Measurement of cAMP Production and Inositol 1,4,5-Trisphosphate (IP3) Concentration

The pituitary NIL cells were seeded at a density of ~1 × 10⁶ cells/2 ml/dish in 35-mm dishes precoated with poly-D-lysine and cultured overnight at 28°C. Following overnight incubation, the old medium was replaced with 1.8 mL HHBSA (Hank’s balanced salts solution with 25 mM HEPES, 0.1% BSA) medium (Gibco) supplemented with IBMX (0.1 mM) and incubated for 30 min before adding 0.2 mL of 10 × stock solutions of GCGb at 28°C. The duration of the GCGb treatment was fixed at 30 min. After that, cAMP production was quantified using a cAMP ELISA kit (EIAab Science Co., Ltd, Wuhan, China) as previously described (32). The assay for cAMP had a detection sensitivity range of 40–2,500 pmol/L. The intra-assay coefficient of variation was 10%. In parallel studies, the production of IP3 was determined on NIL cells using general inositol 1,4,5,-trisphosphate (IP3) ELISA kit according to the manufacturer’s protocol (CusaBio, Wuhan, China) with a sensitivity range of 5–1,000 pg/mL and intra-assay coefficient of variation was less than 15%.