The enzyme 5-lipoxygenase (5-LO) catalyzes the first two steps in the metabolism of arachidonic acid to leukotrienes, substances which play pivotal roles both in normal host defense and in pathologic states of inflammation. Recent studies in myeloid cells have indicated that activation of 5-LO involves its Ca2+-dependent translocation to the membrane. By contrast, little information exists about the molecular regulation of 5-LO in macrophages (m- phi), even though these cells comprise the resident effector cell population of most organs. Preliminary studies from our laboratory suggest that, as compared to myeloid cells, alveolar m-phi (AM) exhibit important differences in the regulation of 5-LO activation and synthesis which are not shared by peritoneal m-phi (PM), and which reflect the fact that protein kinase C (PKC) is constitutively activated in AM. The specific hypotheses are that: 1) Resting AM display a previously unrecognized Ca2+-independent but PKC-dependent pathway for membrane association of 5-LO which results in augmented specific activity; and 2) AM synthesis of 5-LO exceeds that by PM, also as a consequence of PKC activation. The experimental approach entails comparing resident rat AM to resident PM. Leukotriene B4 synthesis by intact cells will be correlated with 5-LO specific activity and immunoreactivity in cytosol and membrane fractions of cell homogenates.
Aim I will utilize intact cells to determine the significance of PKC-dependent membrane association of enzyme, and cell-free mixtures of 5-LO and isolated m-phi membranes to determine the mechanisms responsible for membrane association.
Aim 2 will utilize cell-free systems to determine the relative importance of membrane association per se vs. activation of PKC in the regulation of 5-LO activation in m- phi; possible substrates for phosphorylation by PKC will also be examined.
In Aim 3, rates of synthesis and turnover of 5-LO in AM and PM will be compared, and the role of PKC activation in regulating these processes determined. In addition, the effects on enzyme synthesis of several substances expected to accumulate in the alveolar space under normal and pathophysiologic conditions will be examined. Elucidating the mechanisms by which expression of the m-phi 5-LO pathway is regulated will enhance our understanding of the inflammatory response and our ability to modulate it by pharmacologic and molecular approaches.
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