MECHANISMS OF HUMAN BASOPHIL & MAST CELL DESENSITIZATION

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Basophil and mast cell mediator release is a central feature of both acute and chronic allergic reactions. However, little is known of the biochemical nature of signal transduction in human cells of this type. This proposal focuses on studies which will elucidate some of the important biochemical events in both IgE mediated and univalent hormone induced secretion. The primary goal of these studies is to elucidate the mechanism of desensitization although significant effort will also be applied towards understanding activation events. In particular, the role of phospholipid metabolism and protein kinase C in activation and desensitization will be examined. we have recently demonstrated that IgE-mediated histamine release in both basophils and lung mast cells is accompanied by changes in phosphotidyinositol, increased activity of membrane associated protein kinase C and elevations in cytosolic calcium. It appears that protein kinase C activity increases (with IgE-mediated stimulation) even in the absence of external calcium suggesting that this enzyme may also play a role in the desensitization process of human basophils and mast cells. As such, we hypothesize that protein kinase C phosphorylates a 5'-phosphomonoesterase which in turn becomes active in metabolizing inositol trisphosphate to its inactive form. Since inositol trisphosphate is currently hypothesized to mediate elevations in cytosolic calcium this proposed scheme could account for the phenomenon of desensitization. To substantiate this model of desensitization measurements will be made of phospholipid turnover, in particular phosphotidyinositol, and the two side products of phospholipase C activation, inositol trisphosphate and diacylglercerol in an effort to delineate more accurately their role in activation. Studies will also involve measurements of protein kinase C activation and determine which proteins are phosphorylated by its activation, particularly under calcium-free buffer conditions. Phosphorylation of 5'- phosphomonoesterase will be directly examined as will its activity in desensitized basophils and mast cells.