cAMP Accumulation and βarrestin2 Recruitment

To determine cAMP accumulation and βarrestin2 recruitment by the human MOR, commercial enzyme fragment complementation assays (β-galactosidase) were used (Eurofins-DiscoverX, Fremont, California). PathHunter® CHO-K1 OPRM1 β-Arrestin cells were plated at a density of 5,000 cells per well of a 384-well, white-walled assay microplate in Assay Complete Cell Plating 2 Reagent (DiscoverX) overnight prior to measuring the signal. Cells were treated for 90-180 min with agonist at 37 ^oC and signal determined using the PathHunter Detection Kit to detect functional β-galactosidase. Test concentrations were established in each well with the 5-fold more concentrated samples, and were serial 3-fold reductions of the largest test concentration (100 µM). The resulting increase in luminescence was measured using an Envision^TM (PerkinElmer) microplate reader in relative light units (RLU). The control agonist is [Met]-enkephalin. Percentage activity is computed as 100% x (test agonist mean RLU - vehicle control mean RLU)/(control agonist maximum RLU – vehicle control mean RLU). Maximum control agonist RLU is 24-fold over vehicle control RLU.

cAMP Hunter™ CHO-K1 OPRM1 G_i cells were plated at a density of 10,000 cells per well of a 384-well and incubated as described for βarrestin2 recruitment. Cells were then stimulated for 30 min at 37 ^oC with agonist and 20 µM forskolin, or with 20 µM forskolin only. The same agonist test concentrations were employed as described for βarrestin2 recruitment. Test concentrations were established in each well with the 4-fold more concentrated samples. Following stimulation, signal was detected using the HitHunter cAMP Assay XS+ Detection Kit and luminescence measured as described for βarrestin2 recruitment. Maximum control agonist RLU is 13-fold over vehicle control RLU.