The Key Role of Nitric Oxide in Hypoxia: Hypoxic Vasodilation and Energy Supply

Abstract

Significance: A mismatch between energy supply and demand induces tissue hypoxia with the potential to cause cell death and organ failure. Whenever arterial oxygen concentration is reduced, increases in blood flow—hypoxic vasodilation—occur in an attempt to restore oxygen supply. Nitric oxide (NO) is a major signaling and effector molecule mediating the body's response to hypoxia, given its unique characteristics of vasodilation (improving blood flow and oxygen supply) and modulation of energetic metabolism (reducing oxygen consumption and promoting utilization of alternative pathways). Recent Advances: This review covers the role of oxygen in metabolism and responses to hypoxia, the hemodynamic and metabolic effects of NO, and mechanisms underlying the involvement of NO in hypoxic vasodilation. Recent insights into NO metabolism will be discussed, including the role for dietary intake of nitrate, endogenous nitrite (NO₂ ⁻) reductases, and release of NO from storage pools. The processes through which NO levels are elevated during hypoxia are presented, namely, (i) increased synthesis from NO synthases, increased reduction of NO₂ ⁻ to NO by heme- or pterin-based enzymes and increased release from NO stores, and (ii) reduced deactivation by mitochondrial cytochrome c oxidase. Critical Issues: Several reviews covered modulation of energetic metabolism by NO, while here we highlight the crucial role NO plays in achieving cardiocirculatory homeostasis during acute hypoxia through both vasodilation and metabolic suppression. Future Directions: We identify a key position for NO in the body's adaptation to an acute energy supply–demand mismatch. Antioxid. Redox Signal. 19, 1690–1710.

Introduction

P atients undergoing major surgery or suffering critical illness have increased cellular energy demands yet commonly experience challenges in ensuring an adequate oxygen supply. An oxygen supply–demand imbalance can be immediately life threatening, such as in cardiac arrest or major hemorrhage, or it may cause more insidious damage. As organ function is critically linked to both oxygen availability (30) and adequate utilization, bioenergetic deficiency is likely to be a crucial factor underlying the pathogenesis of cell death (39) and multiple organ failure (18, 202).

The main purpose of the cardiovascular system is to provide oxygen and metabolic substrates at a rate that can both meet and respond rapidly to changes in local demand. Whenever arterial oxygen concentration is reduced, increases in local blood flow—hypoxic vasodilation—occur in an attempt to restore oxygen supply (102). The ability of hypoxia to increase tissue blood flow is a local response that can be demonstrated in isolated organs; thus, it can occur without the involvement of neurally mediated reflex mechanisms. A variety of mediators, such as adenosine (22), adenosine triphosphate (ATP)-sensitive potassium channels (65), and prostaglandins (173), are implicated; however, significant human and animal data point toward nitric oxide (NO) as a major regulator of vascular perfusion and matching of energy supply and demand (24, 25, 76, 119, 147, 190). Nevertheless, considerable controversy persists with respect to its sources and mechanisms of action.

Several reviews have covered modulation of energetic metabolism by NO (33, 219, 232). In this article we highlight the crucial role NO plays in achieving cardiocirculatory homeostasis during acute hypoxia through both vasodilation and metabolic suppression, and the various mechanisms through which NO production is enhanced. We identify a key position for NO in the body's adaptation to an acute energy supply–demand mismatch.

Bioenergetic Reactions and the Role of Oxygen

Living cells constantly perform work to maintain their structures, synthesize cellular components, generate transmembrane ionic gradients, and to undertake their physiologic roles. As most of these metabolic processes are thermodynamically unfavorable, they are coupled directly or indirectly to the hydrolysis of ATP to provide the necessary free energy. ATP supply is maintained through mitochondrial and cytoplasmic reactions that proceed with and without the need for oxygen (32). With its net yield of 2 moles ATP per mole of glucose, glycolysis is generally insufficient to maintain steady-state energy levels in most cell types. Exceptionally, some cells (e.g., erythrocytes) rely solely upon glycolytic production of ATP. Many immune cells use glucose and glutamine as their primary fuel sources, although ketone bodies and fatty acids can be used to a lesser degree (168). Glucose appears to be particularly necessary for cell survival, size, activation, and cytokine production. Resting lymphocytes have low-energy needs and derive most of their ATP from oxidative phosphorylation; switching to the activated state requires a dramatic increase in metabolism, which is mainly derived from rapid upregulation of glycolysis (80). Organs also vary in their predominant energy substrate. Whereas most utilize carbohydrates as their major energy source, some (e.g., cardiomyocytes) predominantly use fatty acids. Notably, this preference can change with a reduction in oxygen availability.

Pathways leading to ATP synthesis have been extensively studied (145). Briefly, the glycolytic end-product pyruvate, and fatty acids enter mitochondria where they are metabolized to acetyl CoA. This enters the tricarboxylic acid (Krebs') cycle whereby oxidation reactions directly produce ATP equivalents and electron donors. Electrons are transported down the respiratory chain, generating a proton gradient across the inner mitochondrial membrane, which serves as the driving force for phosphorylation of adenosine diphosphate to ATP. By comparison with glycolysis, 1 mole of glucose liberates ∼30 moles of ATP. Oxygen is crucially required as the terminal electron acceptor by the last complex of the chain, cytochrome c oxidase (CcO, complex IV) (44). In the steady state, ∼90% of total body O₂ consumption (VO₂) occurs within mitochondria (204), and is primarily directed toward oxidative phosphorylation. A constant O₂ supply is thus critical for continued cell function and survival. When compromised (e.g., supply reduced and/or demand excessively increased), a state of metabolic crisis ensues (231) with potential activation of cell death pathways.

Oxygen and nutrients required by mammalian cells to support metabolism cannot be directly obtained in sufficient quantity by diffusion alone. From an evolutionary perspective, this limitation was resolved by the development of a cardio-respiratory system whose activity is closely regulated such that, in the steady state, ventilation delivers oxygen to the alveolar capillaries at the same rate of delivery by the vasculature to the tissues, and of consumption by metabolic processes within the tissues (233). The quantity of oxygen delivered to tissues (DO₂) depends on arterial oxygen content (mainly carried by hemoglobin [Hb]) and cardiac output. Regulation and distribution of cardiac output is driven by regional VO₂ that proceeds at a rate set by tissue metabolic activity. If DO₂ is reduced, VO₂ is initially maintained by increased O₂ extraction. If delivery is reduced further, a critical point is reached below which tissue extraction cannot increase any further, leading to a fall in VO₂ (212). Several complementary macro- and microcirculatory mechanisms act to prevent the onset of tissue hypoxia in the face of a reduced DO₂. These include a redistribution of blood flow to vital organs, increased recruitment of perfused microvessels to facilitate O₂ availability (233), as depicted in Figure 1, and, as discussed later, a decrease in tissue utilization (metabolism).

FIG. 1.

Oxygen supply–demand relationship. The relationship between oxygen delivery (DO₂) to cells and oxygen consumption (VO₂) is nonlinear. Initial reduction in the amount of DO₂ are compensated by a series of adaptive mechanisms. If delivery is reduced further, a critical point is reached (DO₂ _crit) below which tissue extraction cannot increase any further, leading to a fall in consumption. Several complementary macro- and microcirculatory mechanisms act to prevent the onset of tissue hypoxia in the face of a reduced delivery, and many of them are mediated by nitric oxide (NO).

Sensing Hypoxia

How eukaryotic cells sense reductions in pO₂ remains contentious. Five main mechanisms have been proposed (132) based on, respectively, heme-based proteins, O₂-sensitive ion channels, adenosine monophosphate-activated protein kinase (AMPK), NADPH oxidase, and mitochondria. Thus, hypoxia may be detected by an allosteric shift toward a deoxy-configuration in proteins capable of reversibly binding O₂ at a heme site, or by ion channels affected by local pO₂, as has been shown in carotid body cells where hypoxia can inhibit a specific K⁺ current. Reduced O₂ levels increase the AMP:ATP ratio; thus, at sufficient magnitude, AMPK are induced, modulating cellular metabolism at various levels via target protein phosphorylation. With molecular O₂, NADPH oxidase or other nonmitochondrial enzymes such as xanthine oxidase or flavin-containing dehydrogenases generate superoxide (O₂ ⁻), providing a second messenger that may regulate cellular activity through redox modifications. Finally, increased mitochondrial reactive oxygen species (ROS) production during hypoxia may result in mitochondrial O₂ sensing through changes in redox state of the electron transport chain, though without necessarily affecting respiration (50, 72, 96). Mitochondria may play a critical role in oxygen sensing. This model has been controversial as previous studies, which mainly relied on pharmacologic tools, produced conflicting reports (223). However, recent studies using genetic and biochemical approaches have provided evidence for a role of mitochondrial ROS in oxygen sensing and hypoxia-inducible transcription factor-1α (HIF-1α) activation (38). Indeed, blocking superoxide anion production by suppressing the Rieske iron–sulfur protein of complex III impairs HIF-1α induction by hypoxia, whereas hydrogen peroxide or agents that produce ROS activate HIF-1α during normoxia (95). These data indicate that mitochondria can function as O₂ sensors and stabilize HIF-1α during hypoxia by releasing ROS to the cytosol (96).

