Cellular and Molecular Neurobiology

, Volume 31, Issue 6, pp 901–907 | Cite as

Inhibition of Morphine-Induced cAMP Overshoot: A Cell-Based Assay Model in a High-Throughput Format

  • Menghang Xia
  • Vicky Guo
  • Ruili Huang
  • Sampada A. Shahane
  • Christopher P. Austin
  • Marshall Nirenberg
  • Shail K. Sharma
Original Paper

Abstract

Opiates are not only potent analgesics but also drugs of abuse mainly because they produce euphoria. Chronic use of opiates results in the development of tolerance and dependence. Dr Marshall Nirenberg’s group at the National Institutes of Health (NIH) was the first to use a cellular model system of Neuroblastoma × Glioma hybrid cells (NG108-15) to study morphine addiction. They showed that opiates affect adenylyl cyclase (AC) by two opposing mechanisms mediated by the opiate receptor. Although the cellular mechanisms that cause addiction are not yet completely understood, the most observed correlative biochemical adaptation is the upregulation of AC. This model also provides the opportunity to look for compounds which could dissociate the acute effect of opiates from the delayed response, upregulation of AC, and thus lead to the discovery of non-addictive drugs. To identify small molecule compounds that can inhibit morphine-induced cAMP overshoot, we have validated and optimized a cell-based assay in a high throughput format that measures cellular cAMP production after morphine withdrawal. The assay performed well in the 1536-well plate format. The LOPAC library of 1,280 compounds was screened in this assay on a quantitative high-throughput screening (qHTS) platform. A group of compounds that can inhibit morphine-induced cAMP overshoot were identified. The most potent compounds are eight naloxone-related compounds, including levallorphan tartrate, naloxonazine dihydrochloride, naloxone hydrochloride, naltrexone hydrochloride, and naltriben methanesulfonate. The qHTS approach we used in this study will be useful in identifying novel inhibitors of morphine induced addiction from a larger scale screening.

Keywords

Adenylyl cyclase (AC) Adenosine-3′,5′-monophosphate (cAMP) Quantitative high-throughput screening (qHTS) μ Opioid receptor (morphine receptor) Human embryonic kidney293-μ opioid receptor cell line (HEK-MOR) Homogeneous time-resolved fluorescence (HTRF) 

Notes

Acknowledgments

This research was supported by the Laboratory of Biochemical Genetics (LBG), the National Heart Lung and Blood Institute, the National Institutes of Health, and the Molecular Libraries Initiative of the NIH Roadmap for Medical Research, and the Intramural Research Program of the National Human Genome Research Institute. The authors wish to thank Dr R. Balaban, Scientific Director, at NHLBI for providing the support for this study. SKS was a visiting senior scientist in LBG.

