Mammalian tissues produce nitric oxide (NO) to modify proteins at heme and sulfhydryl sites, thereby regulating vital cell functions. The majority of NO produced is widely assumed to be neutralized into supposedly inert oxidation products including nitrite (NO2(-)). Here we show that nitrite, also ubiquitous in dietary sources, is remarkably efficient at modifying the same protein sites, and that physiological nitrite concentrations account for the basal levels of these modifications in vivo. We further find that nitrite readily affects cyclic GMP production, cytochrome P450 activities, and heat shock protein 70 and heme oxygenase-1 expression in a variety of tissues. These cellular activities of nitrite, combined with its stability and abundance in vivo, suggest that this anion has a distinct and important signaling role in mammalian biology, perhaps by serving as an endocrine messenger and synchronizing agent. Thus, nitrite homeostasis may be of great importance to NO biology.

The low barometric pressure at high altitude causes lower arterial oxygen content among Tibetan highlanders, who maintain normal levels of oxygen use as indicated by basal and maximal oxygen consumption levels that are consistent with sea level predictions. This study tested the hypothesis that Tibetans resident at 4,200 m offset physiological hypoxia and achieve normal oxygen delivery by means of higher blood flow enabled by higher levels of bioactive forms of NO, the main endothelial factor regulating blood flow and vascular resistance. The natural experimental study design compared Tibetans at 4,200 m and U.S. residents at 206 m. Eighty-eight Tibetan and 50 U.S. resident volunteers (18 -56 years of age, healthy, nonsmoking, nonhypertensive, not pregnant, with normal pulmonary function) participated. Forearm blood flow, an indicator of systemic blood flow, was measured noninvasively by using plethysmography at rest, after breathing supplemental oxygen, and after exercise. The Tibetans had more than double the forearm blood flow of low-altitude residents, resulting in greater than sea level oxygen delivery to tissues. In comparison to sea level controls, Tibetans had >10-fold-higher circulating concentrations of bioactive NO products, including plasma and red blood cell nitrate and nitroso proteins and plasma nitrite, but lower concentrations of iron nitrosyl complexes (HbFe II NO) in red blood cells. This suggests that NO production is increased and that metabolic pathways controlling formation of NO products are regulated differently among Tibetans. These findings shift attention from the traditional focus on pulmonary and hematological systems to vascular factors contributing to adaptation to high-altitude hypoxia.circulation ͉ endothelium T he low barometric pressure at high altitude causes lower arterial oxygen content among Tibetan highlanders, who maintain normal levels of oxygen use as indicated by basal and maximal oxygen consumption levels that are consistent with sea level predictions (1-3). Hypothetically, the unavoidably low supply of oxygen in the air and the blood could be offset by increasing blood flow to improve oxygen delivery. Blood flow is determined by numbers, length, and diameter of blood vessels that in turn are largely determined directly or indirectly by levels of NO, a potent vasodilator synthesized in the endothelial cells lining the vessels (4-7). Tibetans have high levels of NO synthesis in the lungs (8), and pulmonary blood flow correlated with NO in a sample studied at 4,200 m (8, 9). This suggests the hypothesis that Tibetan highlanders offset hypoxia with higher systemic blood flow and higher levels of circulating, biologically active metabolites of NO. After synthesis by the endothelium, NO rapidly undergoes reaction in the blood to form products that have circulatory and metabolic effects, including nitrite, nitrate, nitrosothiol proteins (proteins containing NO-cysteine covalent bonds), and ␣-nitrosyl hemoglobin (HbFe II NO), in which NO occupies the heme binding site for o...

