POLYUNSATURATED FATTY ACIDS AND CELL FUNCTION

The overall objective is to determine how polyunsaturated fatty acids (PUFA) influence cell properties and function. The current emphasis concerns the relationship between PUFA, lipid peroxidation, and the response of the intact cell to oxidative stress. Our goals are to identify the cell lipids that are especially susceptible to oxidation, determine if the PUFA content and composition can influence this process, and learn how cells respond to PUFA products generated as a result of lipid peroxidation. The work will be done with cultured cells, primarily the U937 monocyte/macrophage, but also with endothelial and vascular smooth muscle cells. Lipid radical formation will be detected by electron spin resonance (ESR) using spin traps such as alpha-[4]pyridyl 1-oxide]-N-tert-butyl nitrone (POBN). Cultures will be supplemented during growth with n-3 and n-6 PUFA to determine their influence on lipid radical formation. Using this approach, we have detected a spin adduct formed by intact U937 cells enriched with docosahexaenoic acid (DHA, 22:6n-3) in response to oxidative stress. This opens up a new experimental approach to study lipid radical formation by intact, functioning cells. There are five specific aims: (1) Complete characterization of lipid radical formation by U937 cells supplemented with DHA, including the cell lipids and organelles that are involved, and the effectiveness of different reactive oxygen species in initiating lipid peroxidation, (2) Determine whether other PUFA also increase lipid radical formation by U937 cells, with emphasis on the physiologically important n-3 and n-6 PUFA, (3) Determine the responses of the intact cells to lipid peroxidation, characterize the lipid products formed, and develop methods to protect the cells or minimize cell injury, (4) Study the effects of PUFA on the responses of endothelial and smooth muscle cells to oxidant stress, and (5) Test the effects of hydroxyoctadienoic acids (HODEs), and determine how vascular cells process these PUFA oxidation product. The results will provide new insight into the effects of PUFA and their oxidation products on vascular function and the atherogenetic process.