Nitric Oxide Inhibition of Free Radical-Mediated Cholesterol Peroxidation in Liposomal Membranes
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- 作者：Witold Korytowski ; Mariusz Zareba ; and Albert W. Girotti
- 刊名：Biochemistry
- 出版年：2000
- 出版时间：June 13, 2000
- 年：2000
- 卷：39
- 期：23
- 页码：6918 - 6928
- 全文大小：197K
- 年卷期：v.39,no.23(June 13, 2000)
- ISSN：1520-4995
文摘
The ability of nitric oxide (
NO) to inhibit propagative lipid peroxidation was investigatedusing unilamellar liposomes (LUVs) constituted with egg phosphatidylcholine (PC) or 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), [14C]cholesterol (Ch), and a nonregenerable singlet oxygen-derivedprimer, 5
-hydroperoxycholesterol (5-OOH). Exposing LUVs to ascorbate and a lipophilic iron chelateat 37 
C resulted in an exponential decay of 5-OOH and accumulation of free radical-derived 7- and7
-hydroperoxycholesterol (7-OOH), as detected by high-performance liquid chromatography withelectrochemical detection. Thiobarbituric acid-reactive species (TBARS) were generated concurrently inegg PC-containing LUVs. Including the NO donor spermine NONOate (SPNO, 5-50 
M) or S-nitroso-N-acetyl-D,L-penicillamine (SNAP, 50-100 M) in the reaction mixture had no effect on 5-OOH decay(suggesting that iron was not redox-inhibited) but slowed TBARS and 7-OOH accumulation in a stronglydose-dependent fashion. Decomposed SPNO or SNAP had no such effects, implying that NO was theresponsible agent. Accumulation of several [14C]Ch oxidation products, detected by high-performancethin-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 afunction of incubation time and NO donor concentration. RCh.4 material was also generated via directiron/ascorbate reduction of 7-OOH in the presence of NO, consistent with 7-nitrite (7-ONO) identity.However, various other lines of evidence suggest that RCh.4 is not 7-ONO, but rather 5-hydroxycholesterol (5-OH) generated by reduction of 5-ONO arising from 7-ONO rearrangement.5-OH was only detected when NO was present in the reaction system, thus providing indirect evidencefor the existence of nitrosated Ch intermediates arising from NO chain-breaking activity.
NO) to inhibit propagative lipid peroxidation was investigatedusing unilamellar liposomes (LUVs) constituted with egg phosphatidylcholine (PC) or 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), [14C]cholesterol (Ch), and a nonregenerable singlet oxygen-derivedprimer, 5-hydroperoxycholesterol (5-OOH). Exposing LUVs to ascorbate and a lipophilic iron chelateat 37 C resulted in an exponential decay of 5-OOH and accumulation of free radical-derived 7- and7-hydroperoxycholesterol (7-OOH), as detected by high-performance liquid chromatography withelectrochemical detection. Thiobarbituric acid-reactive species (TBARS) were generated concurrently inegg PC-containing LUVs. Including the NO donor spermine NONOate (SPNO, 5-50 M) or S-nitroso-N-acetyl-D,L-penicillamine (SNAP, 50-100 M) in the reaction mixture had no effect on 5-OOH decay(suggesting that iron was not redox-inhibited) but slowed TBARS and 7-OOH accumulation in a stronglydose-dependent fashion. Decomposed SPNO or SNAP had no such effects, implying that NO was theresponsible agent. Accumulation of several [14C]Ch oxidation products, detected by high-performancethin-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 afunction of incubation time and NO donor concentration. RCh.4 material was also generated via directiron/ascorbate reduction of 7-OOH in the presence of NO, consistent with 7-nitrite (7-ONO) identity.However, various other lines of evidence suggest that RCh.4 is not 7-ONO, but rather 5-hydroxycholesterol (5-OH) generated by reduction of 5-ONO arising from 7-ONO rearrangement.5-OH was only detected when NO was present in the reaction system, thus providing indirect evidencefor the existence of nitrosated Ch intermediates arising from NO chain-breaking activity.