Activation profiles of opioid ligands in HEK cells expressing delta opioid receptors

Parham Gharagozlou, Hasan Demirci, J David Clark, Jelveh Lameh, Parham Gharagozlou, Hasan Demirci, J David Clark, Jelveh Lameh

Abstract

Background: The aim of the present study was to characterize the activation profiles of 15 opioid ligands in transfected human embryonic kidney cells expressing only delta opioid receptors. Activation profiles of most of these ligands at delta opioid receptors had not been previously characterized in vitro. Receptor activation was assessed by measuring the inhibition of forskolin-stimulated cAMP production.

Results: Naltrexone and nalorphine were classified as antagonists at delta opioid receptor. The other ligands studied were agonists at delta opioid receptors and demonstrated IC50 values of 0.1 nM to 2 microM, maximal inhibition of 39-77% and receptor binding affinities of 0.5 to 243 nM. The rank order of efficacy of the ligands tested was metazocine = xorphanol > or = fentanyl = SKF 10047 = etorphine = hydromorphone = butorphanol = lofentanil > WIN 44,441 = Nalbuphine = cyclazocine > or = met-enkephalin >> morphine = dezocine. For the first time these data describe and compare the function and relative efficacy of several ligands at delta opioid receptors.

Conclusions: The data produced from this study can lead to elucidation of the complete activation profiles of several opioid ligands, leading to clarification of the mechanisms involved in physiological effects of these ligands at delta opioid receptors. Furthermore, these data can be used as a basis for novel use of existing opioid ligands based on their pharmacology at delta opioid receptors.

Figures

Figure 1
Figure 1
Reproducibility of dose response curves for xorphanol at δ opioid receptors. This graph represents inter-experimental variability for dose response curves. Three independent experiments were carried out in duplicate. Varying concentrations of xorphanol were used to determine the potency and efficacy of xorphanol in inhibiting the effect of 5 μM forskolin in producing cAMP, as described under methods. Maximal cAMP levels were in the range of 400–1000 pmole/well. Data have been normalized as described under methods. Error bars represent standard error of the mean of duplicate measurements for each point.
Figure 2
Figure 2
Dose response curves of representative ligands on δ opioid receptors Varying concentrations of opioid ligands were used to determine the potency and efficacy of each ligand in inhibiting the effect of 5 μM forskolin in producing cAMP, as described under methods. Maximal cAMP levels were in the range of 400–1000 pmole/well. Data presented are the average data from 2 or more experiments carried out in duplicate. Data have been normalized as described under methods. Error bars represent standard error of the mean of the normalized data. (■) Etorphine, (□) Lofentanil, (▼) SKF 10047 and (●) Fentanyl.

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