Abstract
Organic nitrates have been used clinically in the treatment of ischemic heart disease for more than a century. Recently, xanthine oxidase (XO) has been reported to catalyze organic nitrate reduction under anaerobic conditions, but questions remain regarding the initial precursor of nitric oxide (NO) and the link of organic nitrate to the activation of soluble guanylyl cyclase (sGC). To characterize the mechanism of XO-mediated biotransformation of organic nitrate, studies using electron paramagnetic resonance spectroscopy, chemiluminescence NO analyzer, NO electrode, and immunoassay were performed. The XO reducing substrates xanthine, NADH, and 2,3-dihydroxybenz-aldehyde triggered the reduction of organic nitrate to nitrite anion (NO2-). Studies of the pH dependence of nitrite formation indicated that XO-mediated organic nitrate reduction occurred via an acid-catalyzed mechanism. In the absence of thiols or ascorbate, no NO generation was detected from XO-mediated organic nitrate reduction; however, addition of L-cysteine or ascorbate triggered prominent NO generation. Studies suggested that organic nitrite (R-O-NO) is produced from XO-mediated organic nitrate reduction. Further reaction of organic nitrite with thiols or ascorbate leads to the generation of NO or nitrosothiols and thus stimulates the activation of sGC. Only flavin site XO inhibitors such as diphenyleneiodonium inhibited XO-mediated organic nitrate reduction and sGC activation, indicating that organic nitrate reduction occurs at the flavin site. Thus, organic nitrite is the initial product in the process of XO-mediated organic nitrate biotransformation and is the precursor of NO and nitrosothiols, serving as the link between organic nitrate and sGC activation.
Highlights
Organic nitrates have been used clinically in the treatment of ischemic heart disease for more than a century
It has been reported that xanthine oxidase (XO) can catalyze organic nitrate reduction to nitrite anion (NO2Ϫ) under anaerobic conditions [13], but questions remain concerning the initial product, the precursor of nitric oxide (NO), and the link of organic nitrate to the activation of soluble guanylyl cyclase
Inorganic Nitrite Generation from XO-mediated Organic Nitrate Reduction—It has been reported that XO can catalyze organic nitrate reduction to NO2Ϫ [13]
Summary
Materials—Xanthine oxidase from buttermilk (xanthine: oxygen oxidoreductase; EC 1.1.3.22), xanthine, oxypurinol, DPI, sodium nitrite, Griess Reagent, -NADH, bovine serum albumin (BSA), and 2,3-dihydroxybenz-aldehyde (DBA) were obtained from Sigma. NO generated by XO-catalyzed reduction of organic nitrate was purged out using argon to a vessel that contained the spin-trap Fe2ϩ-MGD, as described previously [14]. This setup was designed to isolate the reaction solution from spin trap and avoid any possible perturbation caused by the reaction of (MGD)2Fe2ϩ with nitrite or with the enzyme. Nitrite in the solution is measured from its reduction to NO under conditions of acidic pH in the presence of glacial acetic acid and 1% KI [12]. The measurements of cGMP in the solution were performed using direct cGMP assay kit by immunoassay according to the manufacturer’s product protocol (Assay Designs, Inc.). A p value of Յ0.05 was considered to indicate statistical significance
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