Nitric oxide formation versus scavenging: the ... - Wiley Online Library

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J Physiol 590.20 (2012) pp 4993–5000

SYMPOSIUM REVIEW

Nitric oxide formation versus scavenging: the red blood cell balancing act Benjamin Y. Owusu1 , Ryan Stapley1 and Rakesh P. Patel1,2

The Journal of Physiology

1

Department of Pathology and 2 Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham, AL 35294, USA

Abstract Nitric oxide (NO) is a key modulator of vascular homeostasis controlling critical functions related to blood flow, respiration, cell death and proliferation, and protecting the vasculature from pro-inflammatory and coagulative stresses. Inhibition of NO formation, and/or diversion of NO away from its physiological signalling targets lead to dysregulated NO bioavailability, a hallmark of numerous vascular and pulmonary diseases. Current concepts suggest that the balance between NO formation and NO scavenging is critical in disease development, with the corollary being that redressing the balance offers a target for therapeutic intervention. Evidence presented over the last two decades has seen red blood cells (RBCs) and haemoglobin specifically emerge as prominent effectors in this paradigm. In this symposium review article, we discuss recent insights into the mechanisms by which RBCs may modulate the balance between NO-formation and inhibition. We discuss how these mechanisms may become dysfunctional to cause disease, highlight key questions that remain, and discuss the potential impact of these insights on therapeutic opportunities. (Received 18 April 2012; accepted after revision 6 June 2012; first published online 11 June 2012) Corresponding author R. Patel: Department of Pathology, University of Alabama at Birmingham, 901 19th Street South, BMRII 532, Birmingham, AL 35294, USA. Email: [email protected]

Physiological role of erythrocyte haemoglobin in the regulation of NO bioavailability

Several lines of evidence implicate red blood cells (RBCs) as modulators of NO signalling by effecting both NO formation and inhibition of NO signalling (illustrated in Fig. 1). In the context of mediating NO signalling, the primary investigated function is stimulation of hypoxic dilatation (Singel & Stamler, 2005; Gladwin

et al. 2006; Sprague et al. 2011), although as discussed below, modulation of coagulation and inflammation may also need to be considered (Crawford et al. 2004a; Srihirun et al. 2012). In the context of blood flow, the requirement for hypoxia, or more specifically hypoxaemia (haemoglobin deoxygenation) is key for understanding biological functions and molecular mechanisms. Hypoxic dilatation is a critical physiological process that ensures blood flow increases to tissue beds under hypoxic stress

Benjamin Owusu received his bachelor’s degree from University of Ghana and is currently pursuing his PhD in Biochemistry and Structural Biology at the University of Alabama at Birmingham (UAB). His research interests focus on the role of erythrocytes in vascular and pulmonary nitric oxide signaling. Rakesh Patel received his PhD from the University of Essex, UK in 1996. He moved to pursue post-doctoral studies at UAB, where he is currently a Professor in the Department of Pathology. His research interests have centred on understanding the role of erythrocytes and oxidative/nitrosative intermediates in modulating acute and chronic inflammatory diseases.

This report was presented at The Journal of Physiology Symposium on Red blood cell mechanisms of tissue blood flow control: physiological insights in health and disease, which took place at the Experimental Biology 2012 Meeting, San Diego, CA, USA on 22 April 2012. It was commissioned by the Editorial Board and reflects the views of the author.