The HIF-1 pathway is central to the body's innate response to the stressful condition of hypoxia. HIF is a heterodimer composed of α and β subunits that induces expression of multiple genes that promote adaptation and survival (214). The β subunit is constitutively expressed, while α subunit expression is tightly regulated by the local oxygen tension through the action of prolyl hydroxylase (PHD). When O₂ tension falls below a critical threshold, proline residues cannot be hydroxylated. This prevents ubiquitination, allowing the α-subunit to accumulate and hetero-dimerize with HIF-1β. The heterodimer can then bind to specific DNA regions within the nucleus, exerting its regulatory activities. The PHDs are considered effective O₂ sensors in their own right as their Km values for oxygen are above atmospheric O₂ concentrations (110). This allows small changes in O₂ supply to affect the enzyme's activity.

For responses to occur, a decrease in PO₂ must be detected by an O₂ sensor that activates signaling pathways triggering functional responses. In general, adaptation to acute changes in O₂ concentration (lasting from seconds to minutes) principally occur as a result of alterations of pre-existing proteins (e.g., involving phosphorylation or changes in redox state), whereas chronic changes (lasting from minutes to hours or longer) mainly occur through altered gene expression.

Responding to Hypoxia

Transcriptomic

Given oxygen's essential role in cellular metabolism, a wide array of responses has evolved to cope with situations of oxygen supply–demand mismatch. Functional adaptation occurs at systemic, tissue, and cellular levels, ultimately leading to a new phenotype that enhances the likelihood of survival (211). Such adaptation depends both on the modulation of activity of various enzymatic systems by metabolic messengers (e.g., pH, phosphate potential, redox potential), and on altered gene transcription with increased expression of genes encoding, for example, growth factors (e.g., vascular endothelial growth factor [VEGF], platelet-derived growth factor-β), cytokines (e.g., interleukin [IL]-1, IL-8), endothelin, and adhesion molecules (e.g., vascular cell adhesion molecule-1, intercellular adhesion molecule-1).

The AMPK system is a well-conserved pathway for maintaining the balance between energy production and utilization (103). Triggered by an increase in AMP:ATP ratio, this system switches on an energy-preserving phenotype, both rapidly through phosphorylating metabolic enzymes, and by a longer-term adaptation through regulating gene expression via phosphorylation of transcription factors and co-activators. AMPK targets include carbohydrate homeostasis, lipid metabolism, protein synthesis, mitochondrial biogenesis, cell signaling, proliferation, gene expression, and transmembrane ion transport (128).

Responses to hypoxia that involve induction or repression of gene expression are mainly mediated by HIF-1 (214). Three isoforms of HIFα have been characterized, of which HIF-1α and HIF-2α are the most structurally similar and best studied. HIF-3α can be found as multiple splice variants, some of which can even inhibit activity of HIF-1α and HIF-2α (169). While HIF-1α is expressed ubiquitously in all cells, the other isoforms are only selectively expressed in certain tissues, such as vascular endothelium, lungs, and kidney. Activation of HIF-1 and HIF-2 can regulate expression of many other genes induced by hypoxia, such as VEGF, a potent angiogenic factor that contributes to long-term adaptation to hypoxia through new blood vessel formation (130). However, each HIF isoform may have their unique targets offering different adaptive pathways to hypoxia (160); HIF-1 preferentially induces genes coding for the glycolytic pathway, whereas HIF-2 is involved in regulation of genes important for cell cycle progression and induction of erythropoietin (161). The two HIF isoforms also have distinct and somewhat opposing roles to NO regulation in macrophages. While HIF-1 promotes inducible nitric oxide synthase (iNOS) expression and increases NO production, HIF-2 promotes arginase expression, reducing the amount of arginine available for NO synthesis (229). This may offer a balancing regulatory mechanism for NO homeostasis.

Apart from O₂ tension many other factors govern HIFα stability, including microRNAs and post-translational modifications such as acetylation (91). In addition to O₂, PHDs require Fe²⁺, 2-oxoglutarate, and ascorbate to exert their activity, but may be inhibited by NO, Krebs' cycle intermediates and ROS (126). To date, >200 HIF gene targets have been identified, including those encoding for proteins involved in angiogenesis, energy metabolism, erythropoiesis, cell proliferation and viability, vascular remodeling, and vasomotor responses (214).

MicroRNAs, specific small noncoding RNA sequences, also appear to be involved in the hypoxic response (140). These 19- to 24-ribonucleotide sequences, once transferred to the cytoplasm, inhibit target gene expression by translational repression and/or mRNA degradation. A common characteristic of the different microRNAs involved in hypoxic signaling is their dependence upon HIF (141). HIF may thus be the main modulator of the hypoxic response, either through direct gene induction or by indirect microRNA-mediated gene repression (140). On the other hand, microRNAs may act as positive and negative feedback regulators of HIF-mediated responses (106). miR-210 is consistently upregulated in hypoxia and may play a central role in hypoxic signaling by modulating factors implicated in various pathways, for example, downregulating expression of different components of the mitochondrial electron transport chain and the Krebs' cycle, interfering with membrane trafficking, modulating migration and adhesion, differentiation, and cell cycle (67).

Hemodynamic effects

At the systemic level, adaptation to hypoxemia affects many systems although the most evident changes involve the cardiocirculatory system. This response is composed of essentially unopposed local vasodilation in the heart and brain, and of a balance between the competing effects of locally induced vasodilation and reflex chemoreceptor-sensed, sympathetic-mediated vasoconstriction in other tissues, for example, kidney, and skeletal muscle (206). This response pattern attempts to maintain an adequate O₂ supply–demand ratio, compensating for any reduction in arterial O₂ content, while preserving arterial perfusion pressure. Over 130 years ago, Roy and Brown recognized that interrupting tissue perfusion produced a local, non-neurally mediated increase in blood flow, and that blood vessels could vary their diameter independently in response to local metabolic needs (208). Indeed, the main hemodynamic effect of hypoxia is systemic vasodilation with increases in cardiac output and heart rate, and redistribution of regional perfusion with increased coronary, carotid, and hepatic and decreased renal blood flow. The sympathetic nervous system is activated with increased catecholamine levels but a reduced response to exogenous vasopressors (55, 98, 104, 156, 194, 206, 207). Increases in forearm blood flow, a reduced vasomotor reflex, and a reduced response to exogenous norepinephrine and angiotensin were noted in healthy volunteers made hypoxemic (104).

Respiratory

Haldane noted how hypoxia induced a rapid, shallow type of breathing in humans (101). The predominant ventilatory response is an increase in respiratory rate with a rise in bronchiolar tone (127), a response mainly mediated via hypoxemia-responsive peripheral chemoreceptors in the carotid and aortic bodies.

Renal and endocrine

Hypoxemia redistributes blood flow away from the kidney, which, in response, increases blood volume via an antidiuretic and sodium-sparing effect (20, 183). In volunteers breathing 10.5% O₂, blood pressure fell by 10% and urine output by 30% (109), yet vasopressin and cortisol levels significantly increased while urine osmolality more than doubled.

Metabolic

Mammalian cells undergo multiple adaptive modifications of metabolism in response to changes in O₂ availability (184, 215). Some organs with high metabolic demand (e.g., muscle, liver, brain, heart) cope with the initial energy imbalance through glycogenolysis, phosphocreatine dephosphorylation, and the adenylate kinase reaction (4). Moreover, a switch in fuel selection from lipid to carbohydrate oxidation optimizes the rate of ATP production by taking advantage of the higher ATP yield per mole of O₂ consumed (94, 113).

