Unique Features of the Low Molecular Weight Probe Salicylaldehyde in the Detection of Nanomolar Peroxynitrite Fluxes

Monitoring White Blood Cell Mitochondrial Aldehyde Dehydrogenase Activity: Implications for Nitrate Therapy in Humans

Recent animal data suggest that reduced lipoic acid (LA) prevents oxidative inhibition of the nitrate bioactivating enzyme, the mitochondrial aldehyde dehydrogenase (ALDH-2), and that pentaerythritol tetranitrate (PETN) does not induce nitrate tolerance because of its intrinsic antioxidative properties, thereby preserving ALDH-2 activity. We sought to determine whether ALDH-2 activity in circulating white blood cells (WBCs) can be used to monitor nitrate tolerance and whether LA can prevent nitroglycerin tachyphylaxis in humans. Eight healthy male volunteers received, in randomized order, a single dose of glyceryl trinitrate (GTN; 0.8 mg), PETN (80 mg), or GTN plus LA (600 mg) orally. GTN (…

Endogenous peroxynitrite modulates PGHS-1–dependent thromboxane A2 formation and aggregation in human platelets

Aggregation of activated platelets is considerably mediated by the autocrine action of thromboxane A2 (TxA2) which is formed in a prostaglandin endoperoxide H2 synthase-1 (PGHS-1 or COX-1)-dependent manner. The activity of PGHS-1 can be stimulated by peroxides, an effect termed "peroxide tone", that renders PGHS-1 the key regulatory enzyme in the formation of TxA2. Activated platelets release nitric oxide (*NO) and superoxide (O*2) but their interactions with the prostanoid pathway have been controversially discussed in platelet physiology and pathophysiology. The current study demonstrates that endogenously formed peroxynitrite at nanomolar concentrations, originating from the interaction …

Hydralazine is a powerful inhibitor of peroxynitrite formation as a possible explanation for its beneficial effects on prognosis in patients with congestive heart failure

The hemodynamic and anti-ischemic effects of nitroglycerin (GTN) are rapidly blunted as a result of the development of nitrate tolerance. Hydralazine has been shown to prevent tolerance in experimental and clinical studies, all of which may be at least in part secondary to antioxidant properties of this compound. The antioxidant effects of hydralazine were tested in cell free systems, cultured smooth muscle cells, isolated mitochondria, and isolated vessels. Inhibitory effects on the formation of superoxide and/or peroxynitrite formation were tested using lucigenin and L-012 enhanced chemiluminescence as well as DHE-fluorescence. The peroxynitrite scavenging properties were also assessed by…

Chemical model systems for cellular nitros(yl)ation reactions.

S-nitros(yl)ation belongs to the redox-based posttranslational modifications of proteins but the underlying chemistry is controversial. In contrast to current concepts involving the autoxidation of nitric oxide ( • NO, nitrogen monoxide), we and others have proposed the formation of peroxynitrite (oxoperoxonitrate (1-)) as an essential intermediate. This requires low cellular fluxes of 'NO and superoxide ( • O 2 - ), for which model systems have been introduced. We here propose two new systems for nitros(yl)ation that avoid the shortcomings of previous models. Based on the thermal decomposition of 3-morpholinosydnonimine, equal fluxes of • NO and • O 2 - were generated and modulated by the …

Protein tyrosine nitration and thiol oxidation by peroxynitrite-strategies to prevent these oxidative modifications.

The reaction product of nitric oxide and superoxide, peroxynitrite, is a potent biological oxidant. The most important oxidative protein modifications described for peroxynitrite are cysteine-thiol oxidation and tyrosine nitration. We have previously demonstrated that intrinsic heme-thiolate (P450)-dependent enzymatic catalysis increases the nitration of tyrosine 430 in prostacyclin synthase and results in loss of activity which contributes to endothelial dysfunction. We here report the sensitive peroxynitrite-dependent nitration of an over-expressed and partially purified human prostacyclin synthase (3.3 μM) with an EC50 value of 5 μM. Microsomal thiols in these preparations effectively co…

Formation of 2-nitrophenol from salicylaldehyde as a suitable test for low peroxynitrite fluxes

There has been some dispute regarding reaction products formed at physiological peroxynitrite fluxes in the nanomolar range with phenolic molecules, when used to predict the behavior of protein-bound aromatic amino acids like tyrosine. Previous data showed that at nanomolar fluxes of peroxynitrite, nitration of these phenolic compounds was outcompeted by dimerization (e.g. biphenols or dityrosine). Using 3-morpholino sydnonimine (Sin-1), we created low fluxes of peroxynitrite in our reaction set-up to demonstrate that salicylaldehyde displays unique features in the detection of physiological fluxes of peroxynitrite, yielding detectable nitration but only minor dimerization products. By mean…

Role of Reduced Lipoic Acid in the Redox Regulation of Mitochondrial Aldehyde Dehydrogenase (ALDH-2) Activity

Chronic therapy with nitroglycerin results in a rapid development of nitrate tolerance, which is associated with an increased production of reactive oxygen species. We have recently shown that mitochondria are an important source of nitroglycerin-induced oxidants and that the nitroglycerin-bioactivating mitochondrial aldehyde dehydrogenase is oxidatively inactivated in the setting of tolerance. Here we investigated the effect of various oxidants on aldehyde dehydrogenase activity and its restoration by dihydrolipoic acid. In vivo tolerance in Wistar rats was induced by infusion of nitroglycerin (6.6 microg/kg/min, 4 days). Vascular reactivity was measured by isometric tension studies of iso…