Cytosolic phospholipase A2 mediates arachidonoyl phospholipid hydrolysis in immortalized rat brain endothelial cells stimulated by oxidized LDL.
We tested the hypothesis that oxidized low-density lipoprotein (oxLDL), administered in sublethal doses to the culture medium of immortalized rat brain endothelial cells (ECs, GP8.39), acts as a prooxidant signal to stimulate peroxidation processes and membrane phospholipid hydrolysis. ECs were grown at confluence in a medium with or without native LDL (nLDL) or oxLDL (1.5 mg/dish; up to 350-450 nmol hydroperoxides/mg protein) for two temporally distinct phases (short incubation period up to 1 h, or long incubation period spanning 24 h). Peroxidation parameters (conjugated dienes, MDA, hydroperoxides and LDH release) and arachidonic acid (AA) release were determined. Cell lysates and subcellular fractions were assayed for cPLA(2) while the cytotoxic effect and apoptosis were monitored by morphological changes, trypan blue dye exclusion, MTT reduction test, caspase-3 activity, COMET and laser confocal fluorescence microscopy (LCFM) analyses. Effects of alpha-tocopherol and 85-kDa PLA(2) inhibitor (AACOCF(3)), alone or in combination, were also tested. Immunoblot analysis of cPLA(2) was carried out on cell fraction proteins. After incubation for 1 or 24 h, oxLDL (100-200 microM hydroperoxides), but not nLDL, markedly increased lipid peroxidation, cPLA(2) activity and AA release in a dose-dependent manner. AACOCF(3) and antioxidant alpha-tocopherol (1 mM) strongly inhibited the prooxidant-stimulated AA release. Long-term exposure (24 h) to oxLDL (100 microM) had no effect on the cPLA(2) protein content as tested by Western immunoblot analysis, while showing a sharp cytotoxic effect on the cells. Caspase-3 activity and LCFM analysis indicated that oxLDL (100/200 microM) were able to trigger an apoptotic process. The results suggest that (i) ECs may be the target of extensive oxidative damage caused by oxLDL; (ii) activation of cPLA(2) mediates liberation of AA; (iii) cPLA(2) expression was not stimulated by long-term exposure to oxLDL; (iv) oxidized specific constituents of oxLDL, acting as regulatory signals, increase the ability of ECs to degrade membrane phospholipids, end products of which are linked to the development of atherosclerotic lesions.
Lupo
Gabriella
G
Department of Biochemistry, Faculty of Medicine, University of Catania, Viale Andrea Doria 6, 95125, Catania, Italy.
Assero
Giovanna
G
Anfuso
Carmelina D
CD
Nicotra
Ambra
A
Palumbo
Maddalena
M
Cannavò
Giuseppe
G
Renis
Marcella
M
Ragusa
Nicola
N
Alberghina
Mario
M
D000818:Animals; D001921:Brain / Q000378:metabolism; D002461:Cell Line, Transformed; D003600:Cytosol / Q000378:metabolism; D004727:Endothelium / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006868:Hydrolysis; D015227:Lipid Peroxidation; D008077:Lipoproteins, LDL / Q000378:metabolism*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D010741:Phospholipases A / Q000378:metabolism*; D054467:Phospholipases A2; D010743:Phospholipids / Q000378:metabolism*; D051381:Rats
D008077:Lipoproteins, LDL; D010743:Phospholipids; C099252:oxidized low density lipoprotein; D010741:Phospholipases A; D054467:Phospholipases A2