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 stud ied, using large unilamellar liposomes (LUVs) as target membranes. LUVs con sisting of an oxidizable phosphatidylcholine (PC), [C-14]cholesterol (Ch) a s a reaction probe, and 5 alpha -hydroperoxycholesterol (5 alpha -OOH) as a nonregenerable primer underwent chain peroxidation when exposed to a lipop hilic iron chelate [Fe(HQ)(3), 1 muM] and ascorbate (AH(-), 1 mM) at 37 deg reesC. Reaction progress was monitored by (i) HPLC with reductive-mode elec trochemical detection to assess the decay of 5 alpha -OOH and the formation and/or turnover of free radical-derived 7 alpha- and 7 beta -hydroperoxych olesterol (7 alpha beta -OOH) and (ii) HPTLC with phosphorimaging to track all major C-14-labeled oxidation products (ChOX), including 7 alpha beta -O OH, 7 alpha -OH, 7 beta -OH, and 5,6-epoxide. Three diazeniumdiolate . NO d onors with different half-lives were tested for their ability to interfere with peroxidation: MANO(similar to1 min), PANG (15 min), and SPNO (38 min). At starting concentrations of less than or equal to 200 muM, none of the d onors slowed 5 alpha -OOH exponential decay, ruling out any interference wi th redox-active iron. However, SPNO and to a greater extent PANG (but not t he decomposed donors) decreased both the initial rate and steady state of 7 alpha beta -OOH accumulation in a strong dose-dependent fashion. In contra st, IMANO completely inhibited 7 alpha beta -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 latte r response diminished with increasing Fe(HQ)3 concentration, coincident wit h more rapid 5 alpha -OOH loss. The same general trends with MANO, PANG, an d SPNO were observed when the entire population of [C-14]ChOX species was m onitored. These effects are attributed to interception of Ch- and PC-derive d free radicals by NO, high-flux . NO from MANO acting mainly on 5 alpha -O OH-derived radicals (chain prevention), low-flux . NO from SPNO mainly on d ownstream radicals (chain termination), and intermediate-flux . NO from PAN G by a combination of these mechanisms. Thus, delivery rate can be an impor tant determinant of how . NO inhibits peroxide-induced lipid peroxidation.