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.

There is mounting evidence that the established paradigm of nitric oxide (NO) biochemistry, from formation through NO synthases, over interaction with soluble guanylyl cyclase, to eventual disposal as nitrite͞nitrate, represents only part of a richer chemistry through which NO elicits biological signaling. Additional pathways have been suggested that include interaction of NO-derived metabolites with thiols and metals to form S-nitrosothiols (RSNOs) and metal nitrosyls. Despite the overwhelming attention paid in this regard to RSNOs, little is known about the stability of these species, their significance outside the circulation, and whether other nitros(yl)ation products are of equal importance. We here show that N-nitrosation and heme-nitrosylation are indeed as ubiquitous as S-nitrosation in vivo and that the products of these reactions are constitutively present throughout the organ system. Our study further reveals that all NO-derived products are highly dynamic, have fairly short lifetimes, and are linked to tissue oxygenation and redox state. Experimental evidence further suggests that nitroso formation occurs substantially by means of oxidative nitrosylation rather than NO autoxidation, explaining why S-nitrosation can compete effectively with nitrosylation. Moreover, tissue nitrite can serve as a significant extravascular pool of NO during brief periods of hypoxia, and tissue nitrate͞nitrite ratios can serve as indicators of the balance between local oxidative and nitrosative stress. These findings vastly expand our understanding of the fate of NO in vivo and provide a framework for further exploration of the significance of nitrosative events in redox sensing and signaling. The findings also raise the intriguing possibility that N-nitrosation is directly involved in the modulation of protein function.nitrosothiols ͉ nitrosamines ͉ heme-nitrosyls ͉ ascorbate ͉ oxidative stress T he endogenous production of nitric oxide (NO) by NO synthase (NOS) has been established as playing an important role in vascular homeostasis, neurotransmission, and host defense mechanisms (1). Many of these actions are thought to be mediated by means of stimulation of soluble guanylyl cyclase (sGC) and formation of the second messenger, guanosine 3Ј,5Ј-cyclic monophosphate (cGMP), after which NO is disposed in the form of inactive products, such as nitrite and nitrate. There is mounting evidence, however, that the above scenario represents only part of a broader array of alternative biochemical pathways through which NO can trigger or modulate cell signaling, including interaction with thiols and metals (2). Of those pathways, the best known is the concept of thiol nitrosation, often referred to as ''S-nitrosylation,'' § a posttranslational protein modification that occurs independent of the sGC͞cGMP pathway and could play a critical role in health and disease (3). S-nitroso species (RSNOs) have been implicated in controlling oxygen delivery to tissues, modulating the function or activity of transcription factors, enzymes, memb...

Endothelial NO production results in local formation of adducts that may act as storage forms of NO. Because little is known about their chemical nature, concentrations, and possible role in vascular biology, we sought to characterize those species basally present in rat aorta, using two independent approaches. In the first approach, tissue homogenates were analyzed by using chemiluminescenceand ion-chromatography-based techniques that allow trace-level quantification of NO-related compounds in complex biological matrices. In the second approach, NO stores were characterized by their ability to release NO when illuminated with light and subsequently relax vascular smooth muscle (photorelaxation). The latter included a careful assessment of action spectra for photorelaxation, taking into account the light-scattering properties of the tissue and the storage depletion rates induced by exposure to controlled levels of light. Biochemical analyses revealed that aortic tissues contained 10 ؎ 1 M nitrite, 42 ؎ 7 M nitrate, 40 ؎ 6 nM S-nitroso, and 33 ؎ 6 nM N-nitroso compounds (n ‫؍‬ 4 -8). The functional data obtained suggest that the NO photolytically released in the tissue originated from species with photophysical properties similar to those reported for low-molecular-weight S-nitrosothiols, as well as from nitrite. The relative contribution of these potential NO stores to the extent of photorelaxation was consistent with their concentrations detected biochemically in vascular tissue when their photoactivity was taken into account. We conclude that intravascular nitroso species and nitrite both have the potential to release physiologically relevant quantities of NO independent of enzymatic control by NO synthase. M uch attention has been devoted recently to the role of S-nitrosothiols (RSNO) in plasma and circulating erythrocytes, where they are believed to act as a buffer and transport system for NO that is involved in the regulation of vascular tone and blood flow (1-3). The existence of such transporters may have profound implications for the regulation of tissue perfusion, inasmuch as this system appears to operate independently of local enzymatic control via NO synthase. Considerably less attention has been paid to the presence of NO-related products in extraluminal compartments, such as cells of the vascular wall, where they arise as a consequence of endothelial NO production. It is conceivable that such tissue products could also contribute to local blood flow regulation and provide, e.g., additional antiadhesive protection, if bioactivated to regenerate NO. This may be of particular significance under conditions of endothelial dysfunction and in disease states known to be associated with impaired enzymatic NO production. Alternatively, they may represent useful diagnostic markers of nitrosative stress.First evidence for the existence of stored forms of NO has been derived from experiments on the relaxant effect of light on vascular smooth muscle (4). This phenomenon, known as ''photorelaxation,'' is now beli...

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