NO-synthase independent NO generation in mammals
Introduction
Nitrogen (N2) is the most abundant element in the atmosphere. In the Nitrogen cycle atmospheric nitrogen is fixed into forms usable by living organisms. Bacteria oxidize the ammonia () formed by N2 fixation to nitrite () and nitrate (), in a process termed nitrification. The nitrogen cycle is completed by the denitrification process, wherein nitrate is converted back to N2 in a series of reductions catalyzed by anaerobic bacteria. In this pathway nitric oxide (NO) is an intermediate [1].
While bacteria generate NO by reduction under anaerobic conditions, mammals instead utilize oxidation reactions to produce this gas. Mammalian NO-synthases catalyze an oxygen-dependent five-electron oxidation of the amino acid l-arginine to form NO and l-citrulline. Extensive research during the past two decades has established NO as a critical regulator of vascular homeostasis, neurotransmission, redox signaling, cell respiration, and host defence [2].
Until recently, it was thought that the NO-synthases were exclusively responsible for the formation of NO in mammals. This view has now changed.
Here we discuss a previously unknown mammalian nitrogen cycle, in which the inorganic anions nitrate and nitrite are converted back to NO and other bioactive nitrogen oxides in blood and tissues. Interestingly, commensal bacteria play a central role in the first reductive step converting nitrate to nitrite, and then a number of mammalian enzymes and proteins participate in the formation of NO from nitrite. This nitrate–nitrite–NO pathway has attracted substantial scientific interest and its role in physiological processes, therapy and nutrition is currently under investigation.
Section snippets
Sources of nitrate and nitrite
The nitrate and nitrite that we are exposed to derive from endogenous as well as dietary sources (Fig. 1). The main dietary source of nitrate is vegetables, which typically account for up to 90% of the daily nitrate intake. Green leafy vegetables such as lettuce and spinach are particularly rich in nitrate. Nitrite is also found in some foodstuffs — for example, it is used as a food additive in meat to prevent the growth of Clostridium botulinus and to enhance its taste and appearance. The
The enterosalivary circulation of nitrate
After ingestion, nitrate is rapidly and effectively absorbed proximally from the gastrointestinal tract into the bloodstream, where it mixes with endogenously synthesized nitrate. Peak plasma concentrations are seen within 60 min of nitrate ingestion and the half-life of nitrate in plasma is 5–6 h. For as-yet-unknown reasons, the concentrations of nitrate excreted in saliva are exceptionally high; up to 25% of plasma nitrate is actively taken up by the salivary glands and secreted with saliva [15]
NO-synthase independent generation of nitric oxide
NO-synthase independent generation of nitric oxide was first described in the stomach [16], [17]. In 1994, two independent groups could show that NO and other reactive nitrogen oxides were generated non-enzymatically in large amounts following protonation of nitrite in swallowed saliva. Human stomach NO production was highly pH-dependent and could be almost abolished by proton pump inhibitors. The levels of NO in the stomach were in the range 10–100 ppm, i.e., several orders of magnitude higher
Systemic NO generation from nitrite
Soon after the discovery of gastric NOS-independent NO generation, Zweier et al. demonstrated profound NO generation in rat ischemic heart muscle which could not be effectively blocked by pharmacological NOS inhibitors [29]. They then demonstrated that N15-labeled nitrite was reduced to NO. During global cardiac ischemia tissue pH fell below 6 and under these conditions reduction of nitrite to NO was greatly enhanced. Subsequent studies have demonstrated numerous pathways for nitrite reduction
Mechanisms of nitrite reduction
Clearly, a variety of proteins and enzymes can catalyze nitrite reduction to NO in blood and tissues (Fig. 1). In addition to this, nitrite reduction may be further enhanced by reducing agents such as vitamin C [31], polyphenols [32], and thiocyanate [33]. In blood deoxyhemoglobin has been shown to be an allosterically regulated nitrite reductase [10], [34], [35] and in cardiac muscle deoxymyoglobin can act as a nitrite reductase [36], [37]. Xanthine oxidase (XO) is structurally related to
Vasodilatory effects of nitrite
The vasodilating properties of inorganic nitrite have been known for long. Sodium and potassium nitrite were used in the beginning of the 20th century as antihypertensives and Robert Furchgott used acidified sodium nitrite to relax preconstricted rabbit aortic rings as early as 1953 [53]. However in these early experiments the nitrite concentrations and the acidity used were far outside physiological levels and the mechanism of dilatation (NO generation) was unknown. More recently, Modin et al.
Therapeutic effects of nitrite in ischemia–reperfusion injury
As discussed above the pathways for systemic nitrite reduction are greatly enhanced during hypoxic/ischemic conditions. Several studies in animal models of ischemia and reperfusion indicate that nitrite and nitrate can modulate hypoxic signaling. Administration of nitrite as well as nitrate protect against ischemia–reperfusion injury in liver [59], [60], heart [59], [61], [62], [63], brain [64], kidney [65], and chronic hind-limb ischemia [66] (Table 1).
The mechanism of nitrite-mediated
Bioactivation of inorganic nitrate
As discussed above there are several pathways for the systemic conversion of nitrite to NO and other bioactive nitrogen oxides. While nitrite circulates at nanomolar levels, the levels of nitrate are about 20–40 μM, i.e., 100 times higher [79]. So in theory nitrate would be an even greater circulating pool of potential NO bioactivity than nitrite, provided that mechanisms existed for its reduction. The existence of an in vivo reduction of nitrate is clear from a study by Lundberg and Govoni [6]
Inorganic nitrate and the cardiovascular system
Larsen and colleagues recently demonstrated a reduction in blood pressure in healthy volunteers after three days treatment with inorganic nitrate [85], and the year after they showed that dietary nitrate decreases whole body oxygen consumption in humans during submaximal exercise [86]. This latter finding was recently confirmed [87], [88] but the mechanism is still to be elucidated. In a recent study, Webb and colleagues showed that blood pressure decreases if healthy volunteers ingest a
Nutritional aspects of inorganic nitrate
Hypertension affects approximately 1 billion individuals worldwide and remains the most common risk factor for cardiovascular morbidity and mortality [92]. A diet rich in fruits and vegetables is associated with a lower blood pressure and reduced risk of cardiovascular events. Yet, despite extensive research the active ingredient(s) responsible for these effects has not been pinpointed and trials with single nutrients have been largely unsuccessful. Remarkably, in the aforementioned study by
Conclusions
In recent years, we have witnessed a substantial increase in our understanding of the mammalian nitrate–nitrite–NO pathway. This pathway may now be considered as an important alternative provider of NO besides the “classical”l-arginine–NO pathway. An important feature of the nitrate–nitrite–NO pathway is the augmentation by hypoxia which ensures NO at a wide range of oxygen levels. The redundancy in pathways that convert nitrite to NO indicates its importance in regulating physiological
Acknowledgments
The authors acknowledge the generous support from EUs 7th Framework Program (Flaviola), the Torsten and Ragnar Söderberg Foundation, Vinnova (CIDaT), the Swedish Heart and Lung Foundation, Stockholm City Council (ALF) and the Swedish Research Council.
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