© 2005 American Thoracic Society DOI: 10.1165/rcmb.F299
Nitric Oxide, Hemoglobin, and Hypoxic VasodilationChildren's Hospital of Philadelphia, Philadelphia, Pennsylvania It is a continuing conundrum of nitric oxide (NO) biology that a molecule so chemically simple can possess such a wide range of biological effects. Nowhere is this complexity more apparent than in our understanding of NO's role in the circulation, highlighted by the recent review by Robinson and Lancaster in a previous issue of the Journal (1). In 1994, Dr. Lancaster wrote what was considered the definitive theoretical paper on NO's fate in the vasculature, constructing a series of mathematical models to understand how enough NO could escape scavenging (e.g., by hemoglobin) to generate its physiologic effects in the tissue (2). Significantly, the free radical species was the only form of NO considered relevant in these models (thus this modeling did not account for other biologically relevant forms of NO such as S-nitrosothiols [SNOs] which had been found in the plasma [3]). Around that time it was generally accepted that NO had just two possible reactions with hemoglobin: conversion to nitrate via reaction with oxyhemoglobin's bound oxygen, or irreversible binding to deoxyhemoglobin's unbound heme. However, in the years since Dr. Lancaster's original modeling, we have come to recognize that the biological chemistry of NO–hemoglobin interactions is far more subtle and complex than anyone imagined a decade ago. Indeed, the very fact that this review (1) focuses on the participation of NO and hemoglobin in something as complex as hypoxic vasodilation powerfully shows just how far our understanding has come from a time when it was still unclear whether intravascular NO could merely escape instantaneous scavenging. Before discussing the intricacies and questions emphasized by the Robinson and Lancaster review (1), it would be wise to reflect on how this sea change in our understanding occurred. In 1996, Jia and coworkers identified an entirely novel facet of NO and hemoglobin biology, namely the ability of hemoglobin to form a nitrosothiol on the cysteine residue at position 93 of the ß chain (4). Notably this cysteine is invariant throughout all mammalian and avian hemoglobins; despite decades of research, no functional significance had been ascribed to its chemically reactive side chain. Jia and colleagues, and subsequently others, showed that both the formation and the stability of this SNO were dependent on hemoglobin's conformation (i.e., relaxed versus tense) (4–6). These discoveries led to a number of investigations demonstrating that S-nitrosylated hemoglobin (SNOHb) is capable of dilating blood vessels in a manner dependent upon hemoglobin's conformation (5, 7). Although much debate has surrounded the mechanisms involved in NO and hemoglobin reactions, a few key fundamentals raised by Jia and associates have gained general acceptance, namely that: (1) NO equivalents are transported in the blood; (2) delivery of these equivalents is greatest in areas of hypoxia or restricted blood flow; (3) NO–hemoglobin interactions are considerably more complex than simple binding and consumption; (4) SNOs are a bioactive species in NO biology; and (5) red blood cells dilate blood vessels. Clearly the field of NO–hemoglobin biochemistry has come a long way since 1994. The review by Robinson and Lancaster is one of a number of articles to consider the issue of vascular delivery of NO. It appears to me that these articles raise five key areas of contention: (1) How is SNOHb formed?; (2) Is cooperativity involved in NO–hemoglobin binding?; (3) How are NO equivalents exported from the red blood cell?; (4) How is SNOHb best measured?; and (5) Are the hypoxia-dependent effects of SNOHb on vessel tension dependent on hemoglobin saturation? Much of the confusion surrounding these five central questions arises from an underappreciation of the complexity of the systems involved. How Is SNOHb Formed?
Robinson and Lancaster state that the "[f]ormation of nitrosothiol from NO and thiol requires one-electron oxidation (RS– + NO
One published report's failure to observe NO's heme-to-thiol transfer upon hemogobin's oxygenation (17) illustrates one of the confounding problems that continually besets the field: experiments that ostensibly reproduce original work but that are, in fact, substantially different. The details of some of these experimental differences are highlighted in a review that I recently coauthored (18). To take one salient but somewhat complicated example, we showed in our article on SNOHb formation (10) that NO's heme-to-thiol transfer was critically dependent upon hemoglobin's conformational changes. SNO is not formed, for instance, when NO, rather than O2, drives hemoglobin's relaxed-to-tense transition. Incubation of substoichiometric amounts of NO with deoxyhemoglobin results in a mixture of In the review by Robinson and Lancaster, and others like it, various authors have suggested that SNOHb formation is an artifact of adding concentrated NO to aerobic solutions, resulting in the formation of higher oxides of nitrogen. Aware of this potential artifact, we added NO, in the experiments referred to, "in the manner of titration," i.e., in very small quantities, precisely to avoid such an artifact. However, setting aside this consideration for one moment, such a suggestion ignores the fact that complete mass balance for NO can be achieved (i.e., 100% of the NO can be recovered as bound to the hemoglobin when added at low ratios). In our first paper on SNOHb synthesis, NO was added under deoxygenated conditions. Therefore all of the NO was bound by heme iron (as confirmed by spectroscopy) before oxygen addition. As such, virtually no free NO remained in solution to react with added oxygen and form higher oxides of nitrogen (10). This is also true of the work considering AE1 transfer (21). Therefore, one cannot explain the formation of SNOHb via the formation of higher oxides of nitrogen in solution. Further confirmation of this observation was provided by Herold and Rock, who found increased SNOHb yields with slow mixing and low phosphate concentrations, conditions that disfavor NO reactivity with oxygenated solution (22). Is Cooperativity Involved in NO–Hemoglobin Binding?
