doi: 10.1161/CIRCRESAHA.108.183442
© 2008 American Heart Association, Inc.
Editorials |
Transnitrosation Signals Oxyhemoglobin Desaturation
From the Division of Pediatric Respiratory Medicine (N.M., B.G.), University of Virginia School of Medicine, Charlottesville; and Department of Pediatric Critical Care (A.D.), Washington University School of Medicine, St Louis, Mo.
Correspondence to Benjamin Gaston, MD, Box 800386, University of Virginia School of Medicine, Charlottesville, VA 22908. E-mail bmg3g{at}virginia.edu
See related article, pages 545–553
Key Words: S-nitrosylation • hypoxia • blood flow
Erythrocytes dilate peripheral blood vessels as a function of oxyhemoglobin desaturation.1 This effect increases regional blood flow to hypoxic tissues. The mechanisms underlying the peripheral vasodilatory effects of desaturating erythrocytes are incompletely understood but do not involve activation of local, endothelial NO synthase (eNOS). Indeed, eNOS-derived NO itself primarily relaxes large vessels and does that primarily only in the absence of blood.
In this issue of Circulation Research, Diesen et al confirm that thiols carrying a nitrosonium (NO+) equivalent signal cyclic GMP-dependent vascular smooth muscle relaxation during erythrocytic oxyhemoglobin desaturation.2 These data support paradigm-changing work demonstrating that nitrogen oxides are transported by circulating erythrocytes to signal oxyhemoglobin desaturation through serial NO/NO+ thiol equilibria and transfer reactions (transnitrosation) and that these reactions normally take place at sites remote from NOS activity.1–3 These new data show clearly that this signaling is independent of local NOS activity, of cyclooxygenase, of ATP, and of the effects of hypoxia itself on vascular smooth muscle.
Erythrocytes are endogenously "preloaded" with nitrogen oxides for delivery to vessels in conditions of oxyhemoglobin desaturation.1–3 This signaling links delivery of erythrocytic NO/NO+ groups to oxyhemoglobin desaturation, permitting augmented blood flow to hypoxic tissue. Three mechanisms have been proposed by which transitions in hemoglobin (Hb) conformation may result in nitrogen oxide transfer to blood vessels. (1) In the originally proposed mechanism, Hb deoxygenation (R-to-T transition) permits transnitrosation from Hb β-chain cysteine 93 (βCys93) to erythrocytic carrier thiols (Figure).1–3 This concept is supported by the data obtained by Diesen et al.2 (2) At one time, NO radical was proposed to diffuse away from the Fe (II) heme iron in T state Hb. Although there was an initial enthusiasm for this construct as an NO radical–based alternative to nitrosothiol-based NO+ transfer, it is essentially impossible kinetically; it is now acknowledged to be irrelevant to physiology.1,4,5 (3) More recently, it has been proposed that deoxyhemoglobin serves as an NO2– reductase, forming an NO radical that, again, escapes autocapture by Fe(II) heme to diffuse into (and activate guanylate cyclase in) smooth muscle cells.6 This mechanism is also essentially impossible kinetically but is still referenced. The data from the study by Diesen et al argue against this construct, which is discussed further below.
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It had been suggested that NO+ transfer from βCys93 in deoxygenating Hb might not be relevant to physiology because mice genetically engineered to be deficient in the βCys93 were once suggested to not have had abnormalities in hypoxic responses. However, it turns out that none of the physiological responses to Hb desaturation demonstrated to hinge on transnitrosation has actually been tested in the βCys93-deficient mouse. Specifically, there are 3 such responses; none of the 3 was studied. These are: (1) pulmonary vascular remodeling in chronic hypoxemia7; (2) altered blood flow in vascular beds in response to altered oxyhemoglobin saturation1,8,9; and (3) augmented minute ventilation in response to hypoxia.10 Even assuming that humanized Hb expressed in these mice behaves identically to murine Hb (which it likely does not), the mice were studied only with regard to overall blood pressure responses.6 Blood pressure is not a surrogate for regional blood flow distribution. Furthermore, the work by Diesen et al provides insight into the negative vascular ring study using erythrocytes from these mice. Specifically, βCys93 S-nitrosothiol bonds can be rapidly depleted in wild-type red cells,2,11,13 ablating their ability to relax vascular smooth muscle with Hb desaturation. The pulmonary vascular ring responses in the studies of the βCys93-deficient mouse were minimal: either the control RBCs were S-nitrosothiol depleted (rendering them functionally indistinguishable from the RBCs of βCys93-deficient RBCs) or the vascular rings were essentially unresponsive.
Diesen et al confirm previous studies that transnitrosation from erythrocytic S-nitrosothiols forms low-mass S-nitrosothiols that can signal vascular effects (Figure).4–6,8–10,13 Chronic in vivo transnitrosation from hypoxic erythrocytes to N-acetyl-cysteine (forming S-nitroso-N-acetyl cysteine) causes pulmonary vascular remodeling through upregulation of hypoxia- and S-nitrosothiol–sensitive genes in the pulmonary endothelium.7 Diesen et al now show that erythrocytic transnitrosation to N-acetyl-cysteine and other low-mass thiols potentiates acute vascular smooth muscle effects of erythrocytic S-nitrosothiols in the context of Hb deoxygenation.
