GROWTH-DEPENDANT REGULATION OF LIPID AUTACOID PRODUCTION

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The goal of this project is to understand the mechanisms that regulate growth-dependent production of eicosanoids and platelet-activating factor by endothelial cells. Arachidonic acid metabolites have diverse, potent physiologic and pathophysiologic effects and may play important roles in many disease processes, including vascular disease. Platelet-activating factor (1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine; PAF) is a unique phospholipid that potently activates platelets and neutrophils and has dramatic hemodynamic effects. Production of this lipid by endothelial cells may be a mechanism to target inflammatory cells to an area of Injury. This may serve as a physiologic response to injury, or, if inappropriately regulated, as a mediator of pathologic vascular injury. The production of each of these potent lipid mediators by endothelial cells is tightly regulated and appears to have many common features, but our understanding of the mechanism of that regulation is incomplete. The specific aims of this project are to. characterize the cellular mechanisms by which extracellular stimuli cause release of arachidonic acid and production of PAF and an altered synthesis of these lipid autacoids at different growth states. It is appropriate to study both of these lipid mediators because in endothelial cells release of arachidonic acid occurs in response to the same stimuli that cause production of platelet-activating factor. In addition, the initial step for synthesis of both arachidonate metabolites and platelet-activating factor is catalyzed by a phospholipase A2. These studies will utilize cultured endothelial cells from human umbilical vein and bovine pulmonary artery. These have been chosen because the characteristics and responses of these cells in culture are well established in my laboratory. These responses include the ability to release arachidonate and produce PAF in response to specific mediators, including hormonal stimulation. Importantly, these cells demonstrate an altered regulation of arachidonate release and PAF production at different stages of growth in culture. This is an extremely useful characteristic that I will exploit to determine the mechanisms that regulate production of these two important lipids. I will examine several mechanisms for signal transduction that may be involved in these processes and the intracellular regulatory mechanisms that govern arachidonate release and PAF production. These studies will specifically address the roles of protein kinase C, extracellular calcium entry, changes in intracellular calcium concentration and phospholipase C activity in regulating production and release of these lipid mediators. In addition, because of the changes in lipid synthesis that occur at different growth states, related studies will examine the potential role of these lipids as autocrine regulators of cell growth.