Abstract
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 EMAX 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 EMAX 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 Bmax and increased the Kd in a dose-dependent nonlinear manner. Saturation binding studies with [3H]diprenorphine showed that Salvinorin A (10 and 40 μM) decreased the μ-receptor Bmax and increased the Kd 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.
Footnotes
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This work was supported by the Intramural Research Program of the National Institutes of Health and National Institute on Drug Abuse Grant R01 DA018151-01A2 (to T.E.P.).
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.106.113167.
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ABBREVIATIONS: CHO, Chinese hamster ovary cells; hMOR-CHO, CHO cells expressing the cloned human μ-opioid receptor; DAMGO, Tyr-d-Ala-Gly-N-Me-Phe-Gly-ol; herkinorin, (2S,4aR,6aR,7R,9S,10aS,10bR)-9-(benzoyloxy)-2-(3-furanyl)dodecahydro-6a,10b-dimethyl-4, 10-dioxo-2H-naphtho-[2,1-c]pyran-7-carboxylic acid methyl ester; [35S]GTPγS, guanosine 5′-O-(3-[35S]thio)triphosphate; U50,488H, (±)-trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]-benzeneacetamide; U69,593, 5α,7α,8β-(–)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro(4,5)dec-8-yl]-phenyl-benzeneacetamide; 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; SA, specific activity; MAPK, mitogen-activated kinase protein; KRBG, Krebs-Ringer bicarbonate buffer with glucose; [125I]IOXY, 6β-iodo-3,14-dihydroxy-17-cyclopropylmethyl-4,5α-epoxymorphinon.
- Received August 29, 2006.
- Accepted October 20, 2006.
- The American Society for Pharmacology and Experimental Therapeutics