NO-synthase independent NO generation in mammals

https://doi.org/10.1016/j.bbrc.2010.02.136Get rights and content

Abstract

Inorganic nitrate (NO3-) and nitrite (NO2-) are part of the nitrogen cycle in nature. To the general public these anions are generally known as undesired residues in the food chain with potentially carcinogenic effects. Among biologists, these inorganic anions have merely been viewed as inert oxidative end products of endogenous nitric oxide (NO) metabolism. However, recent studies surprisingly show that nitrate and nitrite can be metabolized in vivo to form nitric oxide (NO) and other bioactive nitrogen oxides. This represents an important alternative source of NO especially during hypoxia when the oxygen-dependent l-arginine–NO pathway can be altered. A picture is now emerging suggesting important biological functions of the nitrate–nitrite–NO pathway with profound implications in relation to the diet and cardiovascular homeostasis. Moreover, an increasing number of studies suggest a therapeutic potential for nitrate and nitrite in diseases such as myocardial infarction, stroke, hypertension, renal failure and gastric ulcers.

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 (NH4+) formed by N2 fixation to nitrite (NO2-) and nitrate (NO3-), 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.

References (93)

  • S. Carlsson et al.

    Effects of pH, nitrite, and ascorbic acid on nonenzymatic nitric oxide generation and bacterial growth in urine

    Nitric Oxide

    (2001)
  • B. Gago et al.

    Red wine-dependent reduction of nitrite to nitric oxide in the stomach

    Free Radic. Biol. Med.

    (2007)
  • T.M. Millar et al.

    Xanthine oxidoreductase catalyses the reduction of nitrates and nitrite to nitric oxide under hypoxic conditions

    FEBS Lett.

    (1998)
  • Z. Zhang et al.

    Generation of nitric oxide by a nitrite reductase activity of xanthine oxidase: a potential pathway for nitric oxide formation in the absence of nitric oxide synthase activity

    Biochem. Biophys. Res. Commun.

    (1998)
  • B.L. Godber et al.

    Reduction of nitrite to nitric oxide catalyzed by xanthine oxidoreductase

    J. Biol. Chem.

    (2000)
  • J.L. Zweier et al.

    Mechanisms of nitrite reduction to nitric oxide in the heart and vessel wall

    Nitric Oxide

    (2010)
  • A.V. Kozlov et al.

    Nitrite reductase activity is a novel function of mammalian mitochondria

    FEBS Lett.

    (1999)
  • R. Tischner et al.

    Mitochondrial electron transport as a source for nitric oxide in the unicellular green alga Chlorella sorokiniana

    FEBS Lett.

    (2004)
  • A. Dejam et al.

    Emerging role of nitrite in human biology

    Blood Cells Mol. Dis.

    (2004)
  • J.E. Baker et al.

    Nitrite confers protection against myocardial infarction: role of xanthine oxidoreductase, NADPH oxidase and K(ATP) channels

    J. Mol. Cell. Cardiol.

    (2007)
  • J.O. Lundberg et al.

    Cardioprotective effects of vegetables: is nitrate the answer?

    Nitric Oxide

    (2006)
  • P.R. Castello et al.

    Mitochondrial cytochrome oxidase produces nitric oxide under hypoxic conditions: implications for oxygen sensing and hypoxic signaling in eukaryotes

    Cell Metab.

    (2006)
  • S. Basu et al.

    Nitrite reductase activity of cytochrome c

    J. Biol. Chem.

    (2008)
  • X. Liu et al.

    Nitric oxide inhalation improves microvascular flow and decreases infarction size after myocardial ischemia and reperfusion

    J. Am. Coll. Cardiol.

    (2007)
  • M. Govoni et al.

    The increase in plasma nitrite after a dietary nitrate load is markedly attenuated by an antibacterial mouthwash

    Nitric Oxide

    (2008)
  • J. Petersson et al.

    Gastroprotective and blood pressure lowering effects of dietary nitrate are abolished by an antiseptic mouthwash

    Free Radic. Biol. Med.

    (2009)
  • J.O. Lundberg et al.

    Nitrate, bacteria and human health

    Nat. Rev. Microbiol.

    (2004)
  • S. Moncada et al.

    The l-arginine–nitric oxide pathway

    N. Engl. J. Med.

    (1993)
  • M. Kelm et al.

    Serum nitrite sensitively reflects endothelial NO formation in human forearm vasculature: evidence for biochemical assessment of the endothelial l-arginine–NO pathway

    Cardiovasc. Res.

    (1999)
  • M.T. Gladwin et al.

    Role of circulating nitrite and S-nitrosohemoglobin in the regulation of regional blood flow in humans

    Proc. Natl. Acad. Sci. USA

    (2000)
  • D.J. Green et al.

    Effect of exercise training on endothelium-derived nitric oxide function in humans

    J. Physiol.

    (2004)
  • T.V. Lewis et al.

    Exercise training increases basal nitric oxide production from the forearm in hypercholesterolemic patients

    Arterioscler. Thromb. Vasc. Biol.

    (1999)
  • L. Jungersten et al.

    Both physical fitness and acute exercise regulate nitric oxide formation in healthy humans

    J. Appl. Physiol.

    (1997)
  • M. Herulf et al.

    Increased nitric oxide in infective gastroenteritis

    J. Infect. Dis.

    (1999)
  • Å. Wennmalm et al.

    Nitric oxide synthesis and metabolism in man

    Ann. NY Acad. Sci.

    (1994)
  • J.O. Lundberg et al.

    Intragastric nitric oxide production in humans: measurements in expelled air

    Gut

    (1994)
  • N. Benjamin et al.

    Stomach NO synthesis

    Nature

    (1994)
  • R. Dykhuizen et al.

    Antimicrobial effect of acidified nitrite on gut pathogens: importance of dietary nitrate in host defence

    Antimicrob. Agents Chemother.

    (1996)
  • R.S. Dykhuizen et al.

    Helicobacter pylori is killed by nitrite under acidic conditions

    Gut

    (1998)
  • H.H. Bjorne et al.

    Nitrite in saliva increases gastric mucosal blood flow and mucus thickness

    J. Clin. Invest.

    (2004)
  • J. Petersson et al.

    Dietary nitrate increases gastric mucosal blood flow and mucosal defense

    Am. J. Physiol. Gastrointest. Liver Physiol.

    (2007)
  • M. Miyoshi et al.

    Dietary nitrate inhibits stress-induced gastric mucosal injury in the rat

    Free Radic. Res.

    (2003)
  • T. Sobko et al.

    Gastrointestinal nitric oxide generation in germ-free and conventional rats

    Am. J. Physiol. Gastrointest. Liver Physiol.

    (2004)
  • H. Bjorne et al.

    Intragastric nitric oxide is abolished in intubated patients and restored by nitrite

    Crit. Care Med.

    (2005)
  • J.L. Zweier et al.

    Enzyme-independent formation of nitric oxide in biological tissues

    Nat. Med.

    (1995)
  • E.E. van Faassen et al.

    Nitrite as regulator of hypoxic signaling in mammalian physiology

    Med. Res. Rev.

    (2009)
  • Cited by (119)

    • Nitrate: The Dr. Jekyll and Mr. Hyde of human health?

      2023, Trends in Food Science and Technology
    View all citing articles on Scopus
    View full text