Nitrite as regulator of hypoxic signaling in mammalian physiology

Abstract

In this review we consider the effects of endogenous and pharmacological levels of nitrite under conditions of hypoxia. In humans, the nitrite anion has long been considered as metastable intermediate in the oxidation of nitric oxide radicals to the stable metabolite nitrate. This oxidation cascade was thought to be irreversible under physiological conditions. However, a growing body of experimental observations attests that the presence of endogenous nitrite regulates a number of signaling events along the physiological and pathophysiological oxygen gradient. Hypoxic signaling events include vasodilation, modulation of mitochondrial respiration, and cytoprotection following ischemic insult. These phenomena are attributed to the reduction of nitrite anions to nitric oxide if local oxygen levels in tissues decrease. Recent research identified a growing list of enzymatic and nonenzymatic pathways for this endogenous reduction of nitrite. Additional direct signaling events not involving free nitric oxide are proposed. We here discuss the mechanisms and properties of these various pathways and the role played by the local concentration of free oxygen in the affected tissue.

Figure 1

Kinetics of the reaction between human deoxyHb (50 μM) and nitrite (10 mM) at pH 7.4 and 37° C. (A) UV/VIS absorption spectra were deconvoluted to determine the percentage of each species as a function of time. DeoxyHb is observed to form equal amounts of metHb and iron-nitrosyl-Hb. Deviation from first order behavior is evident in the curve for decay of deoxyHb, having a sigmoidal shape. (B) The instantaneous rate of the reaction shown in panel A where the negative of the slope of the decay curve for deoxyHb is plotted as a function of time. (C) Nitrite (10 mM) was reacted with Hb (50 μM) at various oxygen tensions. The initial rate of the reaction is plotted. (A, B from (114), C from (111) with permission).

Figure 2

Oxygen-dependence of nitrite reductase activity of hemoglobin. As oxygen tension increases the amount of deoxyHb (blue trace) decreases while the amount of R-state Hb (and free hemes in R-state Hb tetramers, red trace) increases. The nitrire reductase activity (gray) depends on both of these factors and is maximal at the P₅₀. It should be noted that this figure is merely illustrative and the precise oxygen tension dependencies are more complex yet give a similar result. From (7) with permission.

Figure 3

The N₂O₃ forming reaction of nitrite and hemoglobin may regulate the export of NO from the erythrocyte. Hemoglobin deoxygenation (purple) occurs preferentially at the submembrane of the red blood cell as it traverses the arteriole. Nitrite reacts with deoxyHb to metHb and free NO. Much of this NO binds to hemes of deoxyHb or reacts with oxyHb to form nitrate and metHb. MetHb binds nitrite to form an adduct with some Fe²⁺-NO₂ character (Hb- ${\text{NO}}_2^{•}$). This species reacts quickly with NO to N₂O₃ which can diffuse out of the red cell, later forming NO or extracellular S-nitrosothiols. Low molecular weight nitrosothiols may contribute to exportable vasodilatory activity. (from (123) with permission).

Figure 4

Depending on ambient oxygen, myoglobin acts as a dioxygenase or as a nitrite-reductase. Under normoxia, oxymyoglobin acts as an NO-scavenger, protecting the mitochondria from inhibition by NO) (left). Under hypoxia, myoglobin changes its function from a dioxygenase to a nitrite-reductase. Now it converts nitrite to free NO (right), regulating mitochondrial respiration and myocardial function.

Figure 5

Depending on the substrate, nitrite may be reduced to free NO at the Molybdenum site of the XO enzyme. Process I is progressively inhibited by oxygen. Process II continues to operate even under normoxia.

Figure 6

Kinetics of NO formation in a single flask of cultured BEND3 cells under anoxia and normoxia. The MNIC yields are given as function of incubation time of the trapping experiment. The anoxic values (solid squares) were taken from (19) with permission. The accuracy of the MNIC yield is ca 10 %.

Figure 7

Possible sites of nitrite reduction in mitochondria at complexes III and IV of the respiratory chain. Abbreviations: R - Rotenone, M - Myxothiazol, A - Antimycin A, T - Thenoyltrifluoroacetone, CN – Cyanide, UQ – Ubiquinone, UQH2 – ubiquinol, Q^•−- semiquinone radical anion, Cyt – Cytochrome.

Figure 8

Many vegetables including spinach, lettuce and beetroot are extremely rich in inorganic nitrate. Ingested nitrate from dietary sources is rapidly absorbed in the small intestine and in the circulation it mixes with endogenous nitrate from the NO pathway. While much of the circulating nitrate is eventually excreted in urine, up to 25% is actively extracted by the salivary glands and concentrated in saliva. In the mouth commensal anaerobic bacteria effectively reduce nitrate to nitrite by the action of nitrate reductase enzymes. Until recently this entero-salivary circulation of nitrate and bacterial reduction to nitrite has received attention only because of nitrites ability to form potentially carcinogenic nitrosamines. However, the link between dietary nitrate and gastric cancer is still very uncertain despite more than 40 years of active research. More recently it was shown that in the acidic stomach nitrite is spontaneously decomposed to form nitric oxide and other bioactive nitrogen oxides which serve to regulate important physiological functions. Gastric NO helps to kill ingested pathogens and also protects the gastric mucosa against luminal aggressors via stimulation of mucosal blood flow and mucus generation. Moreover, much nitrite is also absorbed intact into the ciculation and can convert back to bioactive NO in blood and tissues. This enterosalivary nitrate circulation and serial reduction to nitrite and NO explains the recently described reduction in blood pressure seen after dietary supplementation with nitrate. It has been suggested (216) that the well-known cardioprotective effects of a diet rich in vegetables is related to their high nitrate content.

Figure 9

Schematic representation of the ranges of oxygenation where the various pathways for reduction of nitrite are active. For cytochrome P₄₅₀ no definite data exist yet, but this pathway is likely to function only in near complete absence of oxygen (cf section 7. D). The two different reaction processes for XO were explained in fig 5. The vertical lines indicate the boundaries between anoxia, hypoxia, normoxia and hyperoxia as defined in table III.