The current proposal is aimed at an understanding of the mechanisms by which resting and stimulated human platelets and neutrophils regulate levels of free arachidonate. It Is generally accepted that cyclooxygenase and lipoxygenase can only utilize free, unesterified arachidonate as substrate. These enzymes oxygenate arachidonate to labile intermediates which can then be converted to biologically active compounds, such as the proaggregatory plateletderived thromboxane A2, involved in hemostasis, thrombosis and atherosclerosis. The regulation of levels of free arachidonate is also important for formation of other biologically active eicosanoids since a) endothelial cells utilize plateletderived arachidonate to generate anti- aggregatory prostacyclin, b) unstimulated neutrophils metabolize 12HETE (the platelet lipoxygenase product) to dihydroxy eicosanoids, and c) stimulated neutrophils produce chemotactic and proinflammatory leukotriene B4 from arachidonate liberated by stimulated platelets. I plan to investigate: 1) pathways of synthesis of arachidonate containing phospholipids (with emphasis on individual molecular species of the phospholipids involved) in resting and stimulated platelets and neutrophils; mechanisms by which neutrophils synthesize additional phospholipids upon stimulation, a process which may be necessary for neutrophil phagocytosis; in which respects platelets and neutrophils differ in their capability to synthesize phospholipids upon cell stimulation; the influence of albumin on i. production of eicosanoids, both by limiting availability of free arachidonate and by increasing the half life of labile intermediates, and ii. (re)synthesis of platelet and neutrophil phospholipids 2) the activity of lysophospholipase(s) in platelets and neutrophils, and how these enzymes may regulate levels of lysophospholipids, which are lytic to cells but can be utilized as substrate for formation of new phospholipids; 3) presence or absence of characteristic plasma membrane and endoplasmic reticulum marker enzymes in neutrophilderived cytoplasts to establish unequivocally whether plasma membrane and cytosol contain all the enzymatic equipment required for the lipid metabolic processes observed in stimulated cells; 4) identify specific subcellular sites of calcium storage and release upon cell activation as related to phosphoinositide metabolism, and the regulation of inositide metabolism. Most of the techniques for these studies (TLC, GLC, HPLC, radiochemical procedures and ultracentrifugation) are operational in the laboratory of the Principal Investigator.
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