Bovine protein kinase a

The cell lines, culture, and cAMP assay are all described in our previous work (Apostol et al., 2021 (link)). Briefly, human PAC1, VPAC1, and VPAC2 receptors expressed in CHO cells were used for all experiments. Cells were grown in 50:50 DMEM-F12 medium with 10% heat-inactivated fetal bovine serum, 1X penicillin-streptomycin supplement, and 500 μg/ml G418 (neomycin) to maintain selection (all from ThermoFisher) at 37°C with 5% CO₂ atmosphere. For the cAMP assay, cells were seeded at 20,000 cells/well in a 96 well plate, recovered overnight, then serum starved for 4 h. The cells were then incubated with 500 μM IBMX for 20 min, followed by agonist concentration curves in 500 μM IBMX media for 10 min. The reaction was terminated, boiled, then collected, followed by competition with 7 μg of bovine protein kinase A (Sigma-Aldrich) and ∼1 pmol of ³H-cAMP (PerkinElmer). The assay was incubated at room temp for 1 h, then collected onto GF/B filter plates and the data read using a MicroBeta2 scintillation counter (PerkinElmer). The resulting data was fit to a 3 variable non-linear regression curve using GraphPad Prism, which resulted in potency (EC₅₀) and efficacy (E_MAX) measurements. Native PACAP_1–27 was used as a positive control, and also used to define an E_MAX level of 100%.

This assay was also carried out as reported in our previous work [31 (link)]. First, 20,000 cells/well were plated in a 96-well plate in growth medium as above for 24 h. Then, cells were serum-starved in DMEM/F12 for 4 h and then incubated with 500 μM IBMX for 20 min. Stimulation buffer contained 500 μM IBMX and 50 μM forskolin, which is a known cAMP inducer. Serial dilutions of SNC80, a known reference DOR agonist, or test compounds were added in stimulation buffer for 10 min. Then, incubation mixtures were halted by adding ice-cold assay buffer and heating the plate at 80 °C for 10 min. The plate was centrifuged at 4,000 rpm for 10 min at 4 °C; then, supernatants were transferred into a new 96-well plate. The supernatants were co-incubated with 1 pmol of ³H-cAMP (PerkinElmer #NET1161250UC) and 7 μg of bovine protein kinase A (Sigma-Aldrich, St. Louis, MO, USA) in 0.05% bovine serum albumin for 1 h at room temperature. Then, the reactions were collected and measured as above. The data were normalized to cAMP suppression caused by vehicle (0%) or 10 μM SNC80 (100%) and fit to a 3-variable (Hill Slope = 1) agonist curve by GraphPad Prism 9.0.