Enzymol 344:42–58
Garcia-Jimenez A, Cowbuurn RF, Winblad B et al (1997) Autoradiographic characterization of [3535]GTPgammaS binding sites in rat brain. Neurochem Res 22:1055–1063
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 47:26–32
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 95:197–207
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 247:721–728
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2011 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Unterwald, E.M. (2011). Upregulating G Protein-Coupled Receptors with Receptor Antagonists. In: Stevens, C. (eds) Methods for the Discovery and Characterization of G Protein-Coupled Receptors. Neuromethods, vol 60. Humana Press. https://doi.org/10.1007/978-1-61779-179-6_20
Download citation
DOI: https://doi.org/10.1007/978-1-61779-179-6_20
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61779-178-9
Online ISBN: 978-1-61779-179-6
eBook Packages: Springer Protocols