© 2004 American Heart Association, Inc.
Letter to the Editor |
How do Red Blood Cells Dilate Blood Vessels?—Reply
Department of Physics, Wake Forest University, Winston-Salem, NC, shapiro{at}wfu.edu
Department of Pathology, University of Alabama, Birmingham, Ala
National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Md
National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Md, mgladwin{at}nih.gov
Department of Biophysics, Medical College of Wisconsin, Milwaukee, Wis
To the Editor:
A recent Letter to the Editor in Circulation Research from Drs. Allen and Piantadosi criticizes some of our recent work and its interpretation.1 We find that the letter contains significant errors in evaluating our work that we would like to address with the hope that we can resolve some of the issues raised.
Allen and Piantadosi claim that a recent editorial by Gladwin and Schechter,2 outlining mechanisms that explain how nitric oxide (NO) is delivered by red blood cells (RBCs), creates a false dichotomy that impedes scientific advance. However, we find that these mechanisms involve fundamentally different biochemical reactivities and differ in both the source of NO and the nature of the oxygen sensing reaction. The first mechanism, proposed by Stamler’s group, suggests that hemoglobin binds NO at the cysteine 93 to form S-nitroso hemoglobin (SNO-Hb), which serves as a storage pool for a circulating S-nitrosothiol; the S-nitrosothiol is released from hemoglobin after a change in hemoglobin quaternary structure from the R (oxygenated) to T (deoxygenated) state. The second mechanism, proposed by our group, suggests that the anion nitrite serves as the storage pool and is converted to NO through heme-based reduction by deoxyhemoglobin.3 The SNO-Hb hypothesis does not invoke nitrite nor heme-based nitrite reduction, and the nitrite reductase hypothesis does not require SNO-Hb as an intermediate. In the interest of real "scientific advancement,"1 we believe that a complete understanding of the nitrite model will require characterization of the nitrite heme reaction, including coordination of the nitrite anion, rates of electron donation from the heme group, allosteric and electronic regulation of heme electron donation, and a chemical understanding of intermediates that escape heme inactivation. On the other hand, advancement of the SNO-Hb hypothesis has a different set of challenges, largely unresolved, such as rates of transnitrosation, stability of S-NO in the reductive environment of the erythrocyte, oxygen affinity of the nitrosated hemoglobin, and mechanism of NO capture and S-NO formation. That there are two competing mechanisms is further evidenced by Allen and Piantadosi, themselves, in their effort to discredit the nitrite reduction hypothesis1 and by the fact that proponents of the SNO-Hb hypothesis have explicitly stated that nitrite has no vasodilatory action under physiological conditions.4,5
The arguments given by Allen and Piantadosi to discredit the nitrite reduction hypothesis are based on some misunderstandings of the original work by Cosby et al.3 Allen and Piantadosi argued that the nitrite effect observed in vivo is too slow to be important, occurring on the order of minutes.1 However, nitrite was infused into the brachial artery and increased blood flow was measured by Cosby et al in the same arm.3 In order for the nitrite to have this effect, it must act across the forearm circulation, before it is diluted in the systemic circulation. Likewise, the difference in NO-bound hemoglobin found across the forearm demonstrates that the nitrite reacts to form iron-nitrosyl-hemoglobin within seconds (detailed reaction kinetics in vivo and in vitro were provided).3 Similarly, Allen and Piantadosi argue that the vasodilatory effects of nitrite on aortic rings was too slow to be of significance, taking hundreds of seconds.1 Actually, although relaxation is observed over several hundred seconds in Figure 5f of Cosby et al, the effect begins within tens of seconds.3 As soon as hemoglobin deoxygenates below its P50, nitrite-dependent relaxation commences. The single most significant result in the article by Cosby et al, that infusion of supraphysiological and near physiological levels of nitrite have vasodilatory effects in vivo, is not addressed by Allen and Piantadosi.
The letter by Allen and Piantadosi contains misleading statements and comments.1 For example, they quote the editorial by Gladwin and Schechter2 as stating "... that experimental data and theoretical considerations ‘compellingly show’ that RBCs do not dilate blood vessels through export of NO bioactivity."1 The actual full sentence reads "... these data and the corresponding theoretical considerations... compellingly show that basal vascular tone is physiologically regulated by ‘on-site’ NO production, as opposed to delivery from red blood cell SNOHb."2 There is a difference between how basal tone is regulated and what vasoactive properties RBCs mediate (ie, hypoxic vasodilation). On the same page of that editorial, Gladwin and Schechter wrote, "Another interesting observation from multiple groups is that red blood cells and plasma ‘loaded’ with NO, whether by exposure to high concentrations of NO in solution or to NO gas or donors, can export NO and induce vasodilation in vitro and in vivo."2 Allen and Piantadosi end their letter suggesting some data in the article by Cosby et al resulted from uncontrolled differences in initial vessel tone or deoxygenation rates,1 yet present no evidence that this was the case.
In 1992, Stamler et al published an article suggesting that blood proteins may transport, rather than destroy, NO activity.6 This idea was developed into the SNO-Hb hypothesis, which has resulted in a wealth of research including our own. Although we repeatedly credit Stamler’s group with the principle of stable NO-species transport in blood, several important aspects of the SNO-Hb hypothesis are not reproducible experimentally, casting substantial doubt on the physiological role of SNO-Hb.2,7,8 Our nitrite reductase hypothesis may also contain aspects in need of revision including potential contribution from other heme proteins. Rather than attempting to dismiss the hypothesis without experimental evidence, it is important that test implications based on our hypothesis are confirmed or refuted by actual measurements in a variety of laboratories.
References
1. Allen BW, Piantodosi, C. A. How do red blood cells dilate blood vessels? Circ Res. 2004; 94: e105.
2. Gladwin MT, Schechter AN. NO contest: nitrite versus S-nitroso-hemoglobin. Circ Res. 2004; 94: 851–855.
3. Cosby K, Partovi KS, Crawford JH, Patel RP, Reiter CD, Martyr S, Yang BK, Waclawiw MA, Zalos G, Xu XL, Huang KT, Shields H, Kim-Shapiro DB, Schechter AN, Cannon RO, Gladwin MT. Nitrite reduction to nitric oxide by deoxyhemoglobin vasodilates the human circulation. Nat Med. 2003; 9: 1498–1505.[CrossRef][Medline] [Order article via Infotrieve]
4. McMahon TJ. Hemoglobin and nitric oxide. N Engl J Med. 2003; 349: 403–403.
5. Pawloski JR. Hemoglobin and nitric oxide. N Engl J Med. 2003; 349: 403–404.
6. Stamler JS, Jaraki O, Osborne J, Simon DI, Keaney J, Vita J, Singel D, Valeri CR, Loscalzo J. Nitric-oxide circulates in mammalian plasma primarily as an S-Nitroso adduct of serum-albumin. Proc Natl Acad Sci U S A. 1992; 89: 7674–7677.
7. Rassaf T, Bryan NS, Maloney RE, Specian V, Kelm M, Kalyanaraman B, Rodriguez J, Feelisch M. NO adducts in mammalian red blood cells: too much or too little? Nat Med. 2003; 9: 481–482.[CrossRef][Medline] [Order article via Infotrieve]
8. Gladwin MT, Lancaster JR, Freeman BA, Schechter AN. Nitric oxide’s reactions with hemoglobin: a view through the SNO-storm. Nat Med. 2003; 9: 496–500.[CrossRef][Medline] [Order article via Infotrieve]
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