Functional Characterization of Opioid Receptor Ligands by Aequorin Luminescence-Based Calcium Assay

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 -opioid agonists endomorphin-1, endomorphin-2, morphiceptin, and their position 3-substituted analogs, as well as the ␦-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 -and ␦-opioid receptor agonists in both functional assays. The highest potency at the -receptor was observed for endomorphin-1, endomorphin-2, and [D-1-Nal 3 ]morphiceptin, whereas deltorphin-II was the most potent ␦-receptor agonist. In the aequorin assay, the -and ␦-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 -and ␦-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.Current methods for the identification and characterization of new ligands at the three main opioid receptor types (, ␦, ) typically use binding assays, where a radioligand with high affinity to one receptor type is displaced by a tested analog. Apart from binding studies, different functional assays can also be used to determine relative potency and efficacy of new analogs at the opioid receptors. The most popular functional assay used for opioid activity determination in vitro is performed on isolated tissue preparations. This assay is based on inhibition of electrically evoked contractions of guinea pig ileum (GPI) and mouse vas deferens (MVD). The opioid effect in the GPI preparations is mainly mediated by the -receptors, whereas the predominant receptors of the MVD are of the ␦-type. The GPI/MVD assay makes it possible to determine the -and ␦-receptor interactions, which is the main focus in standard tests for opioid activity.In the present study, we used an alternative functional assay, which does not involve radioactivity or animal tissues and allows high-throughput screening of opioid peptide analogs. This assay is based on the recombinant mammalian cell line expressing an opioid receptor together with a luminescent reporter protein.Opioid receptors belong to a large family of the G proteincoupled receptors (GPCR), which on stimulation by a ligand induce the activation of the phospholipase C, with the subsequent generation of the primary second messenger metabolites: diacylglycerol and inositol 1,4,5-trisphosphate, followed by a release of calcium from intracellular stores. These agonist-induced receptor-mediated changes in calcium levels can be quantitatively assessed and ...

Opioid-induced regulation of µ-opioid receptor gene expression in the MCF-7 breast cancer cell line

“…Morphine and naloxone hydrochloride were purchased from Sigma-Aldrich (St.Louis, Mo., USA). Endomorphin-1 and endomorphin-2 were synthesized in our laboratory using a standard solid-phase method, described previously (Fichna et al 2006). …”

Natural Products as Tools for Neuroscience: Discovery and Development of Novel Agents to Treat Drug Abuse

“…Currently used cell-based assays are focused on the detection of intracellular Ca 2+ and use various fluorescence probes to measure levels of increased concentrations of calcium called FLIPR 157,158. Another available method uses aequorin-derived luminescence to monitor intracellular Ca 2+ levels 159. Aequorin is a photoprotein isolated from the jelly fish Aequorea victoria that has been used as a calcium indicator for more than three decades 160,161.…”

Characterization of Tachykinin‐related Peptides from Different Insect Species on Drosophila Tachykinin Receptor‐expressing Cell Line

“…The CHO cell line WTA11‐DTKR‐Aeq, permanently expressing both mitochondria‐targeted apoaequorin and the D. melanogaster DTKR, was obtained according to the modified method described earlier (12). The aequorin luminescence‐based calcium assay was performed as previously described (13). Non‐transfected CHO cells were used as control.…”