Stimulation of guanosine-5'-o-(3-[35S]thio)triphosphate binding in digitonin-permeabilized C6 rat glioma cells: evidence for an organized association of mu-opioid receptors and G protein.
The guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS) 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 mu-opioid receptor. The mu-agonist [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO) caused a 3-fold increase in [35S]GTPgammaS binding over basal in a naloxone-sensitive manner. Relative mu-agonist efficacy was DAMGO > fentanyl > or = 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 mu cells were treated with the irreversible mu-antagonist beta-funaltrexamine (10 nM) prior to permeabilization. This reduced the number of mu-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]GTPgammaS binding stimulated by DAMGO was observed, but the t(1/2) for [35S]GTPgammaS 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 mu-receptors.
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Department of Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0632, USA.
McFadyen
I J
IJ
Fan
C D
CD
Woods
J H
JH
Traynor
J R
JR
D000701:Analgesics, Opioid / Q000494:pharmacology*; D000818:Animals; D004072:Digitonin / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)- / Q000494:pharmacology*; D019204:GTP-Binding Proteins / Q000187:drug effects* / Q000378:metabolism; D005910:Glioma; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism*; D007202:Indicators and Reagents / Q000494:pharmacology; D009270:Naloxone / Q000494:pharmacology; D009271:Naltrexone / Q000031:analogs & derivatives / Q000494:pharmacology; D009292:Narcotic Antagonists / Q000494:pharmacology; D051381:Rats; D017450:Receptors, Opioid, mu / Q000187:drug effects* / Q000378:metabolism; D014407:Tumor Cells, Cultured
D000701:Analgesics, Opioid; D007202:Indicators and Reagents; D009292:Narcotic Antagonists; D017450:Receptors, Opioid, mu; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D009270:Naloxone; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D009271:Naltrexone; C024524:beta-funaltrexamine; D019204:GTP-Binding Proteins; D004072:Digitonin