Rat basophilic leukemia (RBL) cells are being employed as a model system to explore the biochemical events involved in IgE receptor mediated exocytosis in mast cells. A major obstacle to studying the molecular events in mast cell activation is the inability to introduce compounds of interest into the cytoplasm in such a way as to preserve intracellular structures and the intracellular concentration of macromolecules. We have developed a method for permeabilizing RBL cells to small molecules (Mr less than 1000) using alpha toxin from S. aureus. The permeabilized cells respond to IgE receptor aggregation by activation of phospholipase C (PLC), resulting in hydrolysis of membrane phosphatidylinositol (PI), and release or histamine from secretory granules. We are characterizing IgE receptor mediated PI hydrolysis and exocytosis in permeabilized cell preparations. Both responses are independent on extracellular Ca2+ in permeabilized cells; however, intact cells require millimolar Ca2+ for both events. Both PI breakdown and exocytosis are inhibited by diisopropylfluorophosphate (DFP), an inhibitor of serine esterases. Using the permeabilized cells, effectors of the various steps involved in IgE receptor mediated exocytosis can be directly introduced into RBL cells. The purpose of this project is to identify events occurring between IgE receptor aggregation and PI hydrolysis, and to examine what role (if any) PI hydrolysis plays in exocytosis.
