The phospholipid composition of the outer leaflet of plasma membranes is a tightly regulated process that controls the cells interactions with its environment. Responses of adjacent cells to membrane phospholipids are modulated by both receptors and lipid metabolizing ecto-enzymes. This proposal will investigate the hypothesis that phosphatidic acid (PA) translocated to the outer surface of stimulated endothelial cell (EC) plasma membranes induces a novel signaling pathway in adjacent neutrophils that results in adherence, directed migration and functional activation. Activation of this pathway, we propose is mediated by cell surface receptors for PA and modulated by an ecto-phosphatidic acid phosphohydrolase (PAPase) on neutrophil plasma membranes. We will characterize neutrophil ecto-PAPase and determine its influence on functional responses of neutrophils to PA and related lipids. Experiments are designed to determine the relation of neutrophil ecto- PAPase to previously cloned plasma membrane PAPases in order to facilitate the development of methods to probe the function of ecto- PAPase in regulatory inflammatory responses. We will further explore the signal transduction pathway activated by PA in neutrophilic leukocytes, and define how initial events, including the activation of lyn, syk and phosphatidylinositol 3' kinase, lead to Ca++ mobilization and consequent metabolic activation. Finally, we will address the hypothesis that endogenously generated PA translocates under certain conditions to the outer leaflet of EC membranes, where it induces functional responses of adjacent neutrophils. We will explore conditions under which phospholipase D (PLD)- generated PA in EC translocates to the outer membrane and explore how neutrophil ecto-PAPase regulates responses to membrane-associated PA. The proposal focuses on the possibility, supported by preliminary data, that PA induces directed, chemotactic migration of neutrophils by promoting the generation of chemotactic gradients. Diacylglycerol generated as this gradient is being formed may regulate other responses, such as priming of the neutrophil superoxide generating system. The results of this study will provide important insights into the role of neutrophil ecto-PAPase in inflammation. They may also provide new insights into the involvement of EC PLD-generated PA in initiating the inflammatory response.
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