This application proposes to continue to investigate how polymorphonuclear leukocyte (PMN) derived inflammatory molecules (IM) alter endothelial (endo) and epithelial (epi) barriers. The goals of the original application were to determine some mechanisms for the reversible effects of oxidants on endo barriers. We have also extended our observations to epithelium and we have investigated two additional groups of PMN IM, neutrophil cationic proteins and chlorinated amines. These observations have led to 2 hypotheses. 1. PMN derived IM initiate phospholipase C hydrolysis of inositol phospholipids in endo and epi cells, and the products of the hydrolysis cause changes in the cells' cytoskeletons that contribute to changes in barrier function. 2. Cytolytic doses of PMN derived IM initiate phospholipase A hydrolysis of phospholipids, increase cell membrane ionic permeability and deplete cell energy stores. The depleted energy stores preclude maintenance of normal intracellular ionic concentrations in the face of increased membrane ionic permeability. The depleted energy stores also preclude reesterification of phospholipids with resultant increases in free fatty acids and lysophosdpholipids which further increase membrane ionic permeability. These events lead to progressive cell dysfunction and cessation of normal activities. We will use monolayers of cells cultured on permeable supports, assays of metabolic pathways in cultured cells, and patch clamping of cultured cells and synthetic lipid bilayers to answer the following questions relevant to the hypotheses. 1. Do PMN derived IM activate phospholipase C hydrolysis of inositol phospholipids in endo and epi cells? How is phospholipase C activated? 2. Do PMN derived IM deplete ATP in endo and epi cells? How is phospholipase C activated? 3. What are some of the effects of PMN derived IM on the cytoskeleton and endo and epi cells? 4. Does accumulation of free fatty acid and lysophospholipid contribute to cell dysfunction in endo and epi cells exposed to PMN derived IM? What is the role of ATP in this process? 5. Do PMN derived IM alter the ionic permeability of endo and epi cell membranes? Do the IM alter the permeability of a synthetic lipid bilayer. Answers to these questions will increase our knowledge of how inflammation alters endo and epi barriers.
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