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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM037758-02
Application #
3293430
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1986-09-01
Project End
1989-08-31
Budget Start
1987-09-01
Budget End
1988-08-31
Support Year
2
Fiscal Year
1987
Total Cost
Indirect Cost
Name
University of Iowa
Department
Type
Schools of Medicine
DUNS #
041294109
City
Iowa City
State
IA
Country
United States
Zip Code
52242
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Roberts, R L; Fine, R E; Sandra, A (1993) Receptor-mediated endocytosis of transferrin at the blood-brain barrier. J Cell Sci 104 ( Pt 2):521-32
Sandra, A; Cai, J (1991) Plasma membrane appearance of phosphatidylethanolamine in stimulated macrophages. J Leukoc Biol 50:19-27
Sandra, A; Marshall, S; Cai, J (1991) Plasma membrane phospholipid translocation in the mouse peritoneal macrophage: differential response to stimulation of eicosanoid production. Cell Mol Biol 37:565-73
Sandra, A; Mishra, B S (1991) Ultrastructural localization of arachidonic acid in stimulated macrophages. Prostaglandins Leukot Essent Fatty Acids 42:131-5
Turner, J W; Sandra, A (1989) Preparation of collagen gel matrices for light and electron microscopy. J Electron Microsc Tech 11:134-6
Bar, R S; Boes, M; Dake, B L et al. (1988) Insulin, insulin-like growth factors, and vascular endothelium. Am J Med 85:59-70