Figure 1.

a, Induction of CREB-dependent gene expression by cAMP requires Ca²⁺. Stimulation of 4xCRE luciferase activity by forskolin (10 μm) or 8-Br-cAMP (3 mm) was abolished by adding 3 mm Bapta-free acid (Bapta-FA) or by pretreating with Bapta-AM (10 μm). EGTA (3 mm) also blocked stimulation of 4xCRE by cAMP (data not shown). In most experiments, Bapta-FA was used instead of EGTA to chelate extracellular Ca²⁺ to avoid acidification, which occurs when 3 mm EGTA is added to Ca²⁺-containing medium. ***Significantly different from forskolin or 8-Br-cAMP alone (p < 0.001). b, Ca²⁺ is not required for stimulation of cAMP production by forskolin. The forskolin-induced increase in cAMP levels, determined by ELISA, was blocked by the PKA antagonist, H89 (10 μm, data not shown) but was not affected by pretreatment with Bapta-AM (10 μm). ns, Not significantly different from forskolin alone.