C 2012 The Authors. The Journal of Physiology C 2012 The Physiological Society

DOI: 10.1113/jphysiol.2012.234906

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B. Y. Owusu and others

to provide nutrients and oxygen to support respiration, exemplified by increased blood flow to exercising skeletal muscle. Physiology studies in animals and humans have shown that hypoxic blood flow especially in the context of exercise does not track with dissolved oxygen tensions, but is directly correlated with the haemoglobin oxygen fractional saturation (Singel & Stamler, 2005; Gladwin et al. 2006), i.e. the amount of oxygen bound by haemoglobin, which in turn is controlled by several allosteric effectors. Within this framework three distinct, haemoglobin deoxygenation-dependent mechanisms have been investigated: S-nitrosohaemoglobin (SNOHb)-dependent bioactivity (Singel & Stamler, 2005), adenosine 5 -triphosphate (ATP) release (Sprague & Ellsworth, 2012) and deoxyhaemoglobin-mediated nitrite reduction to NO (Patel et al. 2011). We focus our discussion here on nitrite-dependent mechanisms, and limit discussion of ATP and SNOHb to the context of potential overlap between mechanisms and roles in disease and therapeutics. We also note that other mechanisms for RBC-dependent stimulation of NO signalling have been proposed and include effects of haematocrit on shear stress-dependent activation of endothelial nitric oxide synthase (eNOS) as well as the presence of an active eNOS within the RBC (Kleinbongard et al. 2006; Salazar Vazquez et al. 2008). However, to our knowledge, a haemoglobin oxygenation dependence to these eNOS-dependent pathways has not been established and is thus not discussed further here.

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Deoxyhaemoglobin-mediated nitrite reduction to NO

Deoxygenated RBCs and haemoglobin reduce nitrite by one electron to produce NO (eqn (1)), which should be contrasted with nitrite oxidation to nitrate upon reaction with oxyhaemoglobin (eqn (2); note this is an autocatalytic reaction and hydrogen peroxide (H2 O2 ) can react further with methaemoglobin (Hb3+ )). 3+ + Hb2+ + NO− + NO + H2 O 2 + H → Hb

(1)

3+ + + 2NO− 2[HbO2 ]2+ + 2NO− 2 + 2H → 2Hb 3 + H2 O2

(2) In this model, as haemoglobin becomes deoxygenated its nitrite reductase function is activated, resulting in NO production in ischaemic environments. NO formation from eNOS will decrease at very low limiting oxygen tensions, and thus the nitrite reductase activity of haemoglobin has been discussed as a mechanism to ensure sufficient NO bioavailability to sustain the multiple signalling roles associated with this free radical in ischaemic tissues (van Faassen et al. 2009). Biochemical studies demonstrate that haemoglobin oxygen sensing is key in controlling nitrite reduction with maximal rates of this reaction occurring at the haemoglobin p50 (oxygen tension at which haemoglobin is 50% bound with oxygen) (Huang et al. 2005; Crawford et al. 2006).In other words the nitrite reductase activity of haemoglobin

Figure 1. Current models for how RBCs can inhibit and stimulate NO signalling Shown is how haemoglobin oxygen sensing may be coupled to NO scavenging (by intact or cell-free haemoglobin), or formation (from deoxyhaemoglobin-mediated nitrite reduction or ATP release and subsequent eNOS activation) to control NO bioavailability in the vasculature. Also shown is the concept that nitrite reduction to elicit NO signalling during hypoxia may occur by tissues independent of RBCs, but RBCs can affect this process by controlling nitrite concentration via oxidation to nitrate. C 2012 The Authors. The Journal of Physiology C 2012 The Physiological Society

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Erythrocytes and NO homeostasis

