NO adducts in mammalian red blood cells: too much or too little?
NO adducts in mammalian red blood cells: too much or too little?
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哺乳动物红细胞中没有加合物：太多还是太少？
DOI：
10.1038/nm0503-481
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发表时间：
2003
期刊：
影响因子：
82.9
通讯作者：
Feelisch,Martin
中科院分区：
文献类型：
--
作者：
Rassaf,Tienush;Bryan,NathanS;Maloney,RonaldE;Specian,Vicky;Kelm,Malte;Kalyanaraman,Balaraman;Rodriguez,Juan;Feelisch,Martin
与这些试剂一起介绍的给编辑的信。尝试使用McMahon等人的方案，用ELISA-2检测人溶血产物中的SNOHb。（检测限为50 nM）未显示可测量的量。通过化学发光法定量相同RBC裂解物等份试样中的亚硝酸盐证实了SNOHb的缺乏。与两个早期的研究6，7，没有HbNO检测到人全血和红细胞裂解物的EPR光谱，尽管灵敏度的限制。尽管红细胞中NO加合物的水平不同，但啮齿类动物和灵长类动物的血浆亚硝酸盐和S-亚硝基白蛋白浓度一致（大鼠、人类和豚鼠分别为198±33、205±21和413±94 nM亚硝酸盐和1.2±0.3、7.2±1.1和25.2±5.1 nM S-亚硝基白蛋白）。动物和人类受试者的实验分别由什里夫波特的路易斯安那州立大学健康科学中心的动物护理和使用委员会和机构审查委员会批准。本翻译研究的一个重要发现是从啮齿动物到灵长类动物RBC中观察到的NO加合物的下降，这密切反映了这些物种的血红蛋白巯基反应性的差异8，9（对于人类，大鼠和豚鼠数据，r= 0.99）。因此，在大鼠和豚鼠红细胞中发现的近微摩尔亚硝基水平可能是啮齿动物血红蛋白中存在快速反应硫醇（Cysβ-125）群体的结果，其浓度和反应性超过谷胱甘肽8，9。相比之下，尽管暴露于相当水平的血浆亚硝酸盐和S-亚硝基白蛋白（以及可能的NO），但灵长类动物RBC中NO加合物的浓度要低得多（< 1 nM），这可能是该血红蛋白中Cysβ-93的巯基反应性较低的结果。我们的研究中一个同样重要和令人困惑的发现是，与Stamler的研究组1报告的结果相比，人RBC中NO加合物的水平有很大差异（低≥ 3个数量级）。尽管关于人红细胞中NO加合物真实水平的争论并不新鲜（总结见参考文献10的表1），但本文报道的亚纳摩尔值将这一争论提高到了一个新的水平，提出了McMahon等人提出的调节作用是否可以在如此低的浓度下发挥作用的问题。可能有人认为，我们的方法只是缺乏灵敏度，或者它使NO加合物不稳定，但这与我们从啮齿动物和加标的人RBC中检测显著NO信号的能力不一致，或者与我们在未处理的全血等分试样和样品处理后分析的全血等分试样之间获得可比信号的能力不一致。同样很难驳回麦克马洪等人的观点。s微摩尔水平作为亚硝酸盐的人工亚硝化，因为我们自己的数据表明，这种物质仅以低得多的浓度存在于血浆（见上文）和RBC（200-300 nM）中。使目前关于人类细胞中真实浓度的分歧更加令人困惑的是，Stamler的方法和我们的方法似乎在大鼠RBC中的SNOHb和HbNO水平上相当一致。那么，是我们的方法检测到的NO太少了，还是真的很少，而Stamler的方法检测到的是人工产生的东西？或者人类血红蛋白具有一种微妙的机制，使其能够承担高和低的NO水平，而不会立即产生生理后果？考虑到这里提供的令人信服的证据表明，在动静脉氧梯度中NO加合物的水平较低，而McMahon等人报告的高水平支持了与此类加合物相关的拟议生理作用和治疗应用。
LETTERS TO THE EDITOR introduced together with these reagents. Attempts to detect SNOHb in human hemolysates with DAF-2 using the protocol of McMahon et al.(detection limit of 50 nM) did not show measurable amounts. Quantification of nitrite in aliquots of the same RBC lysates by chemiluminescence corroborated the lack of SNOHb. In agreement with two earlier studies6, 7, no HbNO was detected in human whole-blood and RBC lysates by EPR spectroscopy, sensitivity limitations notwithstanding. Despite contrasting levels of NO adducts in RBCs, plasma nitrite and S-nitrosoalbumin concentrations were consistent between rodents and primates (198±33, 205±21 and 413±94 nM nitrite and 1.2±0.3, 7.2±1.1 and 25.2±5.1 nM S-nitrosoalbumin in rats, humans and guinea pigs, respectively). Experiments in animals and human subjects were approved by the Animal Care and Use Committee and the Institutional Review Board, respectively, of the Louisiana State University Health Sciences Center in Shreveport. An important finding of the present translational study is the drop in NO adducts observed from rodent to primate RBCs, which closely mirrors the differences in hemoglobin thiol reactivity for these species8, 9 (r= 0.99 for human, rat and guinea pig data). The near-micromolar nitroso levels found in RBCs of rats and guinea pigs may therefore be a result of the presence of a population of fast-reacting thiols (Cysβ-125) in rodent hemoglobin whose concentration and reactivity exceeds that of glutathione8, 9. In contrast, the much lower concentration of NO adducts in primate RBCs (< 1 nM), in spite of exposure to comparable levels of plasma nitrite and S-nitrosoalbumin (and presumably NO), may be a consequence of the lower thiol reactivity of the Cysβ-93 in this hemoglobin. An equally important and perplexing finding from our study is the vastly different (≥ 3 orders of magnitude lower) level of NO adducts in human RBCs as compared with that reported by Stamler’s group1. Although disputes over the true levels of NO adducts in human RBCs are not new (summarized in Table 1 of ref. 10), the sub-nanomolar values reported here heighten this controversy to a new level that raises the question of whether the regulatory role proposed by McMahon et al. could function at such vanishingly low concentrations. It might be argued that our methodology simply lacks sensitivity or that it destabilizes NO adducts, but this is not consistent with our ability to detect significant NO signals from rodent and spiked human RBCs, or to obtain comparable signals between unprocessed wholeblood aliquots and those analyzed after sample processing. It is equally difficult to dismiss McMahon et al.’s micromolar levels as artificial nitrosation by nitrite, as our own data show that this substance is present only at much lower concentrations in plasma (see above) and RBCs (200–300 nM). What makes the present disagreement about the true concentration in human cells even more perplexing is the fact that Stamler’s methodologies and ours appear to agree fairly well on the SNOHb and HbNO levels in rat RBCs. So, is our method detecting too little NO, or is there indeed little there and Stamler’s method detecting something artificially produced? Or does human hemoglobin possess a subtle mechanism that allows it to assume both high and low NO levels without an immediate physiological consequence? Given the compelling evidence provided here for low levels of NO adducts across the arterio-venous oxygen gradient, and the high levels reported by McMahon et al. in support of the proposed physiological role and therapeutic applications related to such adducts in …