ATP produced from fatty acid oxidation is strictly dependent upon the presence of oxygen. In contrast, glucose-derived ATP originates both from oxygen-dependent glycolysis and on glucose oxidation. During hypoxia, both fatty acid and glucose oxidation decrease, thereby increasing the importance of glycolytic-derived ATP, which, in most cell types, only plays a minor role in normoxic conditions (182). However, in order to regenerate NAD⁺, pyruvate produced from glycolysis is converted to lactate rather than being utilized within the mitochondria, a process that ultimately depends on the presence of O₂ as the terminal electron acceptor. Moreover, hydrogen ions generated by the hydrolysis of glycolytic-derived ATP accumulate, as these are not taken up by the mitochondria, and will eventually result in a fall in intracellular pH (210). Hypoxia, besides causing a critical reduction of oxygen availability for oxidative phosphorylation, also affects other mitochondrial processes, including a decrease in Complex I-dependent respiration, and reversal of the direction of operation of the F₀F₁-ATPase (Complex V). This latter effect converts mitochondria into major ATP consumers as they attempt to restore and maintain membrane potential to prevent increased mitochondrial permeability transition and cell death (68).

During hypoxia, expression and activity of carbohydrate transporters, and of enzymes involved in glycogenolysis and glycolysis are increased (215), while pyruvate dehydrogenase activity is inhibited (188). The net effect is a shunting of pyruvate away from mitochondria and an increase in glucose availability and glycolytic flux—the Pasteur effect. Hypoxia also realigns the subunit composition of CcO, improving the efficiency of respiration (82). Furthermore, accumulation of the glycolytic intermediate, fructose 1,6-biphosphate, directly inhibits mitochondrial respiration (69), again linking an increase in glycolytic flux to decreases in VO₂.

Further adaptation to hypoxia is achieved through metabolic suppression, measured as a decrease in mitochondrial VO₂ during hypoxia. This oxygen conformance (111) is recognized, at least to some extent, in human heart and in hepatocytes. It begins at partial pressures of O₂ above the critical level at which diffusion limitation into the mitochondria affects oxidative phosphorylation (31). In addition, a reallocation of cellular energy between essential and nonessential ATP-demanding processes provides further defense against the energy mismatch. ATP-consuming processes are arranged in a hierarchy, with processes less critical for cell survival being first sacrificed (30). This is mainly achieved at the level of the two principal ATP consumers: ion pumps and protein synthesis. Hypoxia reversibly suppresses Na⁺/K⁺ ATPase activity and inhibits mRNA translation through multiple mechanisms (244). Indeed, a modified phenotype for adaptive hypoxia tolerance is expressed in indigenous highlander human populations (Quechuas, Sherpas, and Tibetans), whereby improved coupling between ATP demand and supply pathways protects against imbalances due to environmental O₂ limitation (112). Similarly, NO was increased in lowlanders acclimatizing to altitude; this was associated with changes in microcirculatory blood flow, which increased local tissue DO₂, in agreement with an adaptative role in hypoxia (147).

NO Synthesis and Metabolism

Production by NOSs

A large proportion of NO synthesis occurs through the l-arginine-NO pathway (177), one of the main metabolic pathways for arginine (246) (as depicted in Fig. 2). With these reactions, the semi-essential amino acid l-arginine is metabolized to NO and l-citrulline by a family of NOS (135). These enzymes are dimers formed by two monomers consisting of a flavin-containing reductase domain and a heme-containing oxygenase domain. The NO synthesis reaction requires the presence of two oxygen molecules plus NADPH, flavin adenine dinucleotide, flavin mononucleotide, and tetrahydrobiopterin as coenzymes/cofactors (29). NOS may be uncoupled in the absence (or reduced availability) of either l-arginine or tetrahydrobiopterin, or in the presence of the endogenous NOS inhibitor, asymmetric dimethylarginine (ADMA) (227, 239). Here, electrons flowing from the reductase to the oxygenase domain are diverted to molecular O₂ rather than to l-arginine, eventually resulting in production of superoxide rather than NO (239, 247). Three isoforms of NOS have been characterized (135). The endothelial (eNOS) and neuronal (nNOS) isoforms are constitutively expressed. Although initially isolated in vascular endothelium and the nervous system, they have since been found in skeletal muscle, lung, and liver and, recently, an eNOS-like NO-producing machinery has been described in erythrocytes (134). These constitutive isoforms can rapidly increase NO production. In health, they play important regulatory roles in neurotransmission and the cardiovascular system. Production of the inducible isoform (iNOS) depends on transcription; several hours are needed to reach peak activity. Its gene transcript increases on exposure to pro-inflammatory cytokines and bacterial products. In general, iNOS generates larger (nanomolar vs. picomolar) quantities of NO compared to its constitutive isoforms (5). This is related to the amount of protein expressed. The existence of a specific mitochondrial variant of NOS (mtNOS) has been claimed (88, 142). However, others challenge its existence as no specific mtNOS sequence has yet been found in mitochondrial DNA, nor has any pathway enabling NOS protein transport into these organelles been identified, nor could it be found when specifically sought (142).

FIG. 2.

Metabolic pathways for arginine. Four major metabolic pathways for arginine exist: first, arginine is degraded to urea and ornithine by isoforms of the enzyme arginase. A large part of arginine is used for protein synthesis, and arginine is also involved in the biosynthesis of creatine. Another metabolic pathway of arginine is the synthesis of NO by endothelial, neuronal, or inducible isoforms of the enzyme NO synthase (respectively, eNOS, nNOS, and iNOS) with concomitant formation of citrulline. The NO synthesis reaction requires the presence of two oxygen molecules plus NADPH as coenzymes/cofactors, and proceeds with two steps, a first one in which the intermediate N(G)-hydroxy-l-arginine (N-OH-l-Arg) is formed, and a second one in which NO and citrulline are formed as products.

This picture is further complicated by the ability of most cells to express multiple isoforms in different compartments. In the heart, for instance, nNOS is expressed within the sarcoplasmic reticulum and its activation increases contractility, whereas eNOS is confined to the caveolae and inhibits β-adrenoreceptor-mediated increases in contractility (16). Moreover, constitutively expressed NOS can be modulated by post-translational modifications and phosphorylation (216) or be induced (135), while iNOS may be constitutively expressed at low levels in some tissues (189).

Dietary nitrite/nitrate intake

Although traditionally considered as inert oxidative end products, nitrate (NO₃ ⁻) and nitrite (NO₂ ⁻) may play an important role in NO homeostasis (164). Diet is a major source of NO₃ ⁻ with particularly high levels in leafy green vegetables. An average serving of beetroot contains more NO₃ ⁻ than is endogenously generated per day from NO generated by all three NOS isoforms combined. Once absorbed, most NO₃ ⁻ is ultimately excreted in urine. However, up to 25% is taken up by salivary glands and concentrated in the saliva, where it reaches levels 10-fold higher than in plasma. Facultative anerobe bacteria within the oral cavity then reduce it to NO₂ ⁻ while using it as an alternative electron acceptor to O₂ during respiration (165). Nitrite-rich saliva is then swallowed where, within the acidic milieu of the gastric lumen, NO₂ ⁻ is rapidly protonated to nitrous acid that further decomposes to NO. This is termed the “nitrate-nitrite-NO pathway” (166) (Fig. 3).

FIG. 3.

Multiple pathways of NO synthesis. NO synthesis occurs in blood and tissues through the classical l-arginine-NO pathway, where the semi-essential amino acid l-arginine is metabolized to NO and l-citrulline by a family of NO synthases (NOS) that use oxygen as co-factor. Endogenously formed or dietary-derived nitrite (NO₂ ⁻) in blood and tissues may be reduced to NO. Dedicated NO₂ ⁻ reductases are present in bacteria, but are lacking in humans. Nonetheless, certain mammalian enzymes under hypoxic conditions may show some NO₂ ⁻ reductase activity beyond their normal physiologic function, such as proteins from the heme-globin family or from pterin-based molybdenum enzymes. S-nitrosothiols (RSNO) are other circulating reservoirs and carriers of potential NO bioactivity. Their concentrations vary in different pathophysiologic states, and under hypoxic conditions they may be activated to release NO from their thiol (-SH) group. The most studied of such storage pool is hemoglobin (Hb): a cysteine residue on the β-chain may react with NO to form a nitroso-adduct (S-nitrosohemoglobin [SNO-Hb]) that under hypoxic conditions releases NO bioactivity. Traditionally considered as inert oxidative end products, dietary nitrate (NO₃ ⁻) and NO₂ ⁻ contribute to NO homeostasis through the gastroenteric nitrate-nitrite-NO pathway. Once absorbed from the diet, a significant part of plasma NO₃ ⁻ is concentrated in the saliva. Bacteria within the oral cavity then reduce it to NO₂ ⁻. Nitrite-rich saliva is swallowed, and within the acidic milieu of the gastric lumen NO₂ ⁻ is rapidly converted to NO.

