Fig. 4.

Extracellular Ca²⁺-dependency of agonist-stimulated CREB phosphorylation in SH-SY5Y cells. Representative [Ca²⁺]_i traces for responses stimulated by MCh (100 μM; A) and bradykinin (30 μM; B) in Fura-2AM-loaded cells. Experiments were performed in normal KHB (1.3 mM $\text{C}{\text{a}}_{\text{e}}^{\text{2+}}$) or under conditions where $\text{C}{\text{a}}_{\text{e}}^{\text{2+}}$ was depleted prior to agonist addition. The effects of the manipulation of Ca²⁺ gradients on agonist-stimulated changes in phospho-CREB immunoreactivity were assessed for MCh (1 μM; 1 or 5 min; C) or bradykinin (300 nM; 1 and 5 min; D). For each panel representative phospho-CREB, total CREB and γ-tubulin blots are shown. Responses were normalized with respect to the response to forskolin (10 μM)/IBMX (500 μM) addition for 10 min (Fk) in normal KHB (=100%) and are shown as means ± S.E.M. for at least 3 separate experiments. Statistically significant differences between agonist-stimulated responses under the different Ca²⁺ conditions are indicated as ^*p < 0.05 or ^***p < 0.001.