We now see a picture emerge of elegant regulation: NO+ transfer from deoxyhemoglobin thiols to erythrocytic low-mass and membrane14 thiols permits NO/NO+ transfer to target proteins in the vasculature. Thus, acute hypoxic effects are actually signaled by oxyhemoglobin desaturation rather than low PO2.12 These acute effects include increased minute ventilation and cGMP-dependent vascular smooth muscle relaxation.1,2,10 However, excessive NO/NO+ transfer reactions can signal counterregulatory, protective effects in the endothelium both acutely through S-nitrosylation of metallothionein15 and chronically through upregulation of hypoxia and S-nitrosothiol–dependent genes that cause vascular remodeling.7 Indeed, this construct suggests a unifying hypothesis underlying pulmonary arterial hypertension. Specifically, pulmonary arterial hypertension can be caused by (1) chronic systemic hypoxia (causing excessive S-nitrosothiol–mediated NO/NO+ transfer to thiols in erythrocytes returning to the right heart and pulmonary artery); (2) chronically increased blood flow to the pulmonary artery (increasing the numbers of S-nitrosothiol–bearing erythrocytes to pass through the pulmonary vasculature endothelium, particularly in the context of polycythemia); (3) chronic inflammation (increasing the number of S-nitrosothiols in red cells); and (4) chronic N-acetylcysteine administration (excessively transferring NO/NO+ to the pulmonary vascular endothelium).7,16
In normal human physiology, endogenous NO2– does not appear to signal erythrocyte deoxygenation. Diesen et al show that physiologically relevant concentrations of NO2– have no effect on vascular smooth muscle tone in hypoxia.2 Support for the idea that NO2– reductase function of Hb would serve physiological function has rested in 3 legs. Firstly, it is known that pharmacological (high micromolar, 2 to 3 orders of magnitude higher than physiological) concentrations of NO2–, injected into an artery, will cause weak vasodilatation. This mechanism likely involves oxidation of Fe(II) Hb to Fe(III), with the formation of Fe(III)-NO species in equilibrium with an Fe(II)-NO+ that is capable of modifying thiols through transnitrosation.17,18 Whereas this mechanism may be operative in the formation of S-nitroso-Hb and in response to high (pharmacological) concentrations of NO2–, it is not operative during normal hypoxic signaling by which erythrocytes caused cGMP-dependent vascular smooth muscle responses: neither the concentrations nor the kinetics are in the range of concentrations or blood flow rates in vivo.1,5
Secondly, it has been argued that Hb micropopulations respond at the Hb P50 to form and release NO from NO2–, which escapes autocapture from other heme groups. However, the physiological response under study, hypoxic vasodilatation does not demonstrate a P50 threshold: it is a graded effect that increases at decreasing level of oxyhemoglobin saturation.19 Moreover, NO does not diffuse away from Fe(II) heme-containing erythrocytes in any physiologically relevant concentration.5
Finally, high nanomolar concentrations of NO2– (in the presence of deoxyhemoglobin) were once proposed to cause vascular smooth muscle relaxation.20 Five years later, these data still await confirmation at these concentrations. Diesen et al did not observe the same effect. Note in this regard that some of the effects reported to be NO2–/NO radical–mediated may reflect inorganic NO2– protonation at low pH.21
Confusion regarding erythrocytic NO metabolism has arisen because of the use of iodine-based assays to measure S-nitrosothiols. These assays may misidentify nitrogen oxide species and require sample handling that alters Hb allostery before measurement.4,22 Indeed, what has been reported as erythrocytic NO2– using an iodine-based assay was lost with protein precipitation.22,23
More work remains regarding the effects of S-nitrosothiol depletion and transnitrosation-based augmentation of vascular smooth muscle relaxant effects in vivo, at more physiological hematocrit values and using direct blood flow measurements in hypoxic resistance arterioles. Additionally, virtually all the relevant studies regarding hypoxic signaling remain to be done in the βCys93-deficient mouse.
In conclusion, Diesen et al confirm the S-nitrosothiol–mediated mechanism by which cGMP-dependent vascular smooth muscle relaxation may be signaled by erythrocyte deoxygenation. Moreover, they demonstrate that transnitrosation from an erythrocyte thiol to a target thiol augments relaxation, consistent with a paradigm by which S-nitrosothiols can cause acute vascular effects, as well as vascular remodeling in the face of chronic, excessive S-nitrosothiol "dumping" in the pulmonary arterial bed. Their data thus confirm the novel paradigm in which it is not O2 tension per se that is sensed in hypoxic vascular beds but rather oxyhemoglobin desaturation. This concept has been slow to gain acceptance, primarily because it requires an appreciation that the effector molecules are S-nitrosothiol/NO+ donors, rather than NO radical alone. However, it provides an elegant mechanism to explain a broad range of previously obscure effects in physiology and in disease pathophysiology.
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Acknowledgments |
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Sources of Funding
B.G. and N.M. are supported by NIH grants 3RO1 HL59337, SARP 2RO1, and LH69170; and A.D. is supported by NIH grant 1K08GM069977.
Disclosures
B.G. is a consultant for Galleon Pharmaceuticals Inc.
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Footnotes |
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The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
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References |
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Related Article:
Circ. Res. 2008 103: 545-553.
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