Orally administered NO₃ ⁻ or NO₂ ⁻ can modulate the endogenous NO system in various physiologic and pathophysiologic conditions (41, 241, 249). Significant increases in plasma NO₃ ⁻ and NO₂ ⁻ levels were measured in human volunteers after beetroot juice ingestion, with lowering of arterial BP by about 10 mmHg. This was prevented by interrupting the enterosalivary circulation through nonswallowing of saliva (241), or by selective suppression of the oral microflora with an antiseptic mouthwash (193).

Generation by other pathways

Systemic nitrite reduction

Endogenously formed or dietary NO₂ ⁻ in blood and tissues may be recycled to form NO-like bioactive molecules (Fig. 3). In a human forearm blood flow study (90), Gladwin et al. found that of the various plasma NO-related species, only NO₂ ⁻ had a significant arterial-venous gradient, indicating a degree of consumption during circulatory transit, and thus suggestive of possible bioactivity. This gradient markedly increased with exercise and inhibition of regional NO synthesis, suggesting that NO₂ ⁻ is a plasma carrier of NO bioequivalents that are peripherally converted into bioactive NO. However, these results could not be replicated by Lauer et al. (144), who reported no vasodilatory effect following intra-arterial administration of NaNO₂ into healthy volunteers. This may relate to a shorter duration of NO₂ ⁻ infusion, or the need for NO₂ ⁻ to undergo metabolic conversion before becoming vasoactive. A careful comparison of the route of administration, the concentration, and total dose of NO₂ ⁻ is needed to better understand its role (45).

Various possible in vivo pathways by which NO₂ ⁻ is reduced to NO have been investigated (Fig. 4). NO₂ ⁻ can form NO nonenzymatically under acidic and/or ischemic conditions (251). Using ¹⁵N-labeled NO₂ ⁻ to identify the source of NO, and enzyme inhibitors to exclude other pathways, NO generation was explained by a reaction of spontaneous disproportionation; however, the in vivo relevance of this pathway remains uncertain. Dedicated NO₂ ⁻ reductases are present in bacteria, but are lacking in humans. Nonetheless, certain mammalian enzymes show some NO₂ ⁻ reductase activity beyond their normal physiologic function. As an alternative to nonenzymatic reduction, proteins from the heme-globin family (89) or from pterin-based molybdenum enzymes (149) may catalyze the NO₂ ⁻ reductase reaction to NO. An in vitro reaction of NO₂ ⁻ with human deoxyhemoglobin forms NO and methemoglobin, while an intra-arterial NO₂ ⁻ infusion produces, after several circulation times, a vasodilatory effect in healthy volunteers (60). Myoglobin, both in the heart and in the vasculature (105, 185), also has significant NO₂ ⁻ reductase activity, as do heme protein-containing enzymes such as the mitochondrial electron transport chain cytochromes (17, 49, 138), the cytochrome P450 family of microsomal heme proteins (151), and aldehyde dehydrogenase (ALDH2), a mitochondrial enzyme involved in ethanol inactivation that has also been linked to the bioactivation of organic nitrates (13, 51). Even eNOS and soluble guanylate cyclase (sGC), being heme-based enzymes, may have possible NO₂ ⁻ reductase activity, thereby offering an important alternative source of NO outside the conventional l-arginine pathway (8, 87).

FIG. 4.

Enzymes with putative nitrite-reductase activity. Dedicated NO₂ ⁻ reductases are present in bacteria, but are lacking in humans. Nonetheless, certain mammalian enzymes show some NO₂ ⁻ reductase activity beyond their normal physiologic function. As an alternative to nonenzymatic reduction, proteins from the heme-globin family (such as deoxygenated hemoglobin–HHb, and myoglobin–HMb, the mitochondrial cytochrome bc1 complex–CIII, cytochrome c oxidase–CcO, and cytochrome c–CytC, as well as aldehyde dehydrogenase–ALDH2, the endothelial isoform of NOS–eNOS, and soluble guanylate cyclase–sGC) or from pterin-based molybdenum enzymes (such as xanthyne oxidoreductase–XOR, sulfide oxidase–SO and aldehyde oxidase–AO) may all catalyze the NO₂ ⁻ reductase reaction to NO, especially under hypoxic conditions.

Given that Hb is an effective scavenger of NO, the possibility of a heme-independent pathway of NO synthesis from NO₂ ⁻ merits consideration as the in vivo relevance of NO₂ ⁻ reduction by heme-based enzymes may be challenged by the need for the newly synthesized NO to escape from this scavenging. The two most studied NO₂ ⁻ reducing molybdenum-based enzymes are xanthine oxidoreductase (XOR) and aldehyde oxidase (149). The former plays a critical role in purine and pyrimidine catabolism, catalyzing oxidation of hypoxanthine to xanthine, and xanthine to uric acid. As it also reduces O₂ to H₂O₂ and O₂ ⁻, XOR is a key enzyme in the process of oxidative injury. XOR can catalyze reduction of NO₃ ⁻ to NO₂ ⁻, and NO₂ ⁻ to NO under anerobic conditions; this can be blocked by the XOR-inhibitor, oxypurinol (152). Similarly, aldehyde oxidase, a cytosolic enzyme involved in biotransformation of drugs and xenobiotics, also has significant in vitro NO₂ ⁻ reductase activity (150).

Release from pre-formed storage pools

Attempting to solve the apparent paradox of the NO scavenging process being too rapid and effective to allow this short half-life molecule to exert its physiologic effects, it has been suggested that, depending on the oxygenation state of Hb, NO may either react with oxyHb to be oxidized to NO₃ ⁻, or can bind to the deoxygenated form to generate a nitrosyl-adduct (NO-Hb) that subsequently reacts with thiol groups to produce S-nitrosohemoglobin (SNO-Hb) (121, 191, 225). SNO-Hb is more stable and has a longer half-life than NO, and its administration can evoke a hypotensive response, suggesting that it acts as both carrier and donor of NO bioequivalents (Fig. 3).

Other reservoirs of potential NO bioactivity include S-nitrosoalbumin (224), tissue NO₂ ⁻, S-nitrosothiols (RSNO), N-nitrosamines (RNNO), and dinitrosyl iron complexes. Their concentrations vary in different pathophysiologic states, typically showing marked elevation compared to basal levels in acute inflammation and reduction in the more chronic setting. Although little is known about their in vivo relevance, they may act as signaling molecules or storage forms of NO (43, 73, 237). Similar NO storage forms are found in other compartments such as the vascular wall (203).

Regardless of location, all these compounds may be activated to release NO under certain conditions, and to contribute to the body pool of NO-related metabolites. Opinion still remains divided as to the in vivo importance of these mechanisms (90, 118, 198). A mitochondria-targeted RSNO was recently shown to selectively induce NO production and S-nitrosylation (addition of an NO⁺ group to a protein thiol to form a nitrosothiol) at the mitochondrial level, producing vascular relaxation of precontracted aortic rings and protecting against ischemia-reperfusion (197). Molecules able to enhance trans-nitrosation reactions, transferring NO from one cysteine residue to another, constitute an emerging area of research in the field of drug design (86).

