Involvement of sAC in CRH-mediated cAMP response. (A) sAC was detected by RT-PCR in total mouse brain extracts and different mouse cell lines. HPRT was used as control. (B) Depletion of sAC in HT22-CRHR1 cells transfected with siRNA against GL3 (control) or sAC. Western blot developed with sAC monoclonal antibody and total ERK1/2 as a loading control. (C–E) Time course of FRET changes was measured in single HT22-CRHR1-Epac-SH187 cells. Cells were stimulated at time 0 with CRH (C and E) or isoproterenol (D). tmAC-specific (100 µM ddA) or sAC-specific (20 µM 2-HE) inhibitors were added 1.5 min after stimulation. Traces are representative of three independent experiments (mean ± SEM, 20–25 cells). (E) Cells were transfected with 50 nM siRNA against GL3 (control) or sAC for 72 h. Inset shows efficiency of sAC knockdown assessed by quantitative RT-PCR normalized to HPRT (mean ± SEM, n = 3). ***, P < 0.001 by Student’s t test. (F) Inhibition of cAMP response elicited by CRH or forskolin at the indicated concentrations of ddA or 2-HE. Values represent FRET change 2.5 min after inhibitor addition relative to absence of any inhibitor (mean ± SEM, 15–20 cells).
