Premium
Self‐limiting enhancement by nitric oxide of oxygen free radical‐induced endothelial cell injury: evidence against the dual action of NO as hydroxyl radical donor/scavenger
Author(s) -
AzMa Toshiharu,
Fujii Kohyu,
Yuge Osafumi
Publication year - 1996
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1111/j.1476-5381.1996.tb15694.x
Subject(s) - chemistry , hydroxyl radical , superoxide , superoxide dismutase , xanthine oxidase , spin trapping , nitric oxide , bradykinin , free radical scavenger , peroxynitrite , biochemistry , endothelial stem cell , radical , antioxidant , receptor , organic chemistry , enzyme , in vitro
1 The effects of oxygen free radical scavengers and endothelial cell‐derived nitric oxide (EDNO) on the death of porcine cultured aortic endothelial cells exposed to exogenous superoxide‐ [xanthine (0.4 mM)/xanthine oxidase (0.04 unit ml −1 ) + diethylenetriaminepentaacetic acid (DTPA, 10 μ m )] or hydroxyl radical‐generating system(s) [superoxide generating system + ferric iron (Fe 3+ , 0.1 mM) or peroxynitrite (0–100 μ m )] have been evaluated. 2 Spin trapping studies using 5,5‐dimethyl‐1‐pyrroline‐N‐oxide (DMPO) with electron paramagnetic resonance spectrometry were also conducted to determine qualitatively the oxidant species generated by the oxidant generating systems. 3 Endothelial cell injury provoked by the exogenous superoxide generating system was inhibited by catalase, DTPA and a hydroxyl radical scavenger (dimethyl sulphoxide, DMSO), but not by superoxide dismutase (SOD). Addition of Fe 3+ to the superoxide generating system enhanced the cell injury. These suggested that the direct cytotoxicity of exogenous superoxide is limited, and that endogenous transition metal‐dependent hydroxyl radical formation is involved in the cell injury. 4 An inhibitor of the constitutive NO‐pathway, N G ‐monomethyl‐L‐arginine, did not influence cell injury induced by the superoxide generating system, suggesting that basal NO production is not responsible for the cytotoxicity. 5 Stimulation of endothelial cells with bradykinin enhanced cell injury provoked by the exogenous superoxide generating system, but not by the exogenous hydroxyl radical generating system. The enhancement by bradykinin was inhibited by N G ‐monomethyl‐L‐arginine and bradykinin B 2 ‐receptor antagonist, D‐Arg‐[Hyp 3 , Thi 5, 8 , D‐Phe 7 ] bradykinin, suggesting that an interaction of NO with superoxide is involved in the enhanced cytotoxicity. A possible intermediate of this reaction, peroxynitrite, also caused endothelial cell injury in a concentration‐dependent manner. 6 The modulatory effects of NO on hydroxyl radical‐like activity (= formaldehyde production) from the superoxide generating system was also evaluated in a cell‐free superoxide/NO generating system, consisting of xanthine/xanthine oxidase, DTPA, DMSO, and various amounts of a spontaneous NO generator, sodium nitroprusside (SNP) and were compared with those of Fe 3+ . At doses up to 10 μ m , SNP concentration‐dependently increased the formaldehyde production while the higher concentrations of SNP decreased. The maximum amount of formaldehyde produced by SNP was 5 fold less than that produced by Fe 3+ (0.1 mM). Peroxynitrite‐induced formaldehyde formation was concentration‐dependently inhibited by SNP. 7 We conclude that agonist‐stimulated but not basal NO production acts as cytotoxic hydroxyl radical donor as well as the endogenous transition metal when endothelial cells are exposed to exogenous superoxide anion, while the modulatory effect of EDNO is limited by a secondary reaction with hydroxyl radicals.