NO metabolism

The in vivo fate of NO is highly complex; several catabolic pathways exist (Fig. 5), with varying relevance in different body compartments (27, 131). In vitro, NO rapidly reacts with O₂ to form nitrogen dioxide (NO₂). In the presence of NO at low concentration, the latter reacts with water to form equal amounts of NO₂ ⁻ and NO₃ ⁻. At higher NO concentrations, NO₂ reacts with another NO molecule to form dinitrogen trioxide (N₂O₃), which hydrolyzes to form NO₂ ⁻. In plasma, in the presence of O₂, the principal reaction is formation of NO₂ ⁻ (115). It is currently unclear whether this is through autoxidation of NO, reaction with the plasma multi-copper oxidase ceruloplasmin (220), or oxidation by the mitochondrial CcO in vascular cells (192, 218, 230). The situation differs in whole blood where the relatively high amount of oxyHb favors biotransformation of NO to NO₃ ⁻ with concomitant formation of methemoglobin (27). This Hb reaction is considered by some as the primary catabolic process responsible for NO removal (107, 124). A similar reaction with oxymyoglobin (to generate metmyoglobin and NO₃ ⁻) has been recently proposed as a crucial regulatory step of NO inactivation in muscle (40, 79). However, other studies have shown that this reaction only takes place in conditions of excess NO (192).

FIG. 5.

Pathways of NO metabolism in normoxic conditions. NO rapidly reacts with molecular oxygen (O₂) to form nitrogen dioxide (NO₂). If NO is present at low concentration, the NO₂ reacts with water to form equal amounts of NO₂ ⁻ and NO₃ ⁻. At higher NO concentrations, NO₂ reacts with another NO molecule to form dinitrogen trioxide (N₂O₃), which hydrolyzes to form NO₂ ⁻, or may dimerize to form dinitrogen tetraoxide (N₂O₄). In plasma, the principal fate of NO is formation of NO₂ ⁻, although the exact reaction is unclear (autoxidation, reaction with ceruloplasmin, or oxidation by the mitochondrial CcO). In whole blood NO reacts with oxygenated hemoglobin (oxyHb) to form NO₃ ⁻ with concomitant formation of methemoglobin (MetHb). A similar reaction with oxymyoglobin (oxyMb) happens in tissues. NO can also react with superoxide (O₂ ⁻) to produce peroxynitrite (ONOO⁻); the latter may directly trigger oxidation or nitration reactions with various cellular targets, and is eventually converted into NO₃ ⁻ or NO₂. Highly reactive NO by-products (reactive nitrogen oxide species, i.e., ONOO⁻, NO₂, N₂O₃, N₂O₄), possibly through the formation of nitrosonium equivalents (NO⁺), can react with protein thiol (-SH) groups to form RSNOs, or with amines to generate N-nitrosamines (RNNOs). NO can also directly react with metals (Me^x) to give metal nitrosyls (Me^xNO). In addition to nitrosation, RSNOs may also be produced by oxidative nitrosylation.

NO can also react with superoxide to produce peroxynitrite (ONOO⁻). The rapidity of this reaction, some 3–4-fold faster than O₂ ⁻ dismutation by superoxide dismutase, makes ONOO⁻ formation a major potential disposal pathway of NO reactivity (92), though this does depend on the rate of tissue superoxide production. ONOO⁻ itself may trigger oxidation or nitration reactions with various cellular targets modulating their biological activities, and is eventually converted into NO₃ ⁻ or NO₂ ⁻ (228).

NO₃ ⁻ and NO₂ ⁻ were long considered stable end products of NO catabolism; however, both are now recognized to be subject to further biotransformation (78, 131, 226). Highly reactive NO by-products (reactive nitrogen species) can react with protein thiol (-SH) groups to form RSNOs such as S-nitrosoalbumin, S-nitrosoglutathione, and S-nitroso-hemoglobin (131), or with amines to generate RNNO. The physiologic significance of RNNOs is presently unknown, but concentrations change rapidly in response to an acute oxygen shortage (43). NO itself can directly react with metals to generate metal nitrosyls, for example, NO-Hb. In addition to nitrosation, RSNOs may also be produced by oxidative nitrosylation. This reaction is mediated by generation of thiyl radicals (RS^•) that may be derived from interaction of thiols with oxidants such as ONOO⁻ (43). NO metabolism changes under hypoxic conditions, with greater production of metal nitrosyls, RSNOs, and RNNOs (180). Some of these products share some of the biologic properties of NO (226) and may possess the important biological functions of storing and transporting NO. Moreover, S-nitrosylation of thiol groups is a widespread post-translational redox-based protein modification. Similar to phosphorlyation, this exerts control over many protein classes in various physiologic and pathophysiologic conditions (108, 157).

Half-life of NO

The process of NO transfer to its target remains incompletely understood. NO has high reactivity and a very short half-life, with measured blood levels being too low to likely exert any physiologically relevant effect (159). From in vitro studies, the half-life ranges from as little as 10⁻⁶ s to as much as 11.5 s (100). Mathematical modeling estimates an in vivo half-life of about 2 ms (158). Such a short half-life plus the rapid intravascular scavenging of NO has to be reconciled with the prominent autocrine and paracrine roles this molecule is believed to play in cellular physiology. Liao et al. (154) proposed the existence of intravascular erythrocyte-free zones generated by blood flow that may increase the NO half-life by several orders of magnitude due to reduced local NO scavenging, thus allowing the molecule to exert its biologic functions. Another possibility is the concept of stored NO bioactivity (66, 221), as outlined above.

To summarize, referring to the half-life of free NO may no longer be relevant given the rapidity of its transformation and interchange between different metabolites, many of which are longer lived than the parent molecule. A more complex system appears to be in place, in which the short half-life of NO itself is important in limiting the action of the molecule to its site of formation and to enable local signaling (autocrine and paracrine levels), while downstream biotransformation reactions make economical use of the NO produced as well as contributing to distant signaling (endocrine level).

NO and Cardiovascular Homeostasis

NO as a regulator of vascular homeostasis

NO is a core physiologic regulator of many cardiovascular processes, including platelet aggregation and adhesion, myocardial contractility, vascular permeability, and tone (187, 243). NO is essential for both global regulation and regional distribution of blood flow and pressure. Dysregulation of the NO system thus likely plays a fundamental role in many pathophysiologic conditions ranging from essential hypertension and atherosclerosis to the hypotension seen in acute shock states (243).

NO is a key contributor to new vessel formation: in endothelial cells, VEGF induced NO production via eNOS, which, in turn, mediated angiogenesis (83). iNOS-derived NO may also have a role in angiogenesis (178). Besides being an effector of VEGF activation, NO also enhances growth factor synthesis in numerous cell types, mimicking the classical hypoxic stimulus (71). Thus, NO appears to act both as an upstream and a downstream mediator of VEGF-dependent angiogenesis.

Mechanisms underlying the vasodilatory action of NO

NO regulates blood pressure and flow through its effects on vascular smooth muscle tone. The shear stress generated by flowing blood against the endothelial surface triggers production of NO both basally (114), and in response to mechanostimulation (222). This increase in NO production is nonlinear with respect to shear stress (14). Of note, laminar blood flow (which increases shear stress) increases NO production, whereas disturbed flow (causing low and oscillating shear stress) inhibits release of NO and fails to upregulate NOS (58).

Being small and lipophilic, NO rapidly diffuses across membranes to reach vascular smooth muscle cells. Its main mechanism of action is mediated by nitrosylation of the heme-iron within sGC, leading to increased synthesis of cyclic guanosine monophosphate (cGMP) (243). This, in turn, activates protein kinases that modulate myosin light chain kinase and phosphatase activities, resulting in less phosphorylation of myosin and, eventually, vasorelaxation (143). NO can also cause vasodilation via cGMP-mediated opening of calcium-sensitive (K_Ca) (10) and ATP-sensitive (K_ATP) (179) potassium channels. When these ion channels open, the outward efflux of potassium hyperpolarizes the plasma membrane, reducing vascular tone. NO also activates K_ATP and K_Ca channels in a cGMP-independent manner through direct S-nitrosylation (28, 129). NO may contribute to the regulation of intracellular free Ca²⁺ levels, either via cGMP-dependent inhibition of calcium influx through L-type Ca²⁺ channels (23), and/or via increased Ca²⁺ removal from the cytoplasm. The latter can occur by accelerating the Na⁺/Ca²⁺ exchanger (84), or by increasing sequestration into intracellular stores via the sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase (2, 56). This effect may be important as the constitutive isoforms of NOS are regulated by calcium calmodulin (216). An increase in intracellular Ca²⁺ activates calmodulin, leading to NO synthesis. Thus, it could be postulated that reduced intracellular levels of free calcium resulting from increased NO levels could reduce the amount of iNOS-generated NO, contributing to the fine-tuning of NO levels. (Fig. 6)

FIG. 6.

