CRH responses in AtT20 and 3T3L1-CRHR1 cells. (A) pERK1/2 and total ERK1/2 were measured in AtT20 cells preincubated with vehicle (control) or tmAC-specific (50 µM ddA) or sAC-specific (7.5 µM KH7 or 10 µM 2-HE) inhibitors and stimulated with CRH. (B) pERK1/2 and total ERK1/2 were measured in AtT20 cells transfected with 50 nM siRNA against GL3 (control) or sAC and stimulated with CRH or PACAP38. Results are expressed as the percentage of maximum pERK1/2 (mean ± SEM, n = 3). *, P < 0.05 by Student’s t test. Efficiency of sAC knockdown assessed by quantitative RT-PCR normalized to HPRT is shown (mean ± SEM, n = 3). ***, P < 0.001 by Student’s t test. (C) Response to CRH in AtT20 cells transiently transfected with Pomc–Luc and pretreated with 7.5 µM KH7 or 10 µM 2-HE. Each value was normalized to β-Gal activity (mean ± SEM, n = 4 of one representative of three independent experiments with similar results). ***, P < 0.001; *, P < 0.05 with respect to control by two-way analysis of variance followed by Tukey’s test. (D) pERK1/2 and total ERK1/2 of HT22-CRHR1, AtT20, or 3T3L1-CRHR1 cells treated with 8-CPT-cAMP. (E,F) 3T3L1-CRHR1 cells expressing Epac-S^H187 were stimulated at time 0 with CRH. (F) Cells were transfected with sACt-HA. When indicated, 100 µM ddA or 20 µM 2-HE was added. Time course of FRET changes were measured in single cells. Traces are representative of three independent experiments (mean ± SEM, 20–30 cells).