Nitrite signaling in pulmonary hypertension: mechanisms of bioactivation, signaling, and therapeutics

Abstract

Significance: Pulmonary arterial hypertension (PAH) is a disorder characterized by increased pulmonary vascular resistance and mean pulmonary artery pressure leading to impaired function of the right ventricle, reduced cardiac output, and death. An imbalance between vasoconstrictors and vasodilators plays an important role in the pathobiology of PAH.

Recent advances: Nitric oxide (NO) is a potent vasodilator in the lung, whose bioavailability and signaling pathway are impaired in PAH. It is now appreciated that the oxidative product of NO metabolism, the inorganic anion nitrite (NO(2)(-)), functions as an intravascular endocrine reservoir of NO bioactivity that can be reduced back to NO under physiological and pathological hypoxia.

Critical issues: The conversion of nitrite to NO is controlled by coupled electron and proton transfer reactions between heme- and molybdenum-containing proteins, such as hemoglobin and xanthine oxidase, and by simple protonation and disproportionation, and possibly by catalyzed disproportionation. The two major sources of nitrite (and nitrate) are the endogenous L-arginine-NO pathway, by oxidation of NO, and the diet, with conversion of nitrate from diet into nitrite by oral commensal bacteria. In the current article, we review the enzymatic formation of nitrite and the available data regarding its use as a therapy for PAH and other cardiovascular diseases.

Future directions: The successful efficacy demonstrated in several animal models and safety in early clinical trials suggest that nitrite may represent a promising new therapy for PAH.

FIG. 1.

Radiological imaging in PH. (A) Contrast enhanced-CT image showing enlarged pulmonary artery in PAH patient. (B) CT image obtained from patient with severe PH. The right-sided chambers are dilated, the right ventricle hypertrophied. (C) Apical four-chamber two-dimensional echocardiography image showing enlarged right-sided chambers and small left ventricle. CT, computed tomography; LA, left atrium; LV, left ventricle; PA, pulmonary artery; PAH, pulmonary arterial hypertension; PH, pulmonary hypertension; RA: right atrium; RV, right ventricle.

FIG. 2.

The classical l-arginine-NOS-NO signaling pathway. NO is produced in mammalian cells by an oxygen-dependent oxidation of a guanidine nitrogen of l-arginine (with citrulline as a side product). This multistep reaction is catalyzed by the heme-containing protein NOS, which also requires two flavin molecules and tetrahydrobiopterin as cofactors. In most endothelial cells, the type III isoform is expressed (or eNOS) as is regulated by calcium-dependent binding of calmodulin and by tyrosine phosphorylation. Target tissue effects of NO depend on its quantity. At higher concentrations, NO rapidly reacts with oxygen (and especially with superoxide), forming the highly reactive peroxynitrite. At lower concentrations, NO serves a regulatory role via the activation of soluble guanylate cyclase, resulting in increased cGMP levels in target cells. In vascular smooth muscle, cGMP causes relaxation by reducing the intracellular calcium concentration and by downregulating the contractile apparatus. These actions are mostly (although not exclusively) mediated by type I cGMP-dependent protein kinases. cGMP, cyclic guanosine monophosphate; eNOS, endothelial NOS; NO, nitric oxide; NOS, nitric oxide synthase. (To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars.)

FIG. 3.

The enterosalivary circulation of nitrate in humans. The activity of orally ingested inorganic nitrate (from dietary sources—entry point as a green arrow) is thought to lie in its conversion to nitrite by facultative anaerobic bacteria found on the dorsal surface of the tongue. By swallowing, saliva enters the acidic stomach (around 1 L per day), where much of the nitrate is rapidly protonated to form nitrous acid, which decomposes further to form NO (absorption arrow in blue) and other bioactive nitrogen species. Nitrate and remaining nitrite are then absorbed from the intestine into the circulation and can be converted to bioactive NO in blood (absorption arrow in blue). Later on, NO can again be oxidized to nitrite and/or nitrate in tissue. Although much of the nitrate is excreted in the urine (excretion arrow in red), salivary glands actively concentrate nitrate from plasma that can go back again to the mouth to be reduced to nitrite. (To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars.)

FIG. 4.