is integrated with allosteric mechanisms that control oxygen delivery. Support for a RBC-dependent nitrite reduction mechanism to stimulate hypoxic NO signalling comes from in vitro data showing that a combination of deoxygenated RBCs or cell-free haemoglobin and nitrite produce detectable free NO, which can promote vasodilatation of isolated aortic rings, and inhibit platelet aggregation and mitochondrial respiration (Cosby et al. 2003; Nagababu et al. 2003; Crawford et al. 2006; Roche & Friedman, 2010; Cantu-Medellin et al. 2011; Shiva et al. 2011; Srihirun et al. 2012). Moreover, these effects are reversed by an NO scavenger. Several in vivo studies have complemented insights gained from biochemical studies supporting a role for nitrite-reduction by deoxyhemoglobin in NO homeostasis and include (i) positive arterial–venous nitrite concentration gradients in humans (which is associated with concomitant formation of markers of nitrite–deoxyhaemoglobin reactions in RBCs, the magnitude of which increases with exercise stress), which is associated with increased blood flow (Cosby et al. 2003; Rogers et al. 2007; Maher et al. 2008; Dufour et al. 2010), (ii) nitrite-dependent stimulation of blood flow only in ischaemic, but not in normoxic, murine hind limbs (Kumar et al. 2008), (iii) nitrite-dependent increases in cerebral, pulmonary, intestinal blood flow (Kozlov et al. 2005; Blood & Power, 2007; Rifkind et al. 2007), and (iv) data suggesting that nitrite derived from nitrate therapy improves human exercise performance and decreases blood pressure in normo- and hypertensive individuals (Larsen et al. 2006; Kapil et al. 2010; Kenjale et al. 2011; Lansley et al. 2011). Definitively proving a functional role for the RBCs in vivo remains challenging, however, largely due to the fact that disrupting nitrite reduction by RBCs (through modulating oxygen affinity for example) will also affect oxygen transport (Vitturi & Patel, 2011). This is an area that requires further research. It should also be stated that some studies have failed to observe free NO formation by haemoglobin-mediated nitrite reduction (Mikulski et al. 2010). Central to this issue is the question: how could RBCs stimulate NO-dependent signalling via a mechanism that involves NO formation by erythrocyte haemoglobin? This question should be considered in the context that both deoxy- and oxyhaemoglobin react and inhibit NO bioavailability very rapidly (bimolecular rate constants = 3–6 × 107 M−1 s−1 ) (Kim-Shapiro et al. 2011), which when coupled with high millimolar haem concentrations implies that any nitrite-derived NO formed at the haem should not be able to escape the RBC. Thus a critical question in this area remains to resolve the paradox of stimulation of NO signalling by deoxygenated RBCs and nitrite, versus rapid NO scavenging by haem. Proposed solutions include nitrite–deoxyhaem reactions proceeding via formation of intermediate species that avoid rapid inhibitory reactions C 2012 The Authors. The Journal of Physiology C 2012 The Physiological Society

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by the haem, allowing these to diffuse out of the RBCs and then produce NO to elicit endocrine effects (Robinson & Lancaster, 2005; Basu et al. 2007; Salgado et al. 2011). Regulation of this process may occur by controlling transport of nitrite or intermediates in and out of the RBC as recently suggested (Vitturi et al. 2009; Jensen & Rohde, 2010; de Almeida et al. 2011). One candidate is dinitrogen trioxide (N2 O3 ), which may be formed by a reaction between NO and a nitrite–ferric haem intermediate (Basu et al. 2007; Roche & Friedman, 2010). N2 O3 does not react with haem, and is small and uncharged and could therefore diffuse out of the RBC where its homolysis can generate NO (and the nitrogen dioxide radical). Further support for this mechanism is that N2 O3 is a nitrosating agent, and indeed, nitrosated products (e.g. S-nitrosothiols) are also products of nitrite–deoxyhaemoglobin reactions (Cosby et al. 2003; Vitturi et al. 2009). That said, this remains a working model since theoretical concerns still need to be resolved. For example, N2 O3 also readily hydrolyses in water and its rapid reactions with amines and thiols (which are present in the RBC and circulation) provide effective competitive pathways for homolysis and NO formation. Thus the search for mechanism(s) that can explain how NO is produced from nitrite and RBCs remains intriguing and its elucidation will not only inform on physiological linkages between hypoxia and vascular NO signalling, but also provide insights that may be utilized to develop targeted therapies to improve NO bioavailability in vascular inflammatory diseases. We also note that other RBC-dependent mechanisms for nitrite bioactivity have been proposed including acidic pH-dependent reduction by RBC associated xanthine oxidoreductase (Webb et al. 2008), nitrite-dependent stimulation of ATP release (Cao et al. 2009) and carbonic anhydrase-dependent nitrite reduction (Aamand et al. 2009). Dysfunction in RBC-dependent NO signalling and disease

