Mast cells are the primary storage site in tissues for histamine. The histamine is stored in and released from abundant specific secretory granules. Thus mast cell storage and release of granules are critical in the pathogenesis of allergic diseases such as hay fever, hives, asthma and systemic anaphylaxis. It is the aim of this research to elucidate the mechanism for uptake and storage of histamine in the granules, to specify the molecular events that are responsible for the secretion of the granules and to identify the processes involved in the reconstitution of the cells complement of granules after secretion by which the cell is able to repeatedly secrete histamine and other granule components repeatedly. Proton NMR will be used to analyze the forces that determine uptake and storage of histamine. Imidazole C2-H and C4-H resonances will be studied. The chemical shift and line broadening of the signals from these two protons expressed by intracellular histamine will be correlated with comparable charges in model systems using isolated heparin, native and modified heparin protein complexes, and granule matrices. Studies on the mechanism of secretion will concentrate on protein phosphorylation: the sites of phosphorylated proteins and the kinases responsible. For studies of regulation of discharged mast cells, the cells will be identified and separated by fluorescent-activated cell sorting. The separated mast cells will be studied for the synthesis, structure and distribution of mast cell chymase and heparin, the activity of histidine decarboxylase and differences between histamine binding in regenerating and unstimulated cells. It is anticipated that the information provided by these studies will enhance the design of therapeutic agents capable of interupting the mechanism for release of histamine and other inflammatory mediators.