References

  1. Advokat C (1981) Naltrexone and the tail flick reflex. Pharmacol Biochem Behav 15:677–680PubMedCrossRefGoogle Scholar
  2. Ammer H, Schulz R (1993) Alterations in the expression of G-proteins and regulation of adenylate cyclase in human neuroblastoma SH-SY5Y cells chronically exposed to low-efficacy mu-opioids. Biochem J 295(Pt 1):263–271PubMedGoogle Scholar
  3. Avidor-Reiss T, Bayewitch M, Levy R, Matus-Leibovitch N, Nevo I, Vogel Z (1995) Adenylylcyclase supersensitization in mu-opioid receptor-transfected Chinese hamster ovary cells following chronic opioid treatment. J Biol Chem 270:29732–29738PubMedCrossRefGoogle Scholar
  4. Charles AC, Hales TG (2004) From inhibition to excitation: functional effects of interaction between opioid receptors. Life Sci 76:479–485PubMedCrossRefGoogle Scholar
  5. Collier HO, Roy AC (1974) Morphine-like drugs inhibit the stimulation of E prostaglandins of cyclic AMP formation by rat brain homogenate. Nature 248:24–27PubMedCrossRefGoogle Scholar
  6. El Kouhen R, Kouhen OM, Law PY, Loh HH (1999) The absence of a direct correlation between the loss of [d-Ala2, MePhe4, Gly5-ol]enkephalin inhibition of adenylyl cyclase activity and agonist-induced mu-opioid receptor phosphorylation. J Biol Chem 274:9207–9215PubMedCrossRefGoogle Scholar
  7. Fan P, Jiang Z, Diamond I, Yao L (2009) Up-regulation of AGS3 during morphine withdrawal promotes cAMP superactivation via adenylyl cyclase 5 and 7 in rat nucleus accumbens/striatal neurons. Mol Pharmacol 76:526–533PubMedCrossRefGoogle Scholar
  8. Hepburn MJ, Little PJ, Gingras J, Kuhn CM (1997) Differential effects of naltrindole on morphine-induced tolerance and physical dependence in rats. J Pharmacol Exp Ther 281:1350–1356PubMedGoogle Scholar
  9. Klee WA, Nirenberg M (1974) A neuroblastoma times glioma hybrid cell line with morphine receptors. Proc Natl Acad Sci USA 71:3474–3477PubMedCrossRefGoogle Scholar
  10. Koob GF, Bloom FE (1988) Cellular and molecular mechanisms of drug dependence. Science 242:715–723PubMedCrossRefGoogle Scholar
  11. Koshimizu TA, Tsuchiya H, Tsuda H, Fujiwara Y, Shibata K, Hirasawa A, Tsujimoto G, Fujimura A (2010) Inhibition of heat shock protein 90 attenuates adenylate cyclase sensitization after chronic morphine treatment. Biochem Biophys Res Commun 392:603–607PubMedCrossRefGoogle Scholar
  12. Kritikos PG, Papadaki PS (1967) The history of the poppy and of opium and their expansion in antiquity in the eastern Mediterranean area. Bull Narc 19:22Google Scholar
  13. Law PY, Loh HH (1999) Regulation of opioid receptor activities. J Pharmacol Exp Ther 289:607–624PubMedGoogle Scholar
  14. Law PY, Wong YH, Loh HH (2000) Molecular mechanisms and regulation of opioid receptor signaling. Annu Rev Pharmacol Toxicol 40:389–430PubMedCrossRefGoogle Scholar
  15. Loh HH, Smith AP (1990) Molecular characterization of opioid receptors. Annu Rev Pharmacol Toxicol 30:123–147PubMedCrossRefGoogle Scholar
  16. Nestler EJ, Aghajanian GK (1997) Molecular and cellular basis of addiction. Science 278:58–63PubMedCrossRefGoogle Scholar
  17. O’Brien CP (2005) Anticraving medications for relapse prevention: a possible new class of psychoactive medications. Am J Psychiatry 162:1423–1431PubMedCrossRefGoogle Scholar
  18. Sharma SK, Klee WA, Nirenberg M (1975a) Dual regulation of adenylate cyclase accounts for narcotic dependence and tolerance. Proc Natl Acad Sci USA 72:3092–3096PubMedCrossRefGoogle Scholar
  19. Sharma SK, Nirenberg M, Klee WA (1975b) Morphine receptors as regulators of adenylate cyclase activity. Proc Natl Acad Sci USA 72:590–594PubMedCrossRefGoogle Scholar
  20. Sharma SK, Klee WA, Nirenberg M (1977) Opiate-dependent modulation of adenylate cyclase. Proc Natl Acad Sci USA 74:3365–3369PubMedCrossRefGoogle Scholar
  21. Wang L, Gintzler AR (1994) Bimodal opioid regulation of cyclic AMP formation: implications for positive and negative coupling of opiate receptors to adenylyl cyclase. J Neurochem 63:1726–1730PubMedCrossRefGoogle Scholar
  22. Watts VJ (2002) Molecular mechanisms for heterologous sensitization of adenylate cyclase. J Pharmacol Exp Ther 302:1–7PubMedCrossRefGoogle Scholar
  23. Way EL (1967) Brain uptake of morphine: pharmacologic implications. Fed Proc 26:1115–1118PubMedGoogle Scholar
  24. Xia M, Guo V, Huang R, Inglese J, Nirenberg M, Austin CP (2009a) A cell-based beta-lactamase reporter gene assay for the CREB signaling pathway. Curr Chem Genomics 3:7–12PubMedCrossRefGoogle Scholar
  25. Xia M, Huang R, Guo V, Southall N, Cho MH, Inglese J, Austin CP, Nirenberg M (2009b) Identification of compounds that potentiate CREB signaling as possible enhancers of long-term memory. Proc Natl Acad Sci USA 106:2412–2417PubMedCrossRefGoogle Scholar
  26. Yu VC, Eiger S, Duan DS, Lameh J, Sadee W (1990) Regulation of cyclic AMP by the mu-opioid receptor in human neuroblastoma SH-SY5Y cells. J Neurochem 55:1390–1396PubMedCrossRefGoogle Scholar
  27. Zachariou V, Liu R, LaPlant Q, Xiao G, Renthal W, Chan GC, Storm DR, Aghajanian G, Nestler EJ (2008) Distinct roles of adenylyl cyclases 1 and 8 in opiate dependence: behavioral, electrophysiological, and molecular studies. Biol Psychiatry 63:1013–1021PubMedCrossRefGoogle Scholar
  28. Zhang L, Tetrault J, Wang W, Loh HH, Law PY (2006) Short- and long-term regulation of adenylyl cyclase activity by delta-opioid receptor are mediated by Galphai2 in neuroblastoma N2A cells. Mol Pharmacol 69:1810–1819PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Menghang Xia
    • 1
  • Vicky Guo
    • 2
  • Ruili Huang
    • 1
  • Sampada A. Shahane
    • 1
  • Christopher P. Austin
    • 1
  • Marshall Nirenberg
    • 2
  • Shail K. Sharma
    • 2
  1. 1.NIH Chemical Genomics Center, National Human Genome Research Institute, National Institutes of HealthBethesdaUSA
  2. 2.Laboratory of Biochemical Genetics, National Heart, Lung, and Blood InstituteNational Institutes of HealthBethesdaUSA

Personalised recommendations