sAC effectors in HT22-CRHR1 and AtT20 cells. (A,B,D–F) pERK1/2 and total ERK1/2 were measured in HT22-CRHR1 cells stimulated with CRH for the indicated time points. (A, B, and D) Cells preincubated with vehicle (control), PKA inhibitors (A: 700 nM KT5720; B: 75 µM RpcAMPS), or EPAC-specific inhibitor (5 µM ESI09) were stimulated with CRH. In D, results are expressed as the percentage of maximum pERK1/2 after stimulation under control conditions (mean ± SEM, n = 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001 by Student’s t test. (C) HT22-CRHR1 cells stably expressing AKAR4 were preincubated with tmAC-specific (100 µM ddA) or sAC-specific (20 µM 2-HE) inhibitors and stimulated with CRH at time 0. Time course of FRET changes was measured in single cells. Traces are representative of three independent experiments (mean ± SEM, 20–25 cells). (E and F) Cells were transfected with 50 nM siRNA against GL3 (control), EPAC1, or EPAC2 for 72 h before CRH stimulation. Efficiency of EPAC1 or EPAC2 knockdown assessed by quantitative RT-PCR normalized to HPRT is shown (mean ± SEM, n = 3). ***, P < 0.001 by Student’s t test. (G) Cells were transfected with EPAC1 (control) or dominant negative N-EPAC 48 h before CRH stimulation. (H and I) pERK1/2 and total ERK1/2 were measured in AtT20 cells preincubated with vehicle (control), PKA inhibitor (75 µM RpcAMPS; H), or EPAC inhibitor (10 µM ESI09; I) and stimulated with CRH for the indicated times.