Blood leukocyte phospholipase play a crucial role in inflammation by mediating the generation of second messengers and active lipids. In neutrophils, agonist-stimulated activation of phospholipases A2 (PLA2s) initiates synthesis of leukotrienes, hydroxy fatty acids and platelet- activating factor. Despite the importance of cellular PLA2s, both our understanding of their mechanism(s) of activation and our interpretation of pharmacologic intervention studies are limited by the lack of specific inhibitors. This project examines the unique actio of two enzyme-targeted inhibitors of PLA2: aristolochic acid (a naturally occuring nitrophenanthrene derivative) and PGBx (an oligomer of prostaglandin B1). These agents inhibit the neutral-active, calcium-dependent neutrophil PLA2 in vitro and A23187-stimulated mobilization of cellular arachidonic acid. Neutrophil-like HL-60 cells will be used to compare the effects of the PLA2 inhibitors on: a) mobilization of arachidonic acid from major phospholipid pools, b) other agonist-stimulated lipolytic pathways including phospholipase C and D catalyzed turnover of inositol- and choline- phospholipids, and c) cytosolic free calcium. Key findings will be confirmed using human neutrophils. To determine the mechanism by which cells recover from inhibition by PGBx, the cellular binding and metabolism of PGBx will be examined. To assess specificity, results of cellular studies will be correlated with in vitro studies on phospholipase C and D, 5-lipoxygenase, acyl- and acetyl-transferase, and acetylhydrolase. Neutral- active, calcium-dependant PLA2 from HL-60 cells and neutrophils will be isolated, characterized, used to study interactions of inhibitors with - unified enzymes, and to generate polyclonal antibodies. In vitro and in situ actions of inhibitors will be correlated with their effects on human- PLA2-induced mouse-paw edema. These new PLA2 enzyme-directed probes should prove useful in studying the interrelated pathways of signal transduction and synthesis of active lipids, and role(s) of these events in the inflammatory response of the neutrophil. Specific inhibitors of PLa2 may have broad spectrum anti-inflammatory activity and be useful in the treatment of diverse pathological conditions including arthritis, shock and ischemic injury.
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