Beyond physiological functions, emerging data are beginning to highlight how disruption in the mechanisms by which RBCs affect NO signalling can contribute to haematologic, vascular, and pulmonary inflammatory diseases. This can occur by physical disruption of the RBC itself leading to spillage of cell-free haemoglobin into the circulation, as well as by biochemical and molecular defects in the pathways that couple RBC oxygen sensing to stimulation of NO signalling. In this section we provide a brief overview of these processes and the diseases involved, and highlight potential and novel targets for therapeutic intervention. Haemolysis and acute loss of NO bioavailability. The

longest standing insights into NO–haemoglobin reactions

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are that they are rapid, which combined with the high concentration of haemoglobin and high diffusability of NO, should lead to inactivation of the NO signal (and see above). The question why all NO generated in the vascular compartment is not immediately scavenged by RBCs, but is able to elicit many signalling events despite the presence of millimolar haem in the vicinity, was initially raised by Lancaster and colleagues (Lancaster, 1994). A series of seminal studies by multiple groups have established that physical encapsulation of haemoglobin within the RBC is critical in slowing down haemoglobin–NO reactions by ∼1000-fold (Liao et al. 1999; Liu et al. 2002; Azarov et al. 2011). The biophysical and molecular mechanisms underlying this decrease in rate remain under investigation, but effectively increase NO diffusion barriers into the RBCs. Intriguingly, oxygen fractional saturation of RBC modulates NO diffusion barriers (Azarov et al. 2005), suggesting that preventing inhibition of NO signalling should also be considered as a mechanism in the same context as fractional saturation-dependent formation of NO and stimulation of NO signalling. An additional diffusion barrier is an RBC-free zone, generated adjacent to the endothelial monolayer and created according to the Fahraeus effect (Liao et al. 1999). The focus of most recent studies in this area has been to understand factors intrinsic to the RBC that slow NO diffusion, but little attention has been paid to factors that modulate the RBC free zone. The latter is regulated by the glycocalyx, which is rapidly emerging to be an extracellular structure that is dynamically regulated with respect to composition and size, especially during inflammation (Becker et al. 2010). How RBC free zone-dependent modulation of NO bioavailability changes under different inflammatory stress conditions remains to be elucidated. The importance of haemoglobin encapsulation is best evidenced by observations that haemolysis leads to potent inhibition of NO bioavailability. Without compartmentalization-dependent diffusion barriers, haemoglobin increases blood pressure, pro-coagulation and inflammatory stress – all effects implicated in the pathogenesis of sickle cell disease, sub-arachnoid haemorrhage, pulmonary hypertension, toxicity associated with transfusion of older RBC units (so called RBC storage lesion) and failure of acellular haemoglobins as blood substitutes (Rother et al. 2005; Buehler et al. 2011). How haemolysis occurs and how it could be prevented remains to be determined together with a better understanding of endogenous mechanisms that protect against cell-free haemoglobin will provide much needed insights (Baek et al. 2012). Clearly many questions still remain, but it is evident that haemolysis dramatically disrupts NO homeostasis tilting the scales towards the scavenging side of the balance. Haemolysis is the dramatic conclusion to perturbations in RBC structure. However, it is important not to exclude potential effects of more

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subtle changes in the biophysical properties of RBCs and NO-scavenging kinetics. For example during RBC storage in the blood bank, or in chronic obstructive pulmonary disease, RBC morphology is dramatically altered, being smaller less deformable and more echinocytic in shape. Preliminary data (Stapley et al. submitted) suggest that these changes lead to an intact RBC that also scavenges NO at faster rates. Combined with other recent studies showing that haemoglobin containing microparticles emanating from RBC scavenge NO at similar rates compared to cell-free haemoglobin (Donadee et al. 2011), the emerging paradigm is that a spectrum of changes to the RBCs from the intact discoidal ‘healthy’ shape to haemolysis may disrupt mechanisms that slow NO scavenging. More and more studies are beginning to document potential links to RBC alterations and disease and further insights into this question will provide novel therapeutic targets to replete NO signalling in diseases characterized by deficits in vascular NO bioavailability.