Every hemoglobin ligand that has been studied is subject to the laws of allostery and thermodynamic linkage. Is one supposed to consider NO immune to these laws? The fact that globin structure can affect the heme reactivity of NO has been well established in a variety of hemoglobins. Indeed, a variety of mutant hemoglobins have been constructed to specifically reduce their rates of reaction with NO (23). Using both ultraviolet-visible spectroscopy and EPR, we have detected increased binding of NO at the unoccupied hemes of R-state hemoglobin. These findings were inappropriately dismissed by Joshi and coworkers, who claimed that the reduced methemoglobin yield in our experiments resulted from dissolved NO reacting to form higher oxides (24). What their work fails to consider is that the reduced amount of methemoglobin was accounted for by formation of nitrosylhemoglobin, as shown by ultraviolet-visible spectroscopy and EPR. There are significant methodological differences in these experiments. Most notably, where we added NO in the manner of titration, each step was no more than 200 nM (11), Joshi and colleagues added the NO as a single large bolus such that the added concentration was How Are NO Equivalents Exported from the Red Blood Cell? Significant confusion has arisen in this area of the literature, much of it attributable to false suppositions. It is true that a single, precise mechanism has not been elucidated, although the work of Pawloski and colleagues demonstrated that AE1 was an effective conduit for NO equivalents (21). Indeed, its blockade inhibited the delivery of NO equivalents from red blood cells and prevented PO2-dependent vasorelaxation. There is no reason to presuppose that NO must exit the cell as nitrogen monoxide. Indeed, there is considerable experimental evidence indicating that such an assumption is invalid. However difficult the mathematics become by considering species other than this free radical, it is a biological imperative to include all potentially relevant forms of NO in any model. What cannot be denied is that red blood cells have been demonstrated to produce NO-mediated vasorelaxation. Furthermore, it would appear that this vasorelaxation is at least partially mediated by guanylate cyclase activation, as the work of Datta and coworkers showed inhibition by ODQ (25). Moreover, Lipton and colleagues were able to demonstrate the delivery of NO equivalents from red blood cells to glutathione resulting in S-nitrosoglutathione formation (26). Finally, mice lacking an SNO-glutathione reductase enzyme have been demonstrated to have increased circulating SNOHb and a reduced vascular resistance (27). How Is SNOHb Best Measured? To fully understand the issues surrounding NO-metabolite measurement techniques, it helps to consider the array of chemical entities involved. Nitrogen possesses a redox spectrum ranging from fully oxidized nitrate to fully reduced ammonia. (This redox spectrum is similar to that of oxygen itself, which ranges from molecular oxygen to water.) Except for species at the far ends of the spectrum, the redox interchanges between these molecules are relatively facile. As such, it is very difficult to obtain a "real time" picture of any individual NO-metabolite in a collected sample. Nitric oxide should never be considered as a solitary and discrete chemical entity in any biological system. Experimental and technical variations account for a great deal of the inconsistency among reported in vivo measurements of NO and its metabolites. One would not think of performing a series of complicated purification steps on a sample containing superoxide and expect the final measurement to accurately reflect the in vivo quantity. Likewise, accurately measuring SNOHb is fraught with technical challenges. For example, because SNOHb's stability is critically related to the conformation of the hemoglobin molecule, experimental manipulations (e.g., alterations in PO2, acidification, heme modification) can lead to extensive changes in the profile of detectable NO metabolites.
