Nitric oxide inhibition of free radical-mediated cholesterol peroxidation in liposomal membranes.
The ability of nitric oxide ((*)NO) to inhibit propagative lipid peroxidation was investigated using unilamellar liposomes (LUVs) constituted with egg phosphatidylcholine (PC) or 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), [(14)C]cholesterol (Ch), and a nonregenerable singlet oxygen-derived primer, 5alpha-hydroperoxycholesterol (5alpha-OOH). Exposing LUVs to ascorbate and a lipophilic iron chelate at 37 degrees C resulted in an exponential decay of 5alpha-OOH and accumulation of free radical-derived 7alpha- and 7beta-hydroperoxycholesterol (7alphabeta-OOH), as detected by high-performance liquid chromatography with electrochemical detection. Thiobarbituric acid-reactive species (TBARS) were generated concurrently in egg PC-containing LUVs. Including the (*)NO donor spermine NONOate (SPNO, 5-50 microM) or S-nitroso-N-acetyl-D,L-penicillamine (SNAP, 50-100 microM) in the reaction mixture had no effect on 5alpha-OOH decay (suggesting that iron was not redox-inhibited) but slowed TBARS and 7alphabeta-OOH accumulation in a strongly dose-dependent fashion. Decomposed SPNO or SNAP had no such effects, implying that (*)NO was the responsible agent. Accumulation of several [(14)C]Ch oxidation products, detected by high-performance thin-layer chromatography with phosphorimaging, was similarly diminished by active SPNO or SNAP. Concomitantly, a new band referred to as RCh.4 appeared, the radioactivity of which increased as a function of incubation time and (*)NO donor concentration. RCh.4 material was also generated via direct iron/ascorbate reduction of 7alpha-OOH in the presence of (*)NO, consistent with 7alpha-nitrite (7alpha-ONO) identity. However, various other lines of evidence suggest that RCh.4 is not 7alpha-ONO, but rather 5alpha-hydroxycholesterol (5alpha-OH) generated by reduction of 5alpha-ONO arising from 7alpha-ONO rearrangement. 5alpha-OH was only detected when (*)NO was present in the reaction system, thus providing indirect evidence for the existence of nitrosated Ch intermediates arising from (*)NO chain-breaking activity.
Korytowski
W
W
Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, and Institute of Molecular Biology, Jagiellonian University, Krakow, Poland.
Zareba
M
M
Girotti
A W
AW
D000975:Antioxidants / Q000494:pharmacology; D001205:Ascorbic Acid / Q000494:pharmacology; D002784:Cholesterol / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D005609:Free Radicals / Q000378:metabolism; D007700:Kinetics; D015227:Lipid Peroxidation / Q000187:drug effects*; D008081:Liposomes / Q000378:metabolism*; D015394:Molecular Structure; D009569:Nitric Oxide / Q000494:pharmacology*; D009603:Nitroso Compounds / Q000378:metabolism; D010545:Peroxides / Q000378:metabolism / Q000494:pharmacology; D010743:Phospholipids / Q000378:metabolism; D017392:Thiobarbituric Acid Reactive Substances / Q000032:analysis
D000975:Antioxidants; D005609:Free Radicals; D008081:Liposomes; D009603:Nitroso Compounds; D010545:Peroxides; D010743:Phospholipids; D017392:Thiobarbituric Acid Reactive Substances; D009569:Nitric Oxide; C002843:cholesterol hydroperoxide; D002784:Cholesterol; D001205:Ascorbic Acid