Relative potencies of opioid agonists in inhibiting forskolin-stimulated intracellular cAMP production for the cloned mouse δ-opioid receptor (δ-WT) and the D128N mutant and agonist (1 μM) pretreatment (3 hr) effects on opioid inhibition of forskolin-stimulated cAMP levels for the δ-WT stably expressed in HEK 293 cells
| Ligand | δ-Opioid receptor/mutant | |||||
|---|---|---|---|---|---|---|
| δ-WT | δ-WT agonist-pretreated cells | D128N mutant | ||||
| EC50 | Maximum inhibition | EC50 | Maximum inhibition | EC50 | Maximum inhibition | |
| nm | % | nm | % | nm | % | |
| DSLET | 0.02 ± 0.01 | 81.5 ± 2.2 | 0.4 ± 0.11-a | 64.0 ± 2.11-b | 12.4 ± 7.41-a | 74.3 ± 1.5 |
| DPDPE | 0.09 ± 0.01 | 88.3 ± 1.0 | 2.0 ± 1.21-a | 73.5 ± 4.11-b | 171. ± 961-a | 40.3 ± 7.71-c |
| DADLE | 0.03 ± 0.01 | 80.3 ± 2.4 | 1.5 ± 0.51-b | 54.2 ± 9.61-a | 2.8 ± 0.81-b | 71.8 ± 5.2 |
| Etorphine | 0.10 ± 0.01 | 84.4 ± 2.5 | 0.7 ± 0.11-c | 78.3 ± 3.3 | 0.07 ± 0.01 | 87.0 ± 1.2 |
| Levorphanol | 0.01 ± 0.01 | 89.3 ± 2.0 | 4.4 ± 1.51-b | 96.3 ± 0.31-a | 0.12 ± 0.08 | 85.3 ± 2.9 |
| SIOM | 2.2 ± 1.7 | 54.7 ± 7.2 | 1.2 ± 0.2 | 85.0 ± 6.11-a | 69.8 ± 2.11-c | 68.0 ± 11.4 |
| EKC | 8.7 ± 1.3 | 76.3 ± 1.2 | 40.4 ± 11.21-a | 90.0 ± 0.61-c | 13.1 ± 6.7 | 75.3 ± 2.6 |
| Morphine | 38.0 ± 2.1 | 54.3 ± 4.9 | 12.2 ± 5.61-a | 80.2 ± 1.71-a | 0.02 ± 0.011-c | 72.3 ± 2.41-a |
| Methadone | 41.2 ± 8.6 | 68.6 ± 5.0 | 137 ± 212 | 80.0 ± 5.5 | 4.3 ± 1.31-a | 91.0 ± 0.61-a |
The ability of various opioid agonists to inhibit the forskolin-stimulated intracellular cAMP accumulation and the effects of agonist pretreatment on cAMP accumulation were determined. Cell monolayers were either untreated (control) or treated (pretreated) for 3 hr at 37° with 1 μM concentration of the appropriate ligand (DSLET, DPDPE, DADLE, SIOM, EKC, etorphine levorphanol, morphine, or methadone). After a 30-min incubation with 0.5 mM isobutylmethylxanthine at 37°, cells were incubated with 10 μM forskolin and 1 μM concentration of an appropriate ligand for 5 min at 37° and then assayed for intracellular cAMP levels as described in the text. The pretreatment results were compared with results for cells that did not undergo pretreatment. The EC50 values were determined by nonlinear regression computer analysis of the dose-response curves generated using GraphPAD Prism 2. Maximal inhibition of forskolin-stimulated cAMP accumulation was that obtained at the 1 μM concentration and is expressed as a percentage of the forskolin control. Both treated and untreated results represent the mean ± standard error of at least three separate experiments, each performed and assayed in duplicate. Statistical significance (p < 0.05) was determined by a paired Student’s ttest.