Relative potencies of opioid agonists in inhibiting forskolin stimulated intracellular cAMP production for the cloned mouse μ-opioid receptor (μ-WT) and the D114N and μ-TRUNC mutants stably expressed in HEK 293 cells
| Ligand | MuOpioid Receptor/Mutant | |||||
|---|---|---|---|---|---|---|
| μ-WT | D114N mutant | μ-TRUNC mutant | ||||
| EC50(nM) | Max. inhibition (%) | EC50 (nM) | Max. inhibition (%) | EC50 (nM) | Max. inhibition (%) | |
| Morphine | 2.4 ± 1.1 | 71.7 ± 2.8 | 213. ± 722-a | 18.3 ± 11.42-b | ND | ND |
| Fentanyl | 0.15 ± 0.1 | 81.3 ± 1.9 | 0.8 ± 0.12-a | 54.3 ± 0.32-b | 0.53 ± 0.14 | 87.3 ± 0.32-a |
| Lofentanil | 0.03 ± 0.01 | 82.0 ± 1.8 | 0.3 ± 0.12-a | 74.7 ± 2.3 | 0.08 ± 0.1 | 84.7 ± 2.1 |
| Sufentanil | 0.3 ± 0.1 | 81.0 ± 1.0 | 0.5 ± 0.1 | 76.8 ± 2.4 | 0.002 ± 0.0012-b | 81.7 ± 2.6 |
| Nalbuphine | 1.5 ± 0.8 | 58.0 ± 5.6 | 3.1 ± 1.1 | 47.7 ± 4.6 | 14.4 ± 0.62-b | 42.7 ± 6.4 |
| Levorphanol | 1.0 ± 0.1 | 79.0 ± 2.4 | 25.3 ± 8.12-a | 42.5 ± 6.12-b | 0.38 ± 0.152-a | 77.7 ± 2.6 |
Agonist inhibition of cAMP accumulation by wild-type, D114N andmu-TRUNC mutant mu receptors stably expressed in HEK 293 cells. For generation of cAMP results, cell monolayers were treated for 30 min at 37°C with growth medium containing 0.5 mM IBMX. After treatment, the medium was replaced with growth medium containing agonist over the concentration range 10−12 to 10−6with 10 μM forskolin, incubated for 5 min at 37°C and then assayed for intracellular cAMP levels as described in “Methods.” 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 results represent the mean ± S.E. of at least three separate experiments, each performed and assayed in duplicate. Statistical significance (P < .05) was determined by a paired Student’s t test.
↵2-a P < .05 (Student’s t test, compared to wild-type).
↵2-b P < .01.
ND, Not determined.