The long term goal of this work is to elucidate the interrelated lipolytic pathways by which cells respond to both physiological and pathological stimuli. These pathways are key components of homeostatic mechanisms by which the endothelium regulates vascular function, producing vasodilators, preventing platelet aggregation, and modulating responses between inflammatory cells such as neutrophils and the vessel wall. One ongoing study seeks to test the hypothesis that enrichment of membrane phospholipids with eicosapentaenoate and/or docosohexaenoate will modulate the ability of the endothelium to synthesize vasoactive compounds in response to hormonal stimulation. The ability of endothelial cells to synthesize platelet-activating factor in response to hormones (e.g., bradykinin) or calcium ionophores appears to be modulated both by medium supplementation with polyunsaturated fatty acids and by pretreatment with phorbol esters. The effects of modifications in phospholipid fatty acid composition on PI-specific phospholipase C and PC-specific phospholipase D activity will be determined and correlated with those on PAF synthesis. These experiments will thus extend our understanding of the actions of omega-3 fatty acids on the vascular endothelium. The second ongoing study seeks to characterize the cellular effects of PGBx and aristolochic acid, two novel inhibitors of cellular phospholipases A2. In order to define the molecular mechanism of action of these novel effectors, cellular studies will examine the effects of PGBx and aristolochic acid on a) mobilization of arachidonic acid from major phospholipid pools, b) other agonist-stimulated enzymes including phospholipases C and D, and acetyltransferase, and c) cytosolic free calcium; results will be correlated with parallel in vitro studies on enzyme activities. Characterization of the specificity of these novel inhibitors will permit their use to prove the role of phospholipases A2 in complex lipolytic cascades.
