Nitric oxide inhibition of free radical-mediated cholesterol peroxidation in liposomal membranes

The ability of nitric oxide ((NO)-N-.) to inhibit propagative lipid peroxid ation was investigated using unilamellar liposomes (LUVs) constituted with egg phosphatidylcholine (PC) or 1-palmitoyl-2-oleoylphosphatidylcholine (PO PC), [C-14]cholesterol (Ch), and a nonregenerable singlet oxygen-derived pr imer, 5 alpha-hydroperoxycholesterol (5 alpha-OOH). Exposing LUVs to ascorb ate and a lipophilic iron chelate at 37 degrees C resulted in an exponentia l decay of 5 alpha-OOH and accumulation of free radical-derived 7 alpha- an d 7 beta-hydroperoxycholesterol (7 alpha beta-OOH), as detected by high-per formance liquid chromatography with electrochemical detection. Thiobarbitur ic acid-reactive species (TBARS) were generated concurrently in egg PC-cont aining LUVs. Including the (NO)-N-. donor spermine NONOate (SPNO, 5-50 mu M ) or S-nitroso-N-acetyl-D,L-penicillamine (SNAP, 50-100 mu M) in the reacti on mixture had no effect on 5 alpha-OOH decay (suggesting that iron was not redox-inhibited) but slowed TEARS and 7 alpha beta-OOH accumulation in a s trongly dose-dependent fashion. Decomposed SPNO or SNAP had no such effects , implying that (NO)-N-. was the responsible agent. Accumulation of several [C-14]Ch oxidation products, detected by high-performance thin-layer chrom atography with phosphorimaging, was similarly diminished by active SPNO or SNAP. Concomitantly, a new band referred to as RCh.4 appeared, the radioact ivity of which increased as a function of incubation time and (NO)-N-. dono r concentration. RCh.4 material was also generated via direct iron/ascorbat e reduction of 7 alpha-OOH in the presence of (NO)-N-., consistent with 7 a lpha-nitrite (7 alpha-ONO) identity. However, various other lines of eviden ce suggest that RCh.4 is not 7 alpha-ONO, but rather 5 alpha-hydroxycholest erol (5 alpha-OH) generated by reduction of 5 alpha-ONO arising from 7 alph a-ONO rearrangement. 5 alpha-OH was only detected when (NO)-N-. was present in the reaction system, thus providing indirect evidence for the existence of nitrosated Ch intermediates arising from (NO)-N-. chain-breaking activi ty.