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 thet_1/2 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.