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.

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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...

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