This project combines circuit analysis and patch-clamp techniques, to measure the cell membrane expansion (increase in surface area) associated with exocytosis, in single isolated rat peritoneal mast cells. This study will focus on a quantitative determination of the time course of membrane expansion after stimulation. Exocytosis will be monitored at two levels: i) Macroscopic measurements accurately follow the time course of the overall increase in the membrane area. This time course is represented by three parameters a lag period d, where no sizeable area increase is detected after stimulation, a time constant that represents the secretory granule fusion rate after exocytosis begins and the ratio of the final to initial area, A, that reflects the extent of degranulation. Various activators and inhibitors of exocytosis will be tested for their effect on these parameters and mechanistic models that account for the observed effects will be attempted. ii) Microscopic measurements follow unitary events of secretory granule fusion which are to be represented as a distribution of amplitude, and fusion rates. These data will help establish the nature of the various types of unitary events observed (exocytosis, endocytosis, other). Existing theories of exocytosis claim the involvement of various enzymes like phospholipase A2 (PLA2), polyphosphoinositide phosphodiesterase (PDE) or messengers like Ca++ ions, arachidonic acid, lysolipids or physical factors like osmotic pressure. Since the patch-clamp technique, as used in this project, allows for intracellular as well as extracellular perfusion of the cell under study, enzyme inhibitors and products can be directly applied to the cytosol and their effect quantified as in i) and ii). The same is true for the cell's physical environment where osmotic gradients and temperature are easily controlled. In summary, the project will attempt a quantitative description of exocytosis at the single cell level and use it to test for various theories of exocytosis. These results will help understanding the mechanisms of Antigen induced release of inflammatory mediators as well as the mechanism of exocytosis in general.