Although nitrite (NO 2؊ ) and nitrate (NO 3 ؊ ) have been considered traditionally inert byproducts of nitric oxide (NO) metabolism, recent studies indicate that NO 2 ؊ represents an important source of NO for processes ranging from angiogenesis through hypoxic vasodilation to ischemic organ protection. Despite intense investigation, the mechanisms through which NO 2 ؊ exerts its physiological/pharmacological effects remain incompletely understood. We sought to systematically investigate the fate of NO 2 ؊ in hypoxia from cellular uptake in vitro to tissue utilization in vivo using the Wistar rat as a mammalian model. We find that most tissues (except erythrocytes) produce free NO at rates that are maximal under hypoxia and that correlate robustly with each tissue's capacity for mitochondrial oxygen consumption. By comparing the kinetics of NO release before and after ferricyanide addition in tissue homogenates to mathematical models of NO 2 ؊ reduction/NO scavenging, we show that the amount of nitrosylated products formed greatly exceeds what can be accounted for by NO trapping. This difference suggests that such products are formed directly from NO 2 ؊ , without passing through the intermediacy of free NO. Inhibitor and subcellular fractionation studies indicate that NO 2 ؊ reductase activity involves multiple redundant enzymatic systems (i.e. heme, iron-sulfur cluster, and molybdenumbased reductases) distributed throughout different cellular compartments and acting in concert to elicit NO signaling. These observations hint at conserved roles for the NO 2 ؊ -NO pool in cellular processes such as oxygen-sensing and oxygen-dependent modulation of intermediary metabolism. Nitric oxide (NO)3 is the archetypal effector of redox-regulated signal transduction throughout phylogeny, from microorganisms to plants and animals (1). The conserved influences of NO extend from the regulation of basic cellular processes such as intermediary metabolism (2), cellular proliferation (3), and apoptosis (4) to systemic processes such as hypoxic vasoregulation (5). Mammalian NO production has been attributed to the enzymatic activity of NO synthases, nitrate (NO 3 Ϫ )/nitrite (NO 2 Ϫ ) reductases and non-enzymatic NO 2 Ϫ reduction (6). The NO produced is believed to act directly as a signaling molecule by binding to the heme of soluble guanylyl cyclase or nitrosating peptide/protein cysteine residues (7). More recently, it has become apparent that NO 2 Ϫ , previously considered an inert byproduct of NO metabolism present in plasma (50 -500 nM) and tissues (0.5-25 M), is, under some conditions, also a source of NO/nitrosothiol signaling (6,8). Although the importance of NO 2 Ϫ has received increasing appreciation (9) as being central to processes including exercise (10), hypoxic vasodilation (11), myocardial preconditioning (12, 13), and angiogenesis (14), controversy surrounds the chemistry, kinetics, and tissue specificity of NO 2 Ϫ bioactivity (15, 16). Perhaps the greatest uncertainty pertains to the role of heme moieties in NO 2...

Abstract-Nitrite has recently emerged as an important bioactive molecule, capable of conferring cardioprotection and a variety of other benefits in the cardiovascular system and elsewhere. The mechanisms by which it accomplishes these functions remain largely unclear. To characterize the dose response and corresponding cardiac sequelae of transient systemic elevations of nitrite, we assessed the time course of oxidation/nitros(yl)ation, as well as the metabolomic, proteomic, and associated functional changes in rat hearts following acute exposure to nitrite in vivo. Transient systemic nitrite elevations resulted in: (1) rapid formation of nitroso and nitrosyl species; (2) moderate short-term changes in cardiac redox status; (3) a pronounced increase in selective manifestations of long-term oxidative stress as evidenced by cardiac ascorbate oxidation, persisting long after changes in nitrite-related metabolites had normalized; (4) lasting reductions in glutathione oxidation (GSSG/GSH) and remarkably concordant nitrite-induced cardioprotection, which both followed a complex dose-response profile; and (5) 2,3 Nitrite has been shown to elicit NO-dependent vasodilatation, 4,5 angiogenesis, 6 and cardioprotection. [7][8][9][10][11] Although the mechanisms of nitrite-induced cardioprotection remain elusive, proposals include alterations to mitochondrial respiration by direct modulation of the electron transport chain. 7 Nitrite bioconversion to NO has varyingly been attributed to heme-dependent reductase activity, 7,12,13 to reduction by other enzymes, including aldehyde dehydrogenase (ALDH)2 and xanthine oxidase, 14 and to chemistries favored by low oxygen tension or pH. 15 Cardioprotection by nitrite is manifest in isolated heart preparations, 16 and low doses of nitrite prevent ischemia/reperfusion (I/R) injury in myocardial infarction. 17 Thus, although great potential exists for nitrite-based therapeutics, a number of questions remain.(1) What is the dose-response relationship for nitrite-mediated cardioprotection? (2) What are the immediate (first-hour) and longer-term (24-hour) cardiac sequelae of elevations in plasma nitrite? (3) By what mechanisms does nitrite-induced cardioprotection occur? Using a metabolomic/proteomic approach, we demonstrate here that, following a brief systemic nitrite exposure in vivo, cardiac tissue experiences a rapid, dose-dependent wave of S-, N-, and heme nitros(yl)ation, followed by longer-term redox status alterations and cardiac proteomic changes, all of which may contribute to cardioprotection.