Salvinorin A:: Allosteric interactions at the μ-opioid receptor

Salvinorin A [(2S, 4aR, 6aR, 7R, 9S, 10aS, 10bR)-9-(acetyloxy)-2(3-furanyl)-dodecahydro-6a, 10b-dimethyl-4,10-dioxo-2h-naphtho[2,1-c] pyran-7-carboxylic acid methyl ester] is a hallucinogenic kappa-opioid receptor agonist that lacks the usual basic nitrogen atom present in other known opioid ligands. Our first published studies indicated that Salvinorin A weakly inhibited mu-receptor binding, and subsequent experiments revealed that Salvinorin A partially inhibited mu-receptor binding. Therefore, we hypothesized that Salvinorin A allosterically modulates mu-receptor binding. To test this hypothesis, we used Chinese hamster ovary cells expressing the cloned human opioid receptor. Salvinorin A partially inhibited [H-3] Tyr-D-Ala-Gly-N-Me-Phe-Gly-ol (DAMGO) (0.5, 2.0, and 8.0 nM) binding with E-MAX values of 78.6, 72.1, and 45.7%, respectively, and EC50 values of 955, 1124, and 4527 nM, respectively. Salvinorin A also partially inhibited [H-3] diprenorphine (0.02, 0.1, and 0.5 nM) binding with E-MAX values of 86.2, 64, and 33.6%, respectively, and EC50 values of 1231, 866, and 3078 nM, respectively. Saturation binding studies with [H-3] DAMGO showed that Salvinorin A (10 and 30 mu M) decreased the mu-receptor B-max and increased the K-d in a dose-dependent nonlinear manner. Saturation binding studies with [H-3] diprenorphine showed that Salvinorin A (10 and 40 mu M) decreased the mu-receptor B-max and increased the K-d in a dose-dependent nonlinear manner. Similar findings were observed in rat brain with [H-3] DAMGO. Kinetic experiments demonstrated that Salvinorin A altered the dissociation kinetics of both [H-3] DAMGO and [H-3] diprenorphine binding to mu receptors. Furthermore, Salvinorin A acted as an uncompetitive inhibitor of DAMGO-stimulated guanosine 5'-O-(3-[S-35]thio)-triphosphate binding. Viewed collectively, these data support the hypothesis that Salvinorin A allosterically modulates the mu-opioid receptor.