The experimental plan formulated in this research proposal is aimed at elucidating signalling mechanisms involved in the activation of cellular phospholipase A2 (PLA2) following membrane lipid peroxidation. The plan focuses on the well known Ca2+ requirement of PLA2 and develops from observations where enzyme activity is increased and Ca2+ dependency reduced in the presence of peroxidized membranes. We will concentrate our attention to a specific cytosolic PLA2 that appears to account for most of the hydrolysis of cell phospholipids immediately after induction of membrane lipid peroxidation. Our approach will be to utilize an oxidizing system that induces lipid peroxidation principally by free radical propagation reactions involving membrane phospholipids. The response of PLA2 will then be characterized under conditions where membrane lipid peroxidation is the prevalent oxidative reaction in cells. We will test the hypothesis that peroxidative activation of PLA2 is mediated by Ca2+ dependent signalling events that facilitate protein kinase C (PKC) activity and its directed phosphorylation of cytosolic PLA2. We will attempt to verify that the transient rise in intracellular free Ca2+ levels is responsible for the translocation, membrane binding and stabilization of PKC which in turn phosphorylates PLA2. We will then determine if the binding and activity of PLC is thereby increased and sustained towards membranes that contain oxidized phospholipids.
Specific Aims - Our experimental approach involves measurement of PLA2 phosphorylation and hydrolytic activity as mediated by PKC. This measurement will be made in model membranes subjected to lipid peroxidation and the experimental conditions developed therein extended to cultured endothelial cells. l) Using unilamellar phospholipid vesicles as model membranes we will compare the activity, substrate specificity and Ca2+ dependency of snake venom, pancreatic and cytosolic PLA2 (c-PLA2) against oxidized vs unoxidized vesicles. Using these vesicles we will measure PKC-mediated phosphorylation and activity of c-PLA2 and its Ca2+ dependency and determine if suppression of lipid peroxidation also inhibits PKC activity and/or the activities of the various PLA2's. 2) Using endothelial cells we will ascertain if the Ca2+ requirement of PLA2 is altered after peroxidation and is determined by the level of membrane lipid peroxidation. We will also determine if the transient rise in intracellular free Ca2+ is dependent on inositol trisphosphate and diacylglycerol formation. 3) We will verify that the Ca2+ signalling and enzyme activation process is dependent on lipid peroxidation by modulating the extent of peroxidation in cells via the levels and/or activities of key antioxidants involved in preventing lipid peroxidation, ie. vitamin E and glutathione peroxidase. 4) effects of peroxidation on PKC activity and PLA2 phosphorylation and hydrolytic activity will be studied in endothelial cells from the standpoints of their Ca+ requirement and modulation by key antioxidants.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
2R01HL045206-04
Application #
2222001
Study Section
Pathology A Study Section (PTHA)
Project Start
1991-04-05
Project End
1998-04-30
Budget Start
1994-05-01
Budget End
1995-04-30
Support Year
4
Fiscal Year
1994
Total Cost
Indirect Cost
Name
University of Southern California
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
041544081
City
Los Angeles
State
CA
Country
United States
Zip Code
90089
McLeod, L L; Sevanian, A (1997) Lipid peroxidation and modification of lipid composition in an endothelial cell model of ischemia and reperfusion. Free Radic Biol Med 23:680-94
Rashba-Step, J; Tatoyan, A; Duncan, R et al. (1997) Phospholipid peroxidation induces cytosolic phospholipase A2 activity: membrane effects versus enzyme phosphorylation. Arch Biochem Biophys 343:44-54
Sweetman, L L; Zhang, N Y; Peterson, H et al. (1995) Effect of linoleic acid hydroperoxide on endothelial cell calcium homeostasis and phospholipid hydrolysis. Arch Biochem Biophys 323:97-107
Watson, A D; Navab, M; Hama, S Y et al. (1995) Effect of platelet activating factor-acetylhydrolase on the formation and action of minimally oxidized low density lipoprotein. J Clin Invest 95:774-82
Verity, M A; Sarafian, T; Pacifici, E H et al. (1994) Phospholipase A2 stimulation by methyl mercury in neuron culture. J Neurochem 62:705-14
Pacifici, E H; McLeod, L L; Sevanian, A (1994) Lipid hydroperoxide-induced peroxidation and turnover of endothelial cell phospholipids. Free Radic Biol Med 17:297-309
Pacifici, E H; McLeod, L L; Peterson, H et al. (1994) Linoleic acid hydroperoxide-induced peroxidation of endothelial cell phospholipids and cytotoxicity. Free Radic Biol Med 17:285-95
Salgo, M G; Corongiu, F P; Sevanian, A (1993) Enhanced interfacial catalysis and hydrolytic specificity of phospholipase A2 toward peroxidized phosphatidylcholine vesicles. Arch Biochem Biophys 304:123-32
Zhang, J R; Sevanian, A (1993) The genotoxic effects of arachidonic acid in V79 cells are mediated by peroxidation products. Toxicol Appl Pharmacol 121:193-202
Wratten, M L; van Ginkel, G; van't Veld, A A et al. (1992) Structural and dynamic effects of oxidatively modified phospholipids in unsaturated lipid membranes. Biochemistry 31:10901-7

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