cAMP assay

For the measurement of intracellular cAMP accumulation, 1x10⁵ cells were washed several times with HBSS and incubated in DMEM/F12 supplemented with 100 μM IBMX, 100 mg/ml L-ascorbic acid and different compounds for 1 h at 37°C in a 5% CO₂ atmosphere. The pharmacological agents used were 10⁻⁷ M MLT, 1 μM forskolin, 10⁻⁷ M MLT plus 1 μM forskolin or 1 μM luzindole. To assay MTR coupling to G_i/o, cells were pre-incubated with 5 ng/ml PTX for 3 h at 37°C and then incubated with 1 μM forskolin plus 10^-7M MLT for 1 h at 37°C. After washes with HBSS, cells were incubated with 50 μl of cold perchloric acid 2N for 30 min at room temperature. The pH was then neutralized with 50 μl KOH 1N. Culture dishes were centrifuged at 3,000 × g for 10 min at 4°C. Supernatants were collected and centrifuged at 10,000 × g for 7 min at 4°C. cAMP accumulation in the supernatant was then measured with a radioimmunoassay kit.