PHOSPHOLIPASE D AND MAST CELL ACTIVATION

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The formation and/or exocytosis of inflammatory mediators by mast cell play central roles in immediate hypersensitivity reactions and contribute to the development and/or perpetuation of chronic inflammation associated with both atopic and nonatopic inflammatory diseases. Our long range goal is to understand the function(s) of receptor regulated endogenous bioinformational lipid molecules in initiating, regulating and/or effecting mast cell mediator release. Toward this end, the proposed studies seek to explore the roles of phospholipase D (PLD) and the lipid product it generates, phosphatidic acid (PA), in both signal transduction and the direct regulation of membrane biochemical events involved in the fusion of perigranular membranes with each other and with the plasma membrane. Preliminary data indicate the robust presence in purified rat serosal mast cells of Fcepsilon-RI dependent PLD activity as assessed by two independent assays (one of which is novel). Although 1,2- diacylglycerol (DAG) is not the focus of the current proposal, data from our laboratory strongly suggest that PLD activation is important to the formation of a large fraction of the DAG accumulating as the result of the receptor dependent stimulation of an Indirect Pathway initiated by PLD (PC PLD> PA PA-PHase> DAG). The proposed studies seek to characterize receptor-dependent activation of mast cell PLD and to explore the regulatory importance of this pathway in exocytosis. Specifically, using two independent methods to quantitate cell associated PLD, we propose: i) to characterize in detail the changes in PLD activity in intact mast cells occurring as the result of physiologically relevant stimulation both by Fcepsilon-RI crosslinking and by nonimmunologic agonists; ii) to determine the subcellular site of PLD activation; and iii) to assess the role that PA plays in initiating, perpetuating and/or terminating exocytosis. A major common theme in each of these aims is determining the signal transducing element/second messenger/effector systems that participate in the regulation of PLD activity. Specifically, the role of GTP binding proteins, Ca+2, protein kinase C, cAMP/protein kinase A, tyrosine protein phosphorylation and the fatty acid products of phospholipase A2 will be carefully evaluated using a variety of agonists and antagonists chosen to maximally take advantage of the different mast cell model systems to be employed (intact mast cells, permeabilized cells, cytoplasts and isolated secretory granules and plasma membranes). These studies of the nature, regulation and subcellular location of PLD activation, the resultant formation of PA and the derivative generation of other bioinformationally important metabolites of PA (DAG and lyso-PA) -- processes tightly associated with the mast cell mediator release -- should contribute to a much clearer understanding of immunologic activation of mast cells. This knowledge will help to facilitate the development of novel pharmacologic approaches to the therapy of allergic disorders.