Nitrite anions comprise the largest vascular storage pool of nitric oxide (NO), provided that physiological mechanisms exist to reduce nitrite to NO. We evaluated the vasodilator properties and mechanisms for bioactivation of nitrite in the human forearm. Nitrite infusions of 36 and 0.36 micromol/min into the forearm brachial artery resulted in supra- and near-physiologic intravascular nitrite concentrations, respectively, and increased forearm blood flow before and during exercise, with or without NO synthase inhibition. Nitrite infusions were associated with rapid formation of erythrocyte iron-nitrosylated hemoglobin and, to a lesser extent, S-nitroso-hemoglobin. NO-modified hemoglobin formation was inversely proportional to oxyhemoglobin saturation. Vasodilation of rat aortic rings and formation of both NO gas and NO-modified hemoglobin resulted from the nitrite reductase activity of deoxyhemoglobin and deoxygenated erythrocytes. This finding links tissue hypoxia, hemoglobin allostery and nitrite bioactivation. These results suggest that nitrite represents a major bioavailable pool of NO, and describe a new physiological function for hemoglobin as a nitrite reductase, potentially contributing to hypoxic vasodilation.

Local vasodilation in response to hypoxia is a fundamental physiologic response ensuring oxygen delivery to tissues under metabolic stress. Recent studies identify a role for the red blood cell (RBC), with hemoglobin the hypoxic sensor. Herein, we investigate the mechanisms regulating this process and explore the relative roles of adenosine triphosphate, S-nitrosohemoglobin, and nitrite as effectors. We provide evidence that hypoxic RBCs mediate vasodilation by reducing nitrite to nitric oxide (NO) and ATP release. NO dependence for nitrite-mediated vasodilation was evidenced by NO gas formation, stimulation of cGMP production, and inhibition of mitochondrial respiration in a process sensitive to the NO scavenger C-PTIO. The nitrite reductase activity of hemoglobin is modulated by heme deoxygenation and heme redox potential, with maximal activity observed at 50% hemoglobin oxygenation (P 50 ). Concomitantly, vasodilation is initiated at the P 50 , suggesting that oxygen sensing by hemoglobin is mechanistically linked to nitrite reduction and stimulation of vasodilation. Mutation of the conserved ␤93cys residue decreases the heme redox potential (ie, decreases E 1/2 ), an effect that increases nitrite reductase activity and vasodilation at any given hemoglobin saturation. These data support a function for RBC hemoglobin as an allosterically and redox-regulated nitrite reductase whose "enzyme activity" couples hypoxia to increased NO-dependent blood flow. (Blood. 2006;107:566-574)

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Ischemia/reperfusion (IR) injury in transplanted livers contributes to organ dysfunction and failure and is characterized in part by loss of NO bioavailability. Inhalation of NO is nontoxic and at high concentrations (80 ppm) inhibits IR injury in extrapulmonary tissues. In this prospective, blinded, placebo-controlled study, we evaluated the hypothesis that administration of inhaled NO (iNO; 80 ppm) to patients undergoing orthotopic liver transplantation inhibits hepatic IR injury, resulting in improved liver function. Patients were randomized to receive either placebo or iNO (n = 10 per group) during the operative period only. When results were adjusted for cold ischemia time and sex, iNO significantly decreased hospital length of stay, and evaluation of serum transaminases (alanine transaminase, aspartate aminotransferase) and coagulation times (prothrombin time, partial thromboplastin time) indicated that iNO improved the rate at which liver function was restored after transplantation. iNO did not significantly affect changes in inflammatory markers in liver tissue 1 hour after reperfusion but significantly lowered hepatocyte apoptosis. Evaluation of circulating NO metabolites indicated that the most likely candidate transducer of extrapulmonary effects of iNO was nitrite. In summary, this study supports the clinical use of iNO as an extrapulmonary therapeutic to improve organ function following transplantation.

The aqueous decay and concomitant release of nitric oxide ( ⅐ NO) by nitrolinoleic acid (10-nitro-9,12-octadecadienoic acid and 12-nitro-9,12-octadecadienoic acid; LNO 2 ) are reported. Mass spectrometric analysis of reaction products supports a modified Nef reaction as the mechanism accounting for the generation of ⅐ NO by the aqueous reactions of fatty acid nitroalkene derivatives. Nitrolinoleic acid is stabilized by an aprotic milieu, with LNO 2 decay and ⅐ NO release strongly inhibited by phosphatidylcholine/cholesterol liposome membranes and detergents when present at levels above their critical micellar concentrations. The release of ⅐ NO from LNO 2 was induced by UV photolysis and triiodide-based ozone chemiluminescence reactions currently used to quantify putative protein nitrosothiol and N-nitrosamine derivatives. This reactivity of LNO 2 complicates the qualitative and quantitative analysis of biological oxides of nitrogen when applying UV photolysis and triiodidebased analytical systems to biological preparations typically abundant in nitrated fatty acids. The results reveal that nitroalkene derivatives of linoleic acid are pluripotent signaling mediators that act not only via receptor-dependent mechanisms, but also by transducing the signaling actions of ⅐ NO via pathways subject to regulation by the relative distribution of LNO 2 to hydrophobic versus aqueous microenvironments.Nitrolinoleic acid (10-nitro-9,12-octadecadienoic acid and 12-nitro-9,12-octadecadienoic acid; LNO 2 ) 1 is present in plasma lipoproteins and red blood cell membranes at concentrations of ϳ500 nM, rendering this species the most quantitatively abundant, biologically active oxide of nitrogen in the human vascular compartment (1). Nitrolinoleic acid is a product of nitric oxide ( ⅐ NO)-dependent linoleic acid nitration reactions that predominantly occur at the C-10 and C-12 alkene carbons. The positional isomer distribution of the LNO 2 alkenyl nitro group indicates that in vivo fatty acid nitration is a consequence of nucleophilic (nitronium group (NO 2 ϩ )) and/or radical (nitrogen dioxide ( ⅐ NO 2 )) addition reactions with olefinic carbons.Recent observations reveal that LNO 2 is a pluripotent signaling mediator that acts via both receptor-dependent and receptor-independent pathways. Nitrated fatty acids are specific and high affinity endogenous ligands for peroxisome proliferator-activated receptors (2) and serve to activate receptordependent gene expression at physiological concentrations. LNO 2 also activates cAMP-dependent protein kinase signaling pathways in neutrophils and platelets, serving to down-regulate the activation of these inflammatory cells (3, 4). Finally, LNO 2 induces vessel relaxation in an endothelium-independent manner (5). This LNO 2 -mediated relaxation of phenylephrine-preconstricted aortic rings is 1) a consequence of LNO 2 -induced stimulation of smooth muscle cell and aortic segment cGMP content, 2) inhibitable by the ⅐ NO scavenger oxyhemoglobin, and 3) 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-on...