DIFFERENT PATHWAYS OF INHIBITORY EFFECTS OF WORTMANNIN ON EXOCYTOSIS ARE REVEALED BY VIDEO-ENHANCED LIGHT-MICROSCOPE

Exocytosis of secretory granules, including histamine, in rat basophil ic leukemia (RBL-2H3) cells, which exhibit Ca2+-dependent secretion of granules when stimulated with antigen or Ca2+-ionophore (A23187), was observed under a video-enhanced light microscope. Exocytotic events o f individual granules including fusion, extrusion, and membrane retrie val were visualized in individual cells stimulated with antigen or A23 187. Exocytosis of granules stimulated with A23187 showed two peaks in its time courses. The earlier one of the peaks was inhibited by wortm annin (>100nM), as an inhibitor of myosin light chain kinase (MLCK), a nd the other was not. Exocytosis by antigen-stimulation, however, show ed only one peak, which was inhibited by low concentration of wortmann in (<50nM) as an inhibitor of phosphatidylinositol 3-kinase (PI3-kinas e). These results indicate that quantitative analysis of exocytosis vi sualized by video-enhanced light microscope reveals two different path ways, through P13-kinase and MLCK, of inhibitory effects on exocytosis by wortmannin in RBL-2H3 cells. (C) 1996 Academic Press, Inc.