A wide variety of techniques have been used to measure SNOHb in the circulation. In all cases the groups were able to measure SNOHb (7, 25, 28–32) (although one group was unable to measure SNOHb in their human samples [33]). The quantities of SNOHb have varied between these groups but it has always been detected. One of the principal complaints that has been made about the higher values of measured SNOHb is that they may be contaminated with nitrite. However, in all of our original measurements nitrite (and nitrate) was removed by passage over sephadex G-25. In addition, nitrite is not measured readily by photolysis chemiluminescence ( An alternative technique for measuring hemoglobin-bound NO is chemical reduction via triiodide followed by chemiluminescence. First, nitrite, iron-nitrosyl, and SNO are all reduced. Next, to remove nitrite from the total signal, a number of groups have used acidification and sulfanilamide treatment. Although this method can work with some purified nitrosylated proteins, the addition of excess acid can complicate measurements made in biological samples. This is especially evident with red blood cells, where SNO stability is particularly dependent upon protein conformation. Different SNOHb measurement techniques (e.g., triiodide method versus 3-C system for measurement of SNO stability in the red blood cell [32, 34]) have yielded dramatically different results, and I think it is significant that the lower measurements have all been reported with techniques using acidification to avoid "contaminant nitrite." Regardless, even these low levels of detected SNOHb were sufficient to produce vasorelaxation. Some of the most convincing evidence that a particular process has an important role in physiology arises when derangements are detectable in pathologic states. This has now been shown to be the case for SNOHb in four cardiovascular disease states, namely sickle cell disease, diabetes, sepsis, and congestive heart failure (25, 27, 31, 35, 36). These studies point to the potential importance of SNOHb in the human cardiovascular system. Are the Hypoxia-Dependent Effects of SNOHb on Vessel Tension Dependent on Hemoglobin Saturation? One of the most remarkable observations concerning SNOHb has been its ability to alter vessel diameter in vitro and blood flow in vivo. Stamler and colleagues were able to show that SNOHb in vivo produces changes in blood flow in the brain that were PO2-dependent (5). Since that time a number of publications have demonstrated that red blood cells, both with and without NO pretreatment, dilate blood vessels in a PO2-dependent manner. It has been suggested by some that such measurements may result from the effects of hypoxia on the vessels themselves, but this contention represents a misunderstanding of the experimental setup and the physiology of hypoxic vasodilation. The direct effects of hypoxia are too slow and too insensitive to changes in O2 concentration to explain in vivo responses. Moreover, McMahon and coworkers and Datta and colleagues measured the fold effect of hypoxia on S-nitrosoglutathione-mediated relaxation in their system (2.6) and found it to be much less than that observed with red cells (7.6) (7, 25). Furthermore, James and associates demonstrated that red blood cell–mediated hypoxic vasodilation was hemoglobin-dependent and did not occur with red blood cell ghosts (31). It has also been suggested that these relaxations may be generated by contaminant nitrite. But this idea is not based on experimental evidence. In the work of both McMahon and colleagues and James and coworkers, nitrite was removed from the system before assay (7, 31). Red blood cells were washed free of nitrite. Further, it was shown that addition of nitrite did not alter hypoxic vasodilation mediated by red blood cells. A recent article by Cosby and associates suggesting otherwise should be viewed with skepticism: although hemoglobin-mediated reduction of nitrite was proposed as a mechanism of hypoxic vasodilation, the bioassays performed in this article do not show decrease in vessel tension (rate and amount). Rather, they show "a change in the PO2 at which the vessel starts to relax"—a parameter that the bioassay employed is not designed measure (37). Indeed, this parameter is a function of core muscle PO2, which Cosby and colleagues do not assay. This result of the study by Cosby and coworkers is unrelated to hypoxic vasodilation and distinctly different from the dilation by native red blood cells. Hemoglobin's capacity to act as a nitrite reductase has been well known for over twenty years. This enzymatic function of hemoglobin enables the conversion of nitrite to both iron-nitrosyl heme and SNO. Nitrite is also well known to react with thiols under acidic conditions to produce SNO. Therefore, it is important to consider nitrite not as merely a "stable end product of NO metabolism" but as a bioactive form of NO itself. However, one cannot equate the production of iron-nitrosyl heme, which has no vasodilatory action, with the production of bioactivity. Thus, the data presented so far do not allow the conclusion that nitrite reduction is directly involved in red blood cell–mediated hypoxic vasodilation. In summary, this first decade of NO–hemoglobin investigation has highlighted the novel phenomenon of endogenous red blood cell NO equivalents producing hypoxic vasodilation. As the review by Robinson and Lancaster illustrates, the biochemistry of NO and hemoglobin interaction is complex and one should avoid making overly simplistic statements. However, despite this complexity, it appears that reaction with the "invariant" cysteine at position ß 93 lies very much at the heart of the physiology of NO and the red blood cell. For a more complete review of the points raised here the reader is referred to two recent reviews (18, 20). Footnotes Conflict of Interest Statement: A.J.G. has no declared conflicts of interest. References
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RSNO + e–), for which there is no obvious electron acceptor." This oft-repeated assertion oversimplifies the relevant chemistry and ignores substantial experimental evidence. It has previously been shown that nitrosothiol synthesis can occur via direct reaction with subsequent electron abstraction by oxygen or heme iron (
and ß nitrosylheme; however, over time a pentacoordinate 
25-fold higher (
