Differential nitros(yl)ation of blood and tissue constituents during glyceryl trinitrate biotransformation in vivo

Abstract

Nitric oxide (NO)-derived products may modify tissue constituents, forming S-and N-nitroso adducts and metal nitrosyls implicated in NO signaling. Nitrovasodilator drugs have been in widespread use for more than a century, yet their biotransformation pathways to NO and their effects as NO donors across tissues remain ill defined. By using a metabonomics approach (termed ''NObonomics'') for detailing the global NO-related metabolism of the cornerstone nitrovasodilator, glyceryl trinitrate (GTN; 0.1-100 mg͞kg), in the rat in vivo, we find that GTN biotransformation elicits extensive tissue nitros(yl)ation throughout all major organ systems. The corresponding reaction products remained detectable hours after administration, and vascular tissue was not a major nitros(yl)ation site. Extensive heart and liver modifications involved both S-and N-nitrosation, and RBC S-nitrosothiol formation emerged as a sensitive indicator of organic nitrate metabolism. The dynamics of GTN-derived oxidative NO metabolites in blood did not reflect the nitros(yl)ation patterns in the circulation or in tissues, casting doubt on the usefulness of plasma nitrite͞nitrate as an index of NO͞NO-donor biodynamics. Target-tissue NO metabolites varied in amount and type with GTN dose, suggesting a dose-sensitive shift in the prevailing routes of GTN biotransformation (''metabolic shunting'') from thiol nitrosation to heme nitrosylation. We further demonstrate that GTN-induced nitros(yl)ation is modulated by a complex, tissue-selective interplay of enzyme-catalyzed pathways. These findings provide insight into the global in vivo metabolism of GTN at pharmacologically relevant doses and offer an additional experimental paradigm for the NObonomic analysis of NO-donor metabolism and signaling.nitric oxide ͉ nitrosylheme ͉ nitrosothiols ͉ metabonomics N itric oxide (NO) regulates diverse aspects of mammalian physiology by enhancing cGMP production through soluble guanylyl cyclase activation (1). This classic signal transduction mechanism notwithstanding, NO's highly reactive, diffusible nature fosters an extensive chemical biology in vivo that influences gene expression, mitochondrial respiration, and drug metabolism; induces posttranslational protein modification; and generates numerous NO-derived metabolites (2, 3) (collectively referred to herein as the NO metabonome or ''NObonome'').¶ Some of these metabolites have been implicated in NO's beneficial and pathological effects (2, 3). Recent demonstration that constitutive thiol, amine, and heme nitros(yl)ation in mammalian tissues generates, respectively, S-nitrosothiols (RSNOs), N-nitroso compounds (RNNOs), and nitrosylheme (NO-heme) species suggests new signaling pathways through which nitrogen oxides might influence cell physiology by covalently modifying tissue biomolecules (3,5). Given the mounting evidence that thiol nitrosation modulates cell function (6), it is a matter of great interest to identify the biological targets of NO-dependent modification, the mechanism(s) responsible, and the dis...

Nitric oxide-induced inhibition of smooth muscle cell proliferation involves S-nitrosation and inactivation of RhoA

“…Since glutathione is the most abundant cellular thiol (15-30 nmol/mg protein), formation of GSNO is expected to parallel the activity of NOS. In fact, several studies have confirmed the formation of GSNO in biological systems (15,18,34).…”

Fasting-induced intestinal apoptosis is mediated by inducible nitric oxide synthase and interferon-γ in rat

“…This method is based on the separation of nitrite and nitrate by ion chromatography, followed by online reaction of nitrate to nitrite, postcolumn derivatization with Griess reagent, and detection at 540 nm. Proteins in each sample were removed by centrifugation at 10,000 g for 5 min following methanol precipitation (jejunum-methanol ϭ 1:2 weight/volume, 4°C) (26).…”

Therapeutic Uses of Inorganic Nitrite and Nitrate

“…29 However, the contribution of this or other enzyme systems to the overall vasodilation by these drugs is difficult to assess because multiple metabolic pathways appear to act in concert. 30 In view of the range of organic nitrites and related compounds that act as vasodilators, it is not surprising that potassium and sodium nitrites were tested in this regard. In 1880, Reichert and Mitchell 31 published a very full account of the biological action of potassium nitrite on humans and animals.…”