The goal of this project is to understand how the production of eicosanoids (prostaglandins and leukotrienes) and platelet- activating factor are regulated in several cells relevant to cardiovascular disease. Eicosanoids are oxygenated metabolites of arachidonic acid that have diverse, potent effects on pathophysiological processes. Their production by cardiac cells (including the pericardium, ventricular fibroblasts, and endothelial cells) and by blood cells involved in cardiac injury and repair (neutrophils and monocytes/macrophages) may influence inflammation, blood flow, and electrical and mechanical properties in the diseased heart. Platelet-activating factor (PAF) is a unique phospholipid (1-0-alkyl-2-acetyl-sn-glycero-3- phosphocholine) that potently activates leukocytes and platelets, and has marked hemodynamic effects. Its production by endothelium could serve as a mechanism to target inflammatory cells to an appropriate area or, if incorrectly regulated, could result in vascular injury, thrombosis, and infarction. Both eicosanoids and PAF production are tightly regulated by cells, and the regulation has many common features.
The specific aims of this project are directed at describing the cellular mechanisms by which the initial step in both pathways, phospholipase activation, is achieved and how regulation is exerted at other steps in the pathways. The cells to be studied have been chosen for their relevance to cardiovascular disease and their suitability as experimental models for the relevant biochemical processes. We will examine several mechanisms for signal transduction that have been implicated by us and others in those processes, and will extensively utilize our finding of altered regulation at different times in culture as a probe for the regulatory mechanisms. Specific issues to be examined include the roles of protein kinase C, Ca flux, and G proteins in regulating PAF and eicosanoid production. The studies will use isolated cells and cultured cells that are metabolically labeled, or used as a source for enzymatic assays, followed by analytical procedures including chromatography, immunoassay, spectrometry, and electrophoresis.
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