Effects of increased NO levels during hypoxia in the balancing of oxygen supply and demand. Left panel (A). Metabolic and contractile state of an ideal cell during normoxic conditions. Adenosine triphosphate (ATP) supply is maintained through mitochondrial and cytoplasmic reactions. Glucose (Gluc) is transported from the interstitium via glucose transporter (GLUT) carriers. The vast majority of pyruvate, the end product of glucose breakdown, enters the mitochondria after oxidation by the enzyme pyruvate dehydrogenase (PDH). Here, it is completely oxidized with reactions coupled to the synthesis of ATP. Oxygen (O₂) is crucially required as the terminal electron acceptor of the respiratory chain, CcO. A small part of the pyruvate is reduced to lactate-by-lactate dehydrogenase (LDH). In conditions of excess of substrate availability, glucose is stored as glycogen. The contractile state of the cell is maintained through cycling of myosin from a phosphorylated to a dephosphorylated state, via the enzymes myosin light-chain kinase and phosphatase, respectively (MLCK and MLCP). Right panel (B). Adaptive modifications of metabolism and contractile state during hypoxia. The increased levels of exogenous or endogenously formed NO during hypoxia contribute to the adaptation to the energy supply–demand mismatch. NO reduces mitochondrial metabolic demand by competing with O₂ at CcO. It also limits energy-consuming anabolic processes such as glycogen synthesis and induces energy-producing catabolic pathways via the increased expression and activity of GLUT transporters, and of enzymes involved in glycogenolysis and glycolysis, while PDH activity is inhibited. The net effect is a shunting of pyruvate away from mitochondria and an increase in glucose availability and glycolytic flux. NO also activates the enzyme sGC, leading to increased synthesis of cyclic GMP (cGMP). This, in turn, modulates MLCK and MLCP activities, resulting in less phosphorylation of myosin and, eventually, vasorelaxation. NO can also cause vasodilation via cGMP-mediated opening of calcium-sensitive (K_Ca) and ATP-sensitive (K_ATP) potassium channels. When these ion channels open, the outward efflux of K⁺ hyperpolarizes the plasma membrane, reducing vascular tone. NO may also activate those ion channels in a cGMP-independent manner through direct S-nitrosylation of cysteine (SH) residues. Eventually, NO regulates the intracellular free Ca²⁺ concentration, either via a cGMP-dependent inhibition of calcium influx through L-type Ca²⁺ channels (LTC), and/or via an increased Ca²⁺ removal from the cytoplasm. The latter may occur by accelerating the Na⁺/Ca²⁺ exchanger (SCE), or increasing the sequestration into intracellular stores via the sarcoplasmic/endoplasmic reticulum (SER). The net effects of an increase in NO levels are reduced VO₂, increased glycolytic ATP production, and vasodilation that increases the supply of oxygen and nutrients. Solid arrows indicate pathways induced by NO; dashed arrows indicate inhibition.

In health, eNOS mediates most of the hemodynamic actions of NO, contributing to blood flow regulation between different vascular beds according to their varying metabolic needs (177, 243). NO derived from iNOS is traditionally considered to be primarily responsible for the vascular hyporeactivity and hypotension seen in inflammatory states (235). However, emerging data indicate an important role for nNOS-derived NO in basal microvascular tone regulation, while eNOS-derived NO regulates changes in tone in response to agonists or shear stress (171). This suggests a potentially independent regulation of basal and stimulated blood flow.

Hypoxic Vasodilation to Improve Coupling of DO₂ and Utilization

Evidence for the involvement of NO

Under hypoxemic conditions, a vasodilatory response occurs, augmenting blood flow in an attempt to maintain DO₂. Given its importance in the regulation of cardiovascular homeostasis, NO likely plays a key role. An association between NO and hypoxic vasodilation was first described in 1989 (196). This response was endothelium-dependent and significantly reduced by administration of oxyHb, acting as a potent NO scavenger. Ten-fold higher levels of circulating NO products were found in residents of the Tibetan plateau compared to sea-level dwellers (76). This was associated with increased resting forearm blood flow, suggesting an adaptive role that offsets the O₂ lack caused by high altitude. In volunteers hypoxemia-induced increases in forearm blood flow were blocked by the nonspecific NOS inhibitor, L-NG-monomethylarginine (24). In awake sheep, hypoxemia-induced increases in cerebral blood flow and falls in cerebral vascular resistance were reversed by the sGC inhibitor, methylene blue (119).

Alternative theories

Apart from its role as an energy source for cellular metabolic activity, ATP has important signaling characteristics, particularly in situations of reduced energy supply such as hypoxia. ATP release occurs in all major cell types (162), including endothelial cells, vascular smooth muscle cells, and circulating red blood cells (RBCs). In the blood vessel lumen, ATP levels increase during hypoxia (21) or conditions of increased shear stress (26). Precise mechanisms responsible for release are, at present, incompletely understood. Ellsworth et al. have postulated a key role for the RBC in sensing hypoxia (74) and modulating vascular tone via active release of ATP (75). Blood flow is significantly augmented, either through direct purinergic signaling or by inducing synthesis of vasoactive metabolites such as NO. These pathways do appear to intersect and interact with each other; indeed, NO can inhibit ATP release from RBCs (181) while NO₂ ⁻ enhances erythrocyte ATP synthesis and release during hypoxia (46).

NO and control of metabolism

In tandem with its role in hypoxic vasodilation, NO has potent inhibitory effects upon cellular metabolism that are significantly enhanced under hypoxic conditions (53, 59). NO potently and reversibly reduces mitochondrial membrane potential (213) by competing with O₂ at CcO (34). As less competition occurs in the presence of hypoxia, after an initial rapid (although reversible) inhibition of this enzyme, a potentially irreversible inhibition of Complex I occurs through nitrosylation and nitration that is also accelerated by hypoxia (52, 81). For nitrosothiols and ONOO⁻ to interact with Complex I, prior transition of the enzyme from its active (A) to its deactive (D) state is necessary, as only the D-form is susceptible to inactivation by these agents. Transition of Complex I from A to D preferentially occurs during hypoxia (85). This process may initially confer some degree of protection, reducing the amount of free radicals produced upon re-oxygenation, but may also initiate pathophysiological modifications of mitochondrial activity. Taken together, under conditions of reduced O₂ availability NO mediates an important compensatory response through both enhancing supply and suppressing metabolic demand (Fig. 6).

Other important effects of NO on intermediary metabolism are mediated through ONOO⁻-dependent activation of the AMPK system, or by direct nitrosation of critical thiols of target enzymes (122). The link between NO and the AMPK system involves different levels of regulation; for example, silencing of AMPK caused a decrease in cellular eNOS content (57). In particular, NO can limit energy-consuming anabolic processes such as hepatic gluconeogenesis and glycogen synthesis while inducing energy-producing catabolic pathways via increased expression of transmembrane carbohydrate transporters and a higher glycolytic flux (7, 153). Over a longer time-scale, NO stimulates biogenesis of functionally active mitochondria (54). NO may also be an important modulator of the adaptive response to hypoxia; by redistributing O₂ within cellular compartments and between neighboring cells and interfering with the stabilization process of HIF-1α, it allows fine-tuning of cellular metabolism (99). Altered levels of NO and ROS likely impinge upon oxygen-sensing pathways. In a variety of cell types, NO stabilized HIF-1α protein and provoked HIF-1 target gene expression under normoxia (36). Whether this is due to NO itself or to a reactive intermediate, and whether or not the mechanism is cGMP-dependent is under active investigation (37). It does appear that the ability of NO to stabilize HIF-1α depends to some extent on the formation of co-signals, such as, for instance, superoxide and the consequent production of ONOO⁻.

Notwithstanding the above findings, some authors reported no impact of NO modulation on whole body (63) or myocardial (136) VO₂. By contrast, others did find a modulating effect on whole-body VO₂ (217). The negative studies used nonselective NOS inhibition but did not entertain the possibility that NO may be generated by alternate pathways such as NO₂ ⁻ reduction or release from RSNO. Moreover, the lack of effect after administering the NO donor sodium nitroprusside, or authentic NO, may be explained by the potent scavenging properties of Hb and myoglobin. Conceivably, the effect of NO on DO₂ and consumption may be tissue specific. In the brain, NO synergized with hypoxia to induce necrotic death via CcO inhibition in both neurons (120) and glia (170). NO-mediated inhibition of CcO may thus induce an adaptive state of reduced VO₂ compensated for by increased glycolytic flux, or it may lead to a critical reduction in ATP production and cell death. The overall impact likely depends on the relative contribution of each process; this is turn depends on the extent and rate of metabolic perturbation and, perhaps, the cell type affected.

