dmPGE₂ increases intracellular Ca²⁺ and cAMP concentrations followed by phosphorylation of Akt.

<p>(A) Intracellular calcium mobilization in response to dmPGE₂. SK-N-SH cells were loaded with the calcium fluorescent dye Fluo-4/AM before the addition of 1 µM dmPGE₂ or (B) pre-treatment with 2 mM EGTA before exposure to 1 µM dmPGE₂. The fluorescence intensity was followed using a confocal laser scanning microscope and representative single-cell recordings are shown. The arrows indicate when dmPGE₂ is added. (C) Intracellular accumulation of cAMP in response to dmPGE₂. SK-N-SH cells were incubated overnight in a medium without serum before the addition of 1 µM of dmPGE₂. Pretreatment with 10 µM NF 449, which is a Gαs inhibitor, before the incubation in dmPGE₂ for 10 min inhibited the production of cAMP. Forskolin, 10 µM for 10 min, was used as a positive control. The graph shows mean (±SD) in % of untreated control of three independent experiments. A statistical analysis was performed using 2-sided t-test, P<0.05. (D) PGE₂ induces phosphorlyation of Akt. SK-N-BE(2) and SK-N-SH cells were grown in the presence of serum (Ctr) before 24 h of culturing in the absence of serum (0 h) prior to the addition of 1 µM of dmPGE₂. Cells were further incubated in dmPGE₂ for 1, 2, 4, 6, 12 or 24 h and protein extracts were subjected to western blotting to detect phosphorylated Akt(ser473). An antibody detecting unphosphorylated Akt was used to exclude differences in total protein expression. β-actin was used to control for equal protein loading. The western blots are representative of three independent experiments.