G protein-coupled receptor desensitization and trafficking are important regulators of opioid receptor signaling that can dictate overall drug responsiveness in vivo. Furthermore, different μ-opioid receptor (μOR) ligands can lead to varying degrees of receptor regulation, presumably because of distinct structural conformations conferred by agonist binding. For example, morphine binding produces a μOR with low affinity for β-arrestin proteins and limited receptor internalization, whereas enkephalin analogs promote robust trafficking of both β-arrestins and the receptors. Here, we evaluate μOR trafficking in response to activation by a novel μ-selective agonist derived from the naturally occurring plant product, salvinorin A. It is interesting that this compound, termed herkinorin, does not promote the recruitment of β-arrestin-2 to the μOR and does not lead to receptor internalization. Moreover, whereas G protein-coupled receptor kinase overexpression can promote morphine-induced β-arrestin interactions and μOR internalization, such manipulations do not promote herkinorin-induced trafficking. Studies in mice have shown that β-arrestin-2 plays an important role in the development of morphine-induced tolerance, constipation, and respiratory depression. Therefore, drugs that can activate the receptor without recruiting the arrestins may be a promising step in the development of opiate analgesics that distinguish between agonist activity and receptor regulation and may ultimately lead to therapeutics designed to provide pain relief without the adverse side effects normally associated with the opiate narcotics.

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 κ-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 μ-receptor binding, and subsequent experiments revealed that Salvinorin A partially inhibited μ-receptor binding. Therefore, we hypothesized that Salvinorin A allosterically modulates μ-receptor binding. To test this hypothesis, we used Chinese hamster ovary cells expressing the cloned human opioid receptor. Salvinorin A partially inhibited [³H]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 [³H]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 [³H]DAMGO showed that Salvinorin A (10 and 30 μM) decreased the μ-receptor B_max and increased the K_d in a dose-dependent nonlinear manner. Saturation binding studies with [³H]diprenorphine showed that Salvinorin A (10 and 40 μM) decreased the μ-receptor B_max and increased the K_d in a dose-dependent nonlinear manner. Similar findings were observed in rat brain with [³H]DAMGO. Kinetic experiments demonstrated that Salvinorin A altered the dissociation kinetics of both [³H]DAMGO and [³H]diprenorphine binding to μ receptors. Furthermore, Salvinorin A acted as an uncompetitive inhibitor of DAMGO-stimulated guanosine 5′-O-(3-[³⁵S]thio)-triphosphate binding. Viewed collectively, these data support the hypothesis that Salvinorin A allosterically modulates the μ-opioid receptor.