Figure 10.

Phorbol ester does not rescue the PAO block. Blue bars depict the FM1-43 uptake of fertilized eggs (Strongylocentrotus purpuratus) treated with agatoxin-TK (100 nM), PAO (10 μM), or a combination of both inhibitors, and compares uptake with the uptake observed in control cells. FM1-43 (4 μM) was added at 30 minutes post-fertilization and eggs were incubated in the presence of FM1-43 and inhibitors for an additional 30 minutes. Eggs were then washed with ASW containing trypan blue (1 mg/ml) to quench any residual extracellular FM1-43 fluorescence and uptake was measured. Agatoxin was added with sperm, while PAO was added 2 minutes after the addition of sperm. Red Bars depict the same experiment outlined above but in the presence of 100 nM PMA added 5 minutes before fertilization and present up to the removal of FM1-43 at 60 minutes post-fertilization. All point represent mean±SEM, n=3 using eggs from three different animals each assayed in triplicate. Note the dramatic increase in FM1-43 uptake observed in eggs treated with agatoxin and PMA, and the lack of an increase in eggs treated with PAO and PMA.