The overall goal of this project is to identify and characterize the members of a novel class of biologically active molecules, oxidatively- fragmented phospholipids. We have discovered that oxidation of a prevalent cellular phospholipid generates potent (sub micromolar) activators of leukocyte function. Oxidation of phosphatidylcholine that contains an arachidonic acid residue in the second position, can fragment the arachidonoyl residue at double bonds to create shorter residue. Oxidation can be initiated either enzymatically, by an enzyme (xanthine oxidase) thought to play a major role in a variety of pathological states, or by a well characterized chemical reaction, ozonolysis. We use ozonolysis to generate a single, defined oxidized phospholipid that has an sn-2 residue that is only 5 carbon atoms long terminated with an aldehyde group (5- aldehydic-phosphatidylcholine or [5-AI]PC). We have characterized this product by a variety of physical techniques, so [5-AI]PC serves as our model compound. We have observed that the oxidation of synthetic phosphatidylcholine, or even human serum, byu xanthine oxidase generates new products that are biologically active, like [5-AI]PC itself. The fragment phosphatidylcholines, but not the intact molecule, stimulated neutrophil function and induced hypotension in a rabbit. Furthermore, it appears that the activation is receptor-mediated in that the oxidatively fragmented phosphatidylcholines activated a receptor that previously been considered to be specific for Platelet-activating Factor (PAF is an ether phosphatidylcholine with a short sn-2 acetate residue). The reaction that creates these oxidized phospholipids is an unregulated one that occurs in a variety of pathologic processes such as: injury due to reperfusion of ischemic heart, gut, brain and kidney; hemorrhagic shock; adult respiratory distress syndrome; inhalation injury; and, asbestosis. Oxidative reactions play a significant role in these processes as inhibition of xanthine oxidase, or removing or preventing oxidizing free radicals ameliorates tissue damage. Finally, PAF has been proposed to be involved based on the ability of PAF receptor antagonists to ameliorate these conditions. However, authentic PAF has not been detected in these pathological processes. Since all of these conditions involve oxidative processes, we propose that oxidatively fragmented phosphatidylcholines may be the actual mediators that activate leukocytes to increase tissue damage. In support of this, we find that oxidant-treated human endothelial cells bind PMN by an unknown mechanism that does not involve the synthesis of authentic PAF. There are three specific aims. 1) To identify oxidatively fragmented phosphatidylcholines, other than [5-AI]PC, that can activate leukocytes. We will isolate and identify the products of oxidation of synthetic phospholipids that stimulate leukocytes by a variety of physical methods, especially mass spectrometry. 2) To determine if a single receptor recognizes PAF and the various oxidatively fragmented phospholipids. This will use several structurally unrelated PAF receptor antagonists, and will take advantage of homologous desensitization of the PAF receptor by its agonists. 3) To determine if bioactive oxidatively-fragmented phospholipids occur in plasma, living cells nad tissue. This will rely on HPLC separation and identification of individual species by the methods developed in Aim 1.
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