NO and microRNAs

The interplay between NO and microRNA signaling is intriguing. Such cross talk may connect a very fast and ubiquitous signaling pathway for the acute response to hypoxia with the master regulator of chronic hypoxia. This was recently demonstrated in studies of the mechanisms of ischemic or hypoxic myocardial preconditioning (209). miR-21, a microRNA induced in vascular tissue by shear stress, increased NO availability through phosphorylation of eNOS (242). Conversely, an NO donor modulated production of miR-21 and other microRNAs, thereby regulating smooth muscle cell contraction (137). Brief bursts of myocardial ischemia induced miR-1, miR-21, and miR-24; this, in turn, induced eNOS mRNA and upregulated eNOS protein, whereas no effect was seen on iNOS. This miR-induced, eNOS-derived NO had cardioprotective effects against ischemia-reperfusion injury, possibly by restoring the O₂ supply/demand balance (248).

Very recently, another aspect of miR-mediated regulation of the NO pathway was discovered: a well-known paradox of iNOS-derived NO is that cytokine stimulation can upregulate iNOS gene expression >2000-fold, but, in some tissues, the increase in NO levels was far less (167). miR-939 decreased cytokine-induced iNOS protein expression but with no effect on iNOS mRNA levels or stability, thereby contributing to post-translational silencing through direct binding to the iNOS gene (93). Similarly, increases in miR-146a activity inhibited of lipopolysaccharide-induced iNOS expression and NO production (64). These findings have been interpreted as an endogenous protective mechanism against the untoward consequences of prolonged iNOS overexpression.

NO and the renin–angiotensin system

The renin–angiotensin system is an important regulator of blood flow and pressure through renal, vascular, and central mechanisms (97). The classical pathway involves binding of angiotensin II to the angiotensin II type 1 receptor (AT1), to exert inotropic and vasocontrictor actions through increasing intracellular free calcium. The type 2 receptor (AT2) serves to counterbalance activation of the AT1 pathway; one of the main pathways associated with AT2 activation is stimulation of NO production (47). AT2 activation significantly attenuated mitochondrial respiration, and this was reversed by the NOS inhibitor L-NG-nitroarginine methyl ester (1).

NO levels in hypoxia: balance of synthesis and metabolism

The NO concentration at any given location represents the balance between local synthesis and metabolism/elimination. During hypoxic vasodilation, the rise in NO levels may derive from increased production via NOS isoforms, and/or increased NO synthesis from alternate pathways (e.g., NO₂ ⁻ reduction or RSNO release), and/or reduced elimination, for example, by conversion to NO₂ ⁻ or NO₃ ⁻ (Fig. 7). An alternative (or perhaps concurrent) mechanism is of vasodilation due to other factors such as ATP release by RBCs; the resulting increase in shear stress increases NO production, which then further enhances the vasodilatory response.

FIG. 7.

Effects of hypoxia on NO metabolic pathways. Possible mechanisms of increased NO levels under hypoxic conditions include [1] increased production by NOS, [2] increased release from storage forms such as RSNO, [3] increased reduction from NO₂ ⁻, and [4] reduced metabolism by CcO.

Does hypoxia increase NO production by NOS?

NOS-related NO production increases during hypoxia. In dogs, NOS inhibition reversed hypoxemia-induced tachycardia, hypotension, and increases in cardiac output (12), findings subsequently replicated in human volunteer studies (25, 48, 236). However, NOS inhibition also accentuated the hypoxia-induced rise in pulmonary vascular resistance (25).

Given the rapidity (seconds to minutes) of hypoxic vasodilation, the initial increase in NO synthesis is likely to be primarily mediated by a constitutively expressed NOS isoform. O₂ regulates transcription of eNOS (11) and possibly nNOS (19). With prolonged hypoxia, NO levels progressively rise; in an ex vivo macrophage model iNOS mRNA was detected after 1.5 h of hypoxia (9). HIF-1 may influence with iNOS expression under hypoxic conditions (125). In some cell types, such as macrophages, hypoxia cannot by itself induce iNOS expression, whereas the synergistic combination of hypoxia and interferon-γ was a potent inducer (172). In other cell types (e.g., cardiomyocytes), hypoxic activation of the HIF-1 pathway could upregulate iNOS expression by itself, though this was significantly amplified with interleukin-1β (125). In pulmonary artery endothelial cells, hypoxia alone did not induce iNOS expression, but it significantly modulated cytokine induction of the gene, prolonging the half-life of cytokine-induced iNOS mRNA from 6 to 17 h (250). However, hypoxia itself can induce expression of inflammatory cytokines (133), which then can activate iNOS. Thus, a more complex scenario probably exists in vivo with co-participation of all three isoforms.

The oxygen atom in NO and citrulline is derived from molecular oxygen, regardless of synthesis by constitutive or inducible isoforms of NOS (146). Hypoxia could attenuate the NO component of endothelium-dependent vascular relaxation, likely due to decreased NO production secondary to oxygen depletion (123, 200). The apparent Km values for oxygen were 17, 6, and 5 mmHg for nNOS, eNOS, and iNOS isoforms of NOS, respectively (201). These values are close to the Km values of other enzymes that utilize O₂ as a substrate, for example, CcO. As the neuronal isoform shows a higher Km value for O₂, it is thus more sensitive to the prevailing oxygen concentration.

Despite its apparent simplicity, the likelihood that hypoxic vasodilatation can be predominantly explained by de novo NOS synthesis presents at least three major contradictions. Given that NOS-derived NO synthesis requires molecular O₂, it seems counter-intuitive in situations of O₂ lack to record an increase in NO production. Indeed, some in vitro experiments report reductions in eNOS mRNA expression and decreased NO production during hypoxia (155, 200, 245). These results conflict with those cited above and may relate to the degree of hypoxia (or anoxia) experienced, the varying O₂ sensitivity of the different cells/tissues studied, and/or a possible biphasic nature of events relating to the time course of the overall response (e.g., feedback inhibition of NOS expression secondary to an acute initial increase in NO availability, followed by a gradual increase in expression as hypoxia persists). Nevertheless, these studies raise important concerns regarding the true significance of NOS-derived NO in hypoxia. Secondly, increased arginase activity during hypoxia is a well-known process (139, 163), and this reduces the amount of substrate available for the reaction by NOS.

A more complex regulation of NO synthesis from NOS may exist during hypoxia. In a rodent model of chronic hypoxia, exposure to 10% O₂ significantly increased pulmonary eNOS expression, in addition to an increase in ADMA concentration, reduced dimethylarginine dimethylaminohydrolase (DDAH) (the enzyme responsible for ADMA disposal) and reduced tissue nitrate/nitrite (NOx) concentrations (175). They postulated that hypoxia reduces DDAH activity that, in turn, increases ADMA concentrations that leads to eNOS inhibition and reduced synthesis of NO. Finally, in several studies NOS inhibition could only partially reverse hypoxic vasodilation; thus, other mechanisms must be implicated (35, 148).

Does hypoxia increase NO release from SNO-Hb and other NO storage forms?

Stamler's theory for hypoxic vasodilation demands a central role for erythrocytes in matching blood flow to local metabolic demands. The affinity of Hb for NO is similar to that for O₂, that is, high in the relaxed deoxygenated state, and low in the tense oxygenated state (225). A cysteine residue on the Hb β-chain reacts with NO to form a nitroso-adduct (SNO-Hb) that acts as a carrier of NO bioactivity. Erythrocytes thus act as O₂ sensors to control regional blood flow. Erythrocytes could rapidly relax thoracic aortic rings from both rabbits and mice under hypoxic but not normoxic conditions, though this relaxation could be inhibited by either depletion of SNO-Hb or sGC blockade (70).

Does hypoxia increase reduction of NO₂ ⁻ to NO?

