Figure 1.

Forskolin and GPCR agonists induce CREB-dependent transcription independently of changes in CREB serine-133 phosphorylation. VSMC (n = 3) were transfected with CREB-LUC, serum starved for 4 h before being stimulated for 4 h with indicated doses of forskolin and assayed for luciferase activity (A). VSMC (n = 5) were serum starved for 4 h before being treated with forskolin for 30 min; cell lysates were analysed for phospho-VASP, phospho-CREB and total CREB by western blotting (B) and densitometric analysis (C; n = 5 and D; n = 4). VSMC were transfected with CREB-LUC, serum starved for 4 h before being stimulated for 4 h with indicated concentrations of adenosine (n = 4), BAY60-6583 (BAY, n = 6) or Cicaprost (n = 6) and assayed for luciferase activity (E). VSMC lysates were analysed by western blotting for phospho-VASP, pCREB and total CREB (F). VSMC were transfected with GAL4-NLUC together with either a GAL4, GAL4-CREB_wild-type or GAL4-CREB_S133A expression vector. Cells were serum starved for 4 hours before stimulation with 0.5 µM forskolin for 4 hours and subsequent nano-luciferase measurement (G, n = 5). *Indicates p < 0.05, **indicates p < 0.01, ***indicates p < 0.001 relative to control; one-way repeated measures ANOVA with Student Newman Keuls post-hoc tests.