Uncoupling of intact RBC-dependent activation of NO signalling. Separate studies have implicated dysfunction

in either ATP or SNOHb pathways in various diseases. For example experimental evidence for cause–effect relationships between dysfunctional ATP release from RBCs and pulmonary arterial hypertension (PAH), microvascular flow deficits in diabetes and inflammatory stress during transfusion toxicity has been provided (Sprague et al. 2001, 2006; Zhu et al. 2011). Moreover dysfunctional ATP release has been demonstrated in RBCs isolated from cystic fibrosis patients (Liang et al. 2005). Elegant insights into signalling processes that regulate ATP release from hypoxic RBCs are providing the precise proteins whose function is altered during diabetes that then leads to diminished ATP release. These studies are exciting as they provide therapeutic targets for potentially improving microcirculatory blood flow. Similarly, deficits in SNOHb-dependent signalling have been reported in PAH, diabetes, transfusion toxicity, congestive heart failure and sickle cell disease (Datta et al. 2004; James et al. 2004; McMahon et al. 2005; Pawloski et al. 2005; Bennett-Guerrero et al. 2007; Reynolds et al. 2007). The overlap between diseases implicated by dysfunctional ATP release and SNOHb bioactivity should be noted and underlies in part, we feel, the debate in the literature regarding the SNOHb hypothesis. Our previous data using transgenic mice that express either wild-type human haemoglobin or haemoglobin in which the site of S-nitrosothiol formation, β93cys, has been replaced with an alanine showed no deficit in isolated RBC hypoxic vasodilatation responses (Isbell et al. 2008). Using a combination of approaches including apyrase-dependent ATP degradation, our data suggested that addition of C 2012 The Authors. The Journal of Physiology C 2012 The Physiological Society

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isolated RBCs to hypoxic vessel rings induce vasodilatation via ATP release (Crawford et al. 2006; Isbell et al. 2008). Moreover, the underlying mechanism of dilatation depended on the type of vessel and the species from which it was isolated; whereas rabbit vessels did (Crawford et al. 2006). We note that other data using eNOS knockout aorta, which preclude ATP-dependent effects, show no deficit in isolated RBC-dependent hypoxic vasodilatation supporting a role for SNOHb (Diesen et al. 2008). However, ATP can still promote dilatation of de-endothelialized vessels (Isbell et al. 2008). These data underscore that the experimental system employed plays a critical role in insights gained and suggest that broad conclusions supporting or dismissing hypotheses must be made with all factors considered. Notwithstanding mechanistic questions, strategies that restore controlled ATP release and/or promote nitrosylation of RBC proteins are intriguing possibilities for all the diseases mentioned above. Less is known about if, and if so how, altered nitrite reactions with RBCs could play a role in disease. A clinical study in critically ill patients with sepsis, demonstrated that arterial–venous nitrite gradients were approximately half (∼25 nM) in non-survivors compared to survivors (∼45 nM) or healthy volunteers (∼39 nM) with the association with higher arterial–venous gradients and survival being significant (Morgan et al. 2010). Artery–vein (A-V) nitrite consumption may reflect reactions to produce NO-based dilatory signals from deoxygenated RBCs, although differential consumption by tissues present in the arterial vs. venous circuits cannot be excluded. That said, diminished A-V gradients in sepsis is suggestive of a defect in RBC–nitrite reactions and other studies have shown that RBC are targets for oxidative damage in sepsis indicated by altered antioxidant levels and shape (Crawford et al. 2004b; Spolarics et al. 2004). Another perspective is that since nitrite serves as a source for NO in ischaemia to limit ischaemia–reperfusion injury (higher endogenous or exogenously applied nitrite protects against numerous ischaemia–reperfusion injury insults; Kevil et al. 2011), RBCs may affect inflammatory disease by modulating the concentration of circulating nitrite. Indeed, antioxidants in the RBCs are thought to be important in limiting nitrite oxidation to nitrate. Oxidative damage to RBCs has been documented in several pathologies; it remains to be determined if this leads to increased nitrite oxidation, and if so, whether this predisposes tissues to inflammatory tissue injury. This remains speculation, although our recent studies with stored RBCs, a treatment that decreases cellular antioxidant levels, are demonstrating increased rates of nitrite oxidation and decreased levels of circulating nitrite in vivo (Stapley et al. submitted).


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Summary

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Acknowledgements This work was supported by grants from the National Institutes of Health (HL092624 and HL095468 to R.P.P.).

Conflicts of interest

R.P.P. is a coinventor on a patent for use of nitrite salts for the treatment of cardiovascular conditions.