Nitrite chemistry, physiology, and therapeutics. Nitrite reduction to NO is favored by decreasing physiological oxygen tensions and low pH, via nonenzymatic pathways (in the presence of acid or reducing substrates) or enzymatic pathways catalyzed by metal-containing enzymes. The generated NO modulates critical signal transduction processes, inducing cytoprotection, vasodilation, and inhibition of SMC proliferation (30). SMC, smooth muscle cell. (To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars.)

FIG. 5.

Sites of mitochondrial nitrite reduction. Upper panel: In normoxia, electrons enter the respiratory chain at complex I or II and are shuttled through the Q cycle to complex III. Electrons are then shuttled to cytochrome c and then to complex IV, where oxygen acts as the terminal electron acceptor. Protons are pumped from the matrix to the intermembrane space through the complexes to set up a proton gradient for ATP generation. Lower panel: During hypoxia, nitrite can be reduced at complex III (7) or cytochrome c oxidase (complex IV) (12). If cytochrome c is converted to its pentacoordinate form (through oxidation, nitration, or association with anionic lipid), it can reduce nitrite to NO (6, 72). (To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars.)

FIG. 6.

Neuroglobin acts as a nitrite reductase under oxidative stress conditions. The thiol state of the glutathione is a good indicator of the oxidative stress in vivo conditions and can be modulated in vitro. In normal conditions, cells keep a high concentration of reduced glutathione (GSH) and low oxidized glutathione (GSSG). In these circumstances, the disulfide bond of Ngb is not formed, and the protein has a low nitrite reductase activity (left). As oxidative stress conditions develop (right), reduced glutathione is consumed, and the number of neuroglobin molecules with formed disulfide bonds increases. This leads to increased production of NO from nitrite, causing the inhibition of respiratory enzymes and limiting oxygen consumption and reactive oxygen species-producing reactions [reproduced with permission from (77)]. (To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars.)

FIG. 7.

The nitrite reductase activity of XOR and AO can be inhibited at either molybdenum or flavin site. Oxypurinol or high concentrations of xanthine inhibit nitrite reduction by binding to the molybdenum center of XOR. AO can be inhibited specifically by raloxifene, which binds to the molybdenum site. Electron can be provided at the flavin site, which is transferred via Fe₂S₂ clusters to the molybdenum site. DPI blocks the electron transfer at the flavin site, thus inhibiting the nitrite reductase activity of XOR or AO. The relative distances of the four redox-active centers were taken from the crystal structure of the bovine XOR (PDB code: 1fo4). AO, aldehyde oxidase; DPI, diphenylene iodonium chloride; XOR, xanthine oxidoreductase. (To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars.)

FIG. 8.

Comparison of the physiological effects of nebulized nitrite versus NO. Duration of effect of NO gas inhalation (A) or nitrite nebulization (B) on hemodynamic and metabolic measurements during hypoxic-induced PH. Treatment with nitrite aerosol resulted in a rapid sustained reduction in hypoxic-induced pulmonary vasoconstriction and a graded increase in exhaled NO gas concentration with no change in mean arterial blood pressure. These results are contrasted to the rapid return of pulmonary artery pressure to hypoxic baseline after termination of inhaled NO gas. Methemoglobin concentrations increased after nitrite nebulization from 2.1±0.1% to 2.8±0.8%. Note that exhalated NO concentrations reach the limit of detection during NO inhalation. [reproduced with permission from (38)]. (To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars.)

FIG. 9.

Nitrite-induced SMC proliferation is dependent on XOR. Hypoxia-induced PAH is inhibited by NO generation from nitrite, which is catalyzed by XOR. NO production from nitrite increases expression of p21, which inhibits SMC proliferation. Allopurinol or tungsten diet treatment inhibits XOR activity (85, 86).

FIG. 10.

Nitrite mediates cytoprotection in ischemia/reperfusion injury. Nitrite potently mediates cytoprotection after ischemia/reperfusion (I/R) through the transient inhibition of complex I (via S-nitrosation) and subsequent limitation of oxidative damage. Because oxidants have been shown to sensitize the permeability transition pore, cytoprotective effects of nitrite could in part be caused by nitrite-dependent protection against pore opening after I/R (72). (To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars.)