Different pathways of inhibitory effects of wortmannin on exocytosis are revealed by video-enhanced light microscope

Exocytosis of secretory granules, including histamine, in rat basophilic leukemia (RBL-2H3) cells, which exhibit Ca(2+)-dependent secretion of granules when stimulated with antigen or Ca(2+)-ionophore (A23187), was observed under a video-enhanced light microscope. Exocytotic events of individual granules including fusion, extrusion, and membrane retrieval were visualized in individual cells stimulated with antigen or A23187. Exocytosis of granules stimulated with A23187 showed two peaks in its time courses. The earlier one of the peaks was inhibited by wortmannin ( > 100 nM), as an inhibitor of myosin light chain kinase (MLCK), and the other was not. Exocytosis by antigen-stimulation, however, showed only one peak, which was inhibited by low concentration of wortmannin ( < 50 nM) as an inhibitor of phosphatidylinositol 3-kinase (PI3-kinase). These results indicate that quantitative analysis of exocytosis visualized by video-enhanced light microscope reveals two different pathways, through P13-kinase and MLCK, of inhibitory effects on exocytosis by wortmannin in RBL-2H3 cells.