The role of calcium in BeWo syncytialization.

<p><b>A</b>) Fura red was used as an inverse intracellular calcium indicator and calcium transients were measured in real time utilizing Flow Cytometry. Fura Red was measured with the perCP channel at 488 nm. The top panel shows the effects of ionomycin 1 µM on the calcium transient with a decrease in intensity as intracellular calcium increases. The second panel shows the increase in fura red as calcium levels are decreased by EGTA. In the third and fourth panels, real time decreases in intracellular calcium are seen with both addition of cAMP (8-bromo-cAMP 1.5 mM - third panel) and LY294002 (50 µM - fourth panel). <b>B</b>) Effects of thapsigargin (100 nM) on BeWo fusion were determined by incubating the BeWo cells with thapsigargin in the presence or absence of 30 µM forskolin for 24 h. Thapsigargin completely abolished the presence of double positivity assessed by flow cytometry. C) Cells were incubated with vehicle (control, open bar) or with 30 µM forskolin for 24 hr (black bars). The incubations were repeated in the presence of nifedipine (10 µM) or in the presence of nifedipine (10 µM) and Akt Inhibitor VIII (Akt I at 10 µM). Nifedipine alone did not influence fusion, but nifedipine and Akt Inhibitor VIII increased fusion in both control and forskolin treated cells.