This proposal has two major goals: further understanding of mast cell activation and elucidating the structure and function of mast cell mediators with chemotactic activity. The first major goal will be addressed by developing populations of mast cells which reflect the heterogeneity of this cell type. Connective tissue mast cells will be obtained from rat serosal cavities and mucosal-type mast cells will be cultured from rat and mouse bone marrow utilizing IL-3 rich media to permit analysis of mast cell function and mediators across and within species. These mast cells populations will clarify whether differences noted in mast cell function and mediators are due to species differences or to true mast cell heterogeneity. These mast cells will then be utilized in studies of regulation of mediator release and, along with human lung mast cells, as a source of mast cell derived chemotactic mediators. Studies of mast cell regulation will focus upon the role of ecto Ca/Mg ATPases and cell surface adenosine receptors in modulation of stimulus-secretion coupling. Specific agonist and antagonist probes of ATPases and adenosine receptors will be examined for their ability to affect mast cells as assessed by mediator release, Ca++ homeostasis, Ca++ flux, phospholipid (esp. inositol phosphates) metabolism, protein phosphylation and receptor expression in short term (0-60 min) in vitro and in longer term (1-7d) studies in vivo and in tissue culture. Probes to be utilized include specific ATPase inhibitors such as DIDS (4',4-diisothiocyano - 2', 2 - disulfonic acid stilbene) which inhibits mediator release; ATP which causes mediator release, (as well as various nucleotide analogues); adenosine and its agonist analogues which enhance preformed mediator release; and methylxanthines which inhibit adenosine binding and its action. These studies will aid in developing further insights into the precise sites of action of these compounds and thereby into important elements of mast cell activation. Assessment of mast cell derived chemotactic factors will include their physiochemical characterizations and identification of their target cell specificity. After purification the ability of chemotactic factors to affect leukocyte production of inflammatory mediators will be determined and their ability to induce leukocyte accumulation in vivo assessed.
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