The findings of an increased concentration of NO during hypoxic vasodilation, full reversal of these hemodynamic effects by sGC inhibition but not by NOS blockade, a strict O₂ dependence of the NOS reaction, and the possibility that NO₂ ⁻ can be converted to NO, particularly under acidic conditions, suggest that NO₂ ⁻ itself may act as an NO-equivalent donor during hypoxia (3, 176). cGMP-dependent NO₂ ⁻ vasodilation and the rise in measured NO have been interpreted as evidence for the involvement of an NO-mediated relaxation, rather than a direct NO₂ ⁻ effect. Intravenous infusion of sodium NO₂ ⁻ (1 μmol/min) into healthy volunteers increased forearm blood flow and reduced pulmonary artery pressure only under hypoxemic (12% O₂) conditions, but was not related simply to an increase in plasma NO₂ ⁻ concentration (116). These data are consistent with a direct extravascular metabolism of NO₂ ⁻ to NO to exert hypoxia-associated bioactivity. Whether endogenous NO₂ ⁻ concentrations are sufficient to cause similar hemodynamic effects remains uncertain at present.

Of the many different mammalian molecules with NO₂ ⁻ reductase activity (Fig. 4), the most extensively studied is Hb. Addition of erythrocytes induced a left shift in the vasodilatory dose–response curve and cGMP accumulation in response to NO₂ ⁻ but only under hypoxic conditions (61). This could be inhibited by the NO scavenger, 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (C-PTIO). Thus, NO₂ ⁻ exerted a higher vasodilatory effect when deoxygenated Hb was present, again emphasizing the role of NO-dependent mechanisms in hypoxic vasodilation.

Hb may be an important physiologic O₂ sensor that can modulate vascular tone by (i) scavenging excess NO and (ii) increasing local blood flow through NO generation from NO₂ ⁻ when O₂ content is reduced. This theory remains controversial because of the avid NO-scavenging properties of Hb to the point that it has been argued that this phenomenon has minimal, if any, in vivo relevance (6). The Gladwin group countered with a recent ex vivo study using rat vascular rings (117) wherein the balance between the NO-scavenging and generating properties of Hb was specifically targeted during both normoxia and hypoxia. They found that NO₂ ⁻ displays a particular interaction with deoxyhemoglobin that promotes vasodilation despite its scavenging properties.

Myoglobin can also act as an NO₂ ⁻ reductase, with NO being produced in vitro by reaction between NO₂ ⁻ and myoglobin (199). This conversion was significantly reduced in cardiac tissue taken from a myoglobin knockout mouse model but restored by adding exogenous myoglobin. Under hypoxic conditions in vitro (238) and ex vivo (240) eNOS also displays NO₂ ⁻ reductase activity. As for the pterin-based enzymes, both XOR (174, 240) and aldehyde oxidase (195) are sources of NO₂ ⁻ reduction, particularly during co-existing hypoxia and acidosis.

The apparently conflicting theories of NO₂ ⁻ reduction and RSNOs acting as non-NOS-dependent sources of NO, and as regulators of local blood flow under both physiologic and hypoxic/ischemic conditions, may be reconciled (42, 77). Under physiologic conditions, NO₂ ⁻ is not directly reduced to NO but rather modulates many signaling pathways, including sGC activation. It also induces post-translational modifications normally associated with NO, such as the formation of nitroso- and nitrosyl species (42). NO₂ ⁻ may therefore exert its signaling functions directly, without the need for intermediary formation of free NO. Hypoxia markedly potentiates tissue NO production from NO₂ ⁻ in a dose-dependent manner (77). This occurs particularly in heart, liver, and vascular tissue, with multiple heme, iron–sulfur cluster, and molybdenum-based reductases distributed among distinct subcellular compartments acting in a multifactorial and cooperative manner to catalyze the reaction. Acute hypoxia also reduces NO₂ ⁻ concentrations yet enhances formation of NO metabolites such as RSNOs and RNNOs in an NO-independent manner. This suggests a pathway that generates bioactive NO metabolites directly from NO₂ ⁻. In this paradigm, conversion of NO₂ ⁻ to NO and the storage of NO bioactivity as RSNOs may both be constituents of a more complex regulatory mechanism of interaction of multiple NO-related species. The differences in oxygen dependence of NO₂ ⁻ reductase activity in tissues (exponential) versus RBCs (optimum around the p50) suggest that the regulatory range of the latter may operate at intermediate levels of hypoxia, whereas the former predominates as PO₂ drops further.

Does hypoxia reduce NO metabolism by CcO?

Moncada's group suggested that the hypoxia-induced increase in NO is due to reduced elimination rather than increased production (186, 234). Plausibility has been tested in a computational model of brain O₂ transport and metabolism (205), with description of the kinetic parameters that link decreased NO metabolism by CcO with low O₂ concentrations. They showed in l-arginine-supplemented iNOS-overexpressing cells that the capacity of CcO to metabolize NO was diminished at low O₂ tensions, and that this correlated with both the enzyme's redox state and consequent sGC activation. The same effect was seen when the redox state was altered by cyanide rather than hypoxia. Whereas NOS inhibition was effective at reducing NO levels, NO₂ ⁻ administration had no effect on NO release at any O₂ tension (186, 234). NO production was also similar at all O₂ tensions, regardless of whether CcO was oxidized or reduced (234). They thus concluded that CcO (in its oxidized state) constantly inactivates NO, thus regulating its intracellular concentration. However, in the reduced state seen in hypoxia, impaired inactivation would account for the increase in NO. This leads to local vasodilatation with improvements in DO₂ and a concurrent reduction in VO₂ due to direct inhibition of CcO, thereby facilitating the matching of delivery to needs under hypoxic conditions. Further studies are needed to demonstrate the in vivo relevance of these findings.

Conclusions

Hypoxic vasodilatation is an adaptive response that involves elevations in local NO concentrations in response to an acute reduction in arterial PO₂. This both increases blood flow to restore DO₂ and also modulates local metabolic requirements, thus attempting to re-balance an acute oxygen supply–demand mismatch. Several mechanisms are implicated (Fig. 7), including increased NO synthesis from NOS, increased reduction of NO₂ ⁻ to NO by heme- or pterin-based enzymes, increased release of NO from NO storage forms, and reduced deactivation by mitochondrial CcO. Many of these mechanisms have been shown either in in vitro/ex vivo conditions or by utilization of pharmacologic dosing regimens, so the question remains as to their in vivo (patho)physiologic relevance. While tissue hypoxia can result from decreases in arterial PO₂, blood flow, or Hb concentration (15), the adaptive mechanism for each form of hypoxia need not be identical. A recent animal study (62) demonstrated that NOS inhibition did not blunt the increase in myocardial blood flow during acute normovolemic hemodilution, and suggested, at least in this form of tissue hypoxia, that NO did not mediate vasodilation.

Further work is needed to fully elucidate the multiple and varied roles of NO under hypoxic conditions, and to integrate these into an overall picture.

Footnotes

Acknowledgments

We thank Carlotta Mozzana for the invaluable assistance with the artwork. This work was undertaken at UCLH/UCL, which received a proportion of funding from the Department of Health's NIHR Biomedical Research Centres' funding scheme. M.U. was supported by a Physiology Award from the Accademia Nazionale dei Lincei (Roma)/Royal Society (London).

Author Disclosure Statement

The authors declare no competing financial interests.

Abbreviations Used

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The Key Role of Nitric Oxide in Hypoxia: Hypoxic Vasodilation and Energy Supply–Demand Matching

Abstract

Introduction

Bioenergetic Reactions and the Role of Oxygen

Sensing Hypoxia

Responding to Hypoxia

Transcriptomic

Hemodynamic effects

Respiratory

Renal and endocrine

Metabolic

NO Synthesis and Metabolism

Production by NOSs

Dietary nitrite/nitrate intake

Generation by other pathways

Systemic nitrite reduction

Release from pre-formed storage pools

NO metabolism

Half-life of NO

NO and Cardiovascular Homeostasis

NO as a regulator of vascular homeostasis

Mechanisms underlying the vasodilatory action of NO

Hypoxic Vasodilation to Improve Coupling of DO2 and Utilization

Evidence for the involvement of NO

Alternative theories

NO and control of metabolism

NO and microRNAs

NO and the renin–angiotensin system

NO levels in hypoxia: balance of synthesis and metabolism

Does hypoxia increase NO production by NOS?

Does hypoxia increase NO release from SNO-Hb and other NO storage forms?

Does hypoxia increase reduction of NO2 − to NO?

Does hypoxia reduce NO metabolism by CcO?

Conclusions

Footnotes

Acknowledgments

Author Disclosure Statement

Abbreviations Used

References

Hypoxic Vasodilation to Improve Coupling of DO₂ and Utilization

Does hypoxia increase reduction of NO₂ ⁻ to NO?