Inhibition of free radical-mediated cholesterol peroxidation by diazeniumdiolate-derived nitric oxide: effect of release rate on mechanism of action in a membrane system.
Nitric oxide ((*)NO) flux in relation to antiperoxidant action has been studied, using large unilamellar liposomes (LUVs) as target membranes. LUVs consisting of an oxidizable phosphatidylcholine (PC), [(14)C]cholesterol (Ch) as a reaction probe, and 5alpha-hydroperoxycholesterol (5alpha-OOH) as a nonregenerable primer underwent chain peroxidation when exposed to a lipophilic iron chelate [Fe(HQ)(3), 1 microM] and ascorbate (AH(-), 1 mM) at 37 degrees C. Reaction progress was monitored by (i) HPLC with reductive-mode electrochemical detection to assess the decay of 5alpha-OOH and the formation and/or turnover of free radical-derived 7alpha- and 7beta-hydroperoxycholesterol (7alphabeta-OOH) and (ii) HPTLC with phosphorimaging to track all major (14)C-labeled oxidation products (ChOX), including 7alphabeta-OOH, 7alpha-OH, 7beta-OH, and 5,6-epoxide. Three diazeniumdiolate (*)NO donors with different half-lives were tested for their ability to interfere with peroxidation: MANO ( approximately 1 min), PANO (15 min), and SPNO (38 min). At starting concentrations of < or =200 microM, none of the donors slowed 5alpha-OOH exponential decay, ruling out any interference with redox-active iron. However, SPNO and to a greater extent PANO (but not the decomposed donors) decreased both the initial rate and steady state of 7alphabeta-OOH accumulation in a strong dose-dependent fashion. In contrast, MANO completely inhibited 7alphabeta-OOH formation over the first 5 min of reaction, but thereafter, the peroxide accumulated rapidly, albeit more slowly than without MANO and independently of the MANO dose. The latter response diminished with increasing Fe(HQ)(3) concentration, coincident with more rapid 5alpha-OOH loss. The same general trends with MANO, PANO, and SPNO were observed when the entire population of [(14)C]ChOX species was monitored. These effects are attributed to interception of Ch- and PC-derived free radicals by (*)NO, high-flux (*)NO from MANO acting mainly on 5alpha-OOH-derived radicals (chain prevention), low-flux (*)NO from SPNO mainly on downstream radicals (chain termination), and intermediate-flux (*)NO from PANO by a combination of these mechanisms. Thus, delivery rate can be an important determinant of how (*)NO inhibits peroxide-induced lipid peroxidation.
Korytowski
W
W
Biochemistry Department, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
Zareba
M
M
Girotti
A W
AW
D002784:Cholesterol / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D005609:Free Radicals / Q000737:chemistry*; D006834:Hydrazines / Q000737:chemistry*; D015227:Lipid Peroxidation; D008081:Liposomes / Q000737:chemistry; D009569:Nitric Oxide / Q000737:chemistry*; D009589:Nitrogen Oxides; D013096:Spermine / Q000031:analogs & derivatives / Q000737:chemistry
C105785:1-hexanamine-6-(2-hydroxy-1-methyl-2-nitrosohydrazine)-N-methyl; D005609:Free Radicals; D006834:Hydrazines; D008081:Liposomes; D009589:Nitrogen Oxides; C112653:PAPA NONOate; C091861:spermine nitric oxide complex; D013096:Spermine; D009569:Nitric Oxide; D002784:Cholesterol