CRH-triggered calcium response regulates cAMP. (A and B) HT22-CRHR1 cells cotransfected with Epac-SH187 and REX-GECO1 were stimulated with 100 nM CRH. (A) Representative images for each signal. Bar, 5 µm. (B) Time course of FRET changes (black trace) or REX-GECO1 fluorescence (orange trace). Data: mean ± SEM, 14 cells. (C) pERK1/2 and total ERK1/2 were measured in HT22-CRHR1 cells transfected with 50 nM siRNA against GL3 (control) or AC1 and stimulated with CRH. 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 by Student’s t test. Efficiency of AC1 knockdown assessed by quantitative RT-PCR normalized to HPRT is shown (mean ± SEM, n = 3). **, P < 0.01 by Student’s t test. (D) HT22-CRHR1-Epac-SH187 cells preincubated with vehicle (control), 5 µM BAPTA-AM, or 500 µM EGTA were stimulated at time 0 with CRH. Time course of FRET changes was measured in single cells (mean ± SEM, n = 3). (E) Calcium response was determined in HT22-CRHR1 cells pretreated with tmAC-specific (100 µM ddA) or sAC-specific (20 µM 2-HE) inhibitors, loaded with Fluo-4-AM, and stimulated at time 0 with 100 nM CRH (mean ± SEM, n = 3).
