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Research ArticleCELLULAR AND MOLECULAR

Stimulation of Guanosine-5′-O-(3-[35S]thio)triphosphate Binding in Digitonin-Permeabilized C6 Rat Glioma Cells: Evidence for an Organized Association of μ-Opioid Receptors and G Protein

Andrew Alt, Iain J. McFadyen, Charles D. Fan, James H. Woods and John R. Traynor
Journal of Pharmacology and Experimental Therapeutics July 2001, 298 (1) 116-121;
Andrew Alt
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Iain J. McFadyen
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Charles D. Fan
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James H. Woods
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John R. Traynor
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Abstract

The guanosine-5′-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding assay for the determination of relative opioid efficacy has been adapted to measure G protein activation in digitonin-permeabilized C6 rat glioma cells expressing a cloned μ-opioid receptor. The μ-agonist [d-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO) caused a 3-fold increase in [35S]GTPγS binding over basal in a naloxone-sensitive manner. Relative μ-agonist efficacy was DAMGO > fentanyl ≥ morphine > buprenorphine. Nalbuphine showed no efficacy. G protein activation by receptors has been predicted to occur by random encounter. In this model a reduction in the number of receptors will decrease the rate of G protein activation but not the maximum number of G proteins activated. To test this model C6 μ cells were treated with the irreversible μ-antagonist β-funaltrexamine (10 nM) prior to permeabilization. This reduced the number of μ-opioid receptors determined with [3H]diprenorphine to 23 ± 3% of control with no change in affinity. A commensurate reduction (to 29 ± 10% of control) in the level of [35S]GTPγS binding stimulated by DAMGO was observed, but thet1/2 for [35S]GTPγS binding remained unchanged. Thus, random encounters of receptor and G protein failed to occur in this permeabilized cell preparation. A model that assumes an organized association of G proteins with receptors better describes the activation of G proteins by opioid μ-receptors.

Footnotes

  • This work was supported by National Institutes of Health Grants R01 DA02265 and DA00254.

  • Abbreviations:
    G protein
    GTP-binding protein
    GTPγS
    guanosine-5′-O-(3-thio)triphosphate
    DAMGO
    [d-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin
    β-FNA
    β-funaltrexamine
    CI
    confidence interval
    • Received November 2, 2000.
    • Accepted March 16, 2001.
  • The American Society for Pharmacology and Experimental Therapeutics
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Journal of Pharmacology and Experimental Therapeutics: 298 (1)
Journal of Pharmacology and Experimental Therapeutics
Vol. 298, Issue 1
1 Jul 2001
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Research ArticleCELLULAR AND MOLECULAR

Stimulation of Guanosine-5′-O-(3-[35S]thio)triphosphate Binding in Digitonin-Permeabilized C6 Rat Glioma Cells: Evidence for an Organized Association of μ-Opioid Receptors and G Protein

Andrew Alt, Iain J. McFadyen, Charles D. Fan, James H. Woods and John R. Traynor
Journal of Pharmacology and Experimental Therapeutics July 1, 2001, 298 (1) 116-121;

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Research ArticleCELLULAR AND MOLECULAR

Stimulation of Guanosine-5′-O-(3-[35S]thio)triphosphate Binding in Digitonin-Permeabilized C6 Rat Glioma Cells: Evidence for an Organized Association of μ-Opioid Receptors and G Protein

Andrew Alt, Iain J. McFadyen, Charles D. Fan, James H. Woods and John R. Traynor
Journal of Pharmacology and Experimental Therapeutics July 1, 2001, 298 (1) 116-121;
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