Mast cells perform important functions in immediate-type hypersensitivity and inflammatory reactions. In addition to their role in pathological conditions, these cells may be essential in the maintenance of homeostasis of vascular and mucosal tissues. The details of how mast cells perform these functions are poorly understood. One approach to a clearer understanding is to characterize, in detail, the properties of individual substances stored in the cell's secretory granules. Thus, the involvement of these cells in hypersensitivity reactions has been firmly established by identifying the cell as a major source of vasoactive histamine and leukotrienes. Two other major mast cell products in the rat are distinct chymotrypsin-like proteases, RMCP I and RMCP II found uniquely in peritoneal and mucosal mast cells, respectively. The proposed research is designed to characterize the specificities of these proteases and ultimately identify their physiological substrates and inhibitors. Specifically, the study will test the hypothesis, based on experimental observations, that RMCP I has an intracellular function within peritoneal mast cells, perhaps in exocytosis and RMCP II, secreted by mucosal mast cells, functions to promote an increase in the permeability of epithelial barriers to macromolecules and cells, especially in parasite infections. The methodology used will include investigating the effect of highly effective and selective peptide chloromethylketone inhibitors of the proteases on mast cell exocytosis and mucosal permeability. Initially, studies will be performed in vitro, followed by in vivo experiments in rats. If positive results are obtained, experiments will be performed to identify, isolate, and characterize actual physiological targets of these proteases within mast cell membranes and on epithelial cells. Also, detailed substrate specificity studies will be performed with these enzymes using well characterized proteins and including type IV collagen of basement membrane and fibronectin. In addition, the presence of inactive precursor forms of the proteases will be investigated by recombinant DNA approaches in an attempt to understand how the proteolytic activities of these enzymes are regulated. The results of these studies should provide information leading to a clearer understanding of how mast cells function and, perhaps, ultimately to ways of controlling these cells in hypersensitivity and inflammatory secretions.
