Relationship between rate and extent of G protein activation: comparison between full and partial opioid agonists.

Opioid agonists acting at their receptors alter intracellular events by initiating activation of various types of Gi/Go proteins. This can be measured by the binding of the stable GTP analog [(35)S]guanosine-5'-O-(3-thio)triphosphate ([(35)S]GTPgammaS). In this study agonist efficacy is defined by the degree to which an opioid stimulates the binding of [(35)S]GTPgammaS. This allows for a definition of full and partial agonists; a full agonist causing a greater stimulation of [(35)S]GTPgammaS binding than a partial agonist. The hypothesis that the rate of agonist-stimulated [(35)S]GTPgammaS binding is dependent upon agonist efficacy was tested using membranes from C6 glioma cells expressing mu- or delta-opioid receptors. At maximal concentrations the rate of agonist-stimulated [(35)S]GTPgammaS binding followed the efficacy of mu-agonists in stimulating [(35)S]GTPgammaS binding, i.e., [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin > morphine > meperidine > butorphanol > nalbuphine. At submaximal concentrations of mu- or delta-full agonists the [(35)S]GTPgammaS association rate was also reduced, such that the rate of [(35)S]GTPgammaS binding correlated with the extent of [(35)S]GTPgammaS bound, whether this binding was stimulated by a full agonist or a partial agonist. Agonists also stimulated [(35)S]GTPgammaS dissociation, showing that binding of this stable nucleotide was reversible. Comparison of the delta-agonists [D-Ser(2),Leu(5)]-enkephalin-Thr and (+/-)-4-((alpha-R*)-alpha-((2S*,5R*)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-hydroxylbenzyl)-N,N-diethylbenzamide, a compound with slow dissociation kinetics, showed the measured rate of G protein activation was not influenced by the agonist switching between receptors. The results are consistent with the idea that the active state(s) of the receptor induced by full or partial agonists is the same, but the number of activated receptors determines the rate of G protein activation.