Abstract
A functional assay, based on aequorin-derived luminescence triggered by receptor-mediated changes in intracellular calcium levels, was used to examine relative potency and efficacy of the μ-opioid agonists endomorphin-1, endomorphin-2, morphiceptin, and their position 3-substituted analogs, as well as the δ-agonist deltorphin-II. The results of the aequorin assay, performed on recombinant cell lines, were compared with those obtained in the functional assay on isolated tissue preparations (guinea pig ileum and mouse vas deferens). A range of nine opioid peptide ligands produced a similar rank order of potency for the μ- and δ-opioid receptor agonists in both functional assays. The highest potency at the μ-receptor was observed for endomorphin-1, endomorphin-2, and [d-1-Nal3]morphiceptin, whereas deltorphin-II was the most potent δ-receptor agonist. In the aequorin assay, the μ- and δ-agonist-triggered luminescence was inhibited by the opioid antagonists naloxone and naltrindole, respectively. We can conclude that the use of the aequorin assay for new μ- and δ-receptor-selective opioid analogs gives pharmacologically relevant data and allows high-throughput compound screening, which does not involve radioactivity or animal tissues. This is the first study that validates the application of this assay in the screening of opioid analogs.
Footnotes
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This work was supported by a grant for Bilateral Scientific Cooperation between Flanders and Poland (BIL03/18). In addition, the authors gratefully acknowledge the Belgian Interuniversity Attraction Poles Programme (IUAP/PAI P5/30, Belgian Science Policy) and the Fonds voor Wetenschappelijk Onderzoek—Vlaanderen (FWO) for financial support. J. Poels is supported by the FWO as a postdoctoral research associate.
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doi:10.1124/jpet.105.099986.
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ABBREVIATIONS: GPI, guinea pig ileum; MVD, mouse vas deferens; GPCR, G protein-coupled receptor(s); CHO, Chinese hamster ovary; TFA, trifluoroacetic acid; BSA, bovine serum albumin.
- Received December 20, 2005.
- Accepted February 17, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
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