sAC-generated cAMP has a specific role in CRHR1 signaling. (A–C) pERK1/2 and total ERK1/2 were measured in HT22-CRHR1 cells stimulated with CRH or isoproterenol for the indicated time points. Results are expressed as the percentage of maximum pERK1/2. (A) Cells were preincubated with vehicle (control) or sAC-specific (7.5 µM KH7) or tmAC-specific (100 µM ddA) inhibitors (mean ± SEM, n = 4). Two-way analysis of variance followed by Tukey’s test: *, P < 0.05; **, P < 0.01; ***, P < 0.001 with respect to control; #, P < 0.05; ###, P < 0.01 between indicated treatments. (B) Time course of FRET changes was measured in single HT22-CRHR1-Epac-SH187 cells. Cells were stimulated at time 0 with CRH. When indicated, tmAC-specific (100 µM ddA) or sAC-specific (20 µM 2-HE) inhibitors were added. Traces are representative of three independent experiments (mean ± SEM, 20–25 cells). (C) Cells were transfected with 50 nM siRNA against GL3 (control) or sAC for 72 h before stimulation with CRH or isoproterenol (mean ± SEM, n = 3). Student’s t test was performed for each time point. *, P < 0.05; ***, P < 0.001. 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. (D) CRH neurogenic effect in HT22-CRHR1 cells after 20-h treatment was evaluated in the presence of vehicle (control) or tmAC-specific (50 µM ddA) or sAC-specific (7.5 µM KH7) inhibitors. A representative photograph is shown. Bar, 50 µm. Neurite outgrowth was quantified by repeated-measures one-way analysis of variance followed by Tukey’s test (n = 3). ***, P < 0.001; **, P < 0.01 with respect to basal.
