The formation and/or exocytosis of inflammatory mediators by mast cells play central roles in immediate hypersensitivity reactions and contribute to 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 phosphatidic acid phosphohydrolase (PA-PHase) and phospholipase D (PLD) and the lipids they generate 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 during exocytosis. Data strongly suggest that the increased mass of the second messenger 1,2-diacylglycerol (DAG) after stimulating Fc- epsilon-RI in mast cells is largely derived from phosphatidylcholine (PC). We have also demonstrated the robust presence Fc-epsilon-RI dependent PLD activity. These observations are linked by data from our lab that strongly encourage the view that activation of PLD may culminate in an increase in mast cell associated DAG as the result of the receptor dependent stimulation of an Indirect Pathway involving both PLD and PH-PHase (PC-->PA-- >DAG). The proposed studies seek to characterize certain aspects of this pathway. Specifically, we propose studies seeking to: i) clone PLD using an expression based approach that takes advantage of a novel technical development made by our lab that permits detection of PLD in extremely low quantity and ii) to explore in biochemical, functional and pharmacologic terms the activity of mast cell PA- PHase and its role as the second step in the two step Indirect Pathway of receptor dependent DAG formation. With regard to PA- PHase, the direct or indirect effects of a variety of second messengers will be evaluated for their contributions to short term regulation, while the role of the varying cytokine milieu in regulating long term expression of PA-PHase will also be explored. These studies will generate insights pertaining to key elements of the Indirect Pathway of DAG formation and their subcellular location, regulation and likely roles in controlling physiologically important aspects of mast cell activation and/or the process of exocytosis. This knowledge will help to facilitate development of novel pharmacologic approaches to the therapy of allergic disorders.
