MECHANISMS THAT REGULATE ENZYMES THAT METABOLIZE CIS UNSATURATED FATTY ACIDS

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Summary of Work: The goal is to develop an understanding of the biochemical mechanisms responsible for the metabolism of arachidonic acid and linoleic acid by these enzymes. Nitric oxide (NO) is reported to enhance prostaglandin formation by enhancement of PGHS activity. We found that NO is a substrate for the peroxidase of PGHS-1 and -2 but it does not enhance the cyclooxygenase activity in vitro. In macrophages, NO does not stimulate the expression of PGHS or alter LPS dependent PGHS-2 expression. Furthermore, NO did not enhance prostaglandin formation in intact cells. We propose that peroxynitrite, a reaction product of NO and superoxide oxide is responsible. Other studies with PGHS-1 indicate that a tyrosyl radical is formed which could be the reactive intermediate that initiates arachidonic acid oxygenation. Recently, we have shown that NO reacts with the tyrosyl radical converting the radical to a new radical characterized by ESR analysis as an iminoxyl radical. This leads to the formation of nitrotyrosine which is often used as an indicator of oxidative stress. The metabolism of arachidonic acid and linoleic acid was studies in several human breast cell lines. Linoleic acid was metabolized to 13(S)-HpODE by BT-20 breast cells after treatment of serum starved cells with TGFa. Molecular and sequence analysis confirm the presence of human15-lipoxygenase in these cells. Since 13(S)-HpODE up- regulates the EGFR pathway, these findings suggest a role of 15- lipoxygenase in the regulation of breast cell growth. Understanding the biochemical mechanisms which regulate the activities and expression of these enzymes will provide new insights into potentially controlling or preventing these disease conditions.