The initiation and control of prostaglandin production by the macrophage represents a fundamental problem in such important health-related areas as defense against infectious agents, inflammation and tissue repair, immunological reactions, and arteriosclerosis. Although this basic biological problem is being extensively studied from the biochemical point of view, little is known about the cell biology of prostaglandin production including the intracellular translocation, metabolism, and fate of membrane phospholipids which are a major source of the arachidonic acid used for prostaglandin production. The experimental approaches described in this proposal are directed at applying new methodology to this problem and involves the specific labeling of the cultured mouse peritoneal macrophage plasma membrane with well-characterized, defined phospholipid probes. These lipids, analogues of phosphatidylethanolamine, are labeled with a fluorescent and/or radioactive fatty acid, namely C6-NBD and (3H) arachidonic acid, respectively. Upon the introduction of such lipids into the macrophage plasma membrane by liposome-cell phospholipid exchange, the translocation and subsequent metabolism of the phospholipid will be studied in living and fixed cells and correlated with the stimulation and localization of 6-Keto-PGF1alpha, the stable metabolite of prostacyclin, a major prostaglandin produced by these cells. After macrophage are triggered to produce prostaglandins, living cells will be monitored and analyzed by fluorescence microscopy. Correlative electronmicroscopic localization of specific prostaglandin production will utilize autoradiography and immunohistochemistry of frozen, thin sectioned cells. The final objective of this study is to determine the fate and turnover of the exogenously introduced plasma membrane phospholipid under basal and stimulated condition by using membrane impermeant probes and biochemical analysis to assess the kinetics of the surface appearance of newly synthesized or recycled membrane phospholipid.
