Nitric Oxide Transport By Hemoglobin

正常志愿者的一氧化氮(NO)输注和实验动物的NO输注和吸入的结果证实，NO可以作为一种激素运输，因此具有潜在的药理作用(即药物)。我们认为，我们的镰状细胞患者缺乏血管效应是由于循环血红蛋白的存在，这有助于本病和其他慢性和急性溶血综合征的病理生理，特别是肺高压并发症，我们发现这种并发症在老年患者中非常严重和频繁。在最近的研究中，我们将亚硝酸盐注入正常人类志愿者的臂动脉，并表明这可以增加血液流量，这表明亚硝酸盐可以作为NO的生理来源发挥作用，并可以在药学上使用。然而，我们发现，亚硝酸盐输注对血管特性和高铁血红蛋白形成的影响相对持久，并建议将亚硝酸盐分配到不同的组织中。我们发现，在体外，脱氧红细胞和亚硝酸盐导致主动脉环准备物扩张，这表明亚硝酸盐被脱氧血红素蛋白激活的机制。我们还发现，低氧新生绵羊吸入亚硝酸盐会导致肺动脉压下降和NO呼出；在这些动物中注入亚硝酸盐会导致平均动脉压下降。 我们目前正在研究血液中亚硝酸盐的形成和分区，特别是红细胞中亚硝酸盐的形成和区隔，以及亚硝酸盐水平是否可能是人类心血管风险的标志。这些研究旨在使我们能够开始对正常人和患有各种缺血性(包括镰状细胞性贫血)疾病的人进行亚硝酸盐输注。我们已经证明，亚硝酸盐产生的NO的最大值出现在血红蛋白的p50附近，并且依赖于血红蛋白的变构构象。我们还开发了精确测量人类血液中亚硝酸盐水平的方法，并发现血液中的大部分亚硝酸盐都包含在红细胞中。在最近的报告期间，我们测量了人体红细胞对亚硝酸盐的摄取作为氧分压、pH、温度和其他变量的函数。这种摄取似乎是控制亚硝酸盐在血管内转化为NO的总速率的重要因素。此外，我们还发现，红细胞中的脱氢抗坏血酸可以催化亚硝酸铁血红蛋白(NbNO)的氧化释放NO，然后NO在细胞内转化为亚硝酸盐，而不是硝酸盐。这一反应可能是红细胞亚硝酸盐形成的主要机制，我们认为红细胞亚硝酸盐是生物活性NO在体内的主要储存部位之一，并导致我们建立了抗坏血酸/脱氢抗坏血酸与红细胞内NO/亚硝酸盐循环相互作用的模型。我们还检验了一种假设，即在低氧条件下，无细胞血红蛋白可能是NO的另一个内分泌来源。在这项研究中，我们构建了一个多细胞模型来表征亚硝酸盐与红细胞内和游离血红蛋白反应产生的NO的量，并发现通过这种机制产生的NO水平可能非常显著，与血管壁本身产生的NO相当。我们还详细研究了亚硝酸盐与氧合和脱氧血红蛋白的反应机理，发现在生理和药理上可能获得的条件下--特别是关于亚硝酸根与血红蛋白分子的比例--自由基(包括血红蛋白的铁基形式)的形成不太可能在很大程度上发生。这一点很重要，因为自由基的形成一直与亚硝酸盐(或NO)药理学的发展有关。我们认为，上述研究将有助于我们理解人类红细胞在调节NO生物活性中的作用，特别是通过亚硝酸盐中间体，并促进亚硝酸盐作为心血管病理有用药物的发展。在最近的工作中，我们一直在调查红细胞储存过程中与一氧化氮相关的物种的变化，以确定这些变化是否会导致血液，特别是红细胞，输血的并发症，即所谓的“储存损伤”。我们发现，正如我们之前的工作所预期的那样，亚硝酸盐水平在静脉切开后迅速下降，但随后令人惊讶的是，在长达42天的时间里，亚硝酸盐水平稳定在初始值的约四分之一。我们没有发现其他相关的NO变化的证据，目前正在研究亚硝酸盐变化是否有助于红细胞诱导的病理，以及同样重要的是，控制和稳定红细胞亚硝酸盐水平的机制。 在过去的一年里，我们已经证明，在生理亚硝酸盐浓度下，我们可以产生足够的NO来抑制血小板聚集；我们现在正在研究这些结果的生理和药理学意义，这些结果似乎涉及亚硝酸盐与循环中的红细胞的相互作用，并可能有助于循环中血小板反应性的生理和病理生理调节。我们还表明，在体外储存期间，血小板中的亚硝酸盐水平略有下降，如果这是由于NO的形成，可能有助于保持血小板的功能，以供输血。近几个月来，我们已经能够证明，我们可以通过血栓弹性测定法来测量红细胞、亚硝酸盐和血液凝固的相互作用，这种方法比单独测量血小板聚集或表面标志物测量更多的凝血步骤。使用这一新仪器，我们可能能够将我们关于红细胞产生NO的工作扩展到临床评估各种生理和疾病状态下的血液凝结。这项工作与我们关于亚硝酸盐潜在疗法的报告中描述的工作密切相关。

The results of nitric oxide (NO) infusions in normal volunteers and NO infusions and inhalation in experimental animals confirms that NO can be transported as a hormone and thus has the potential to be a pharmacological agent (i.e., a drug). We believe that the lack of vascular effects in our sickle cell patients is due to the presence of circulating hemoglobin and that this contributes to the pathophysiology of this and other chronic and acute hemolytic syndromes, especially the pulmonary hypertension complications which we have found to be severe and of high frequency in older patients. In recent studies we have infused nitrite into the brachial arteries of normal human volunteers and have shown that this increases blood flow, suggesting that nitrite could function physiologically as a source of NO and could be used pharmacologically. However, we find that the effects of nitrite infusion, both on vascular properties and on methemoglobin formation, are relatively long lasting and suggest partition of the nitrite into various tissues. We find that in vitro deoxyerythrocytes and nitrite cause aortic ring preparations to dilate, suggesting a mechanism of nitrite activation by deoxyheme proteins. We also find that nitrite inhalation in hypoxic newborn sheep lead to decreased pulmonary artery pressures and exhalation of NO; nitrite infusions in these animals leads to decreases in mean arterial blood pressure. We are currently studying the formation and compartmentalization of nitrite in the blood, in erythrocytes in particular, and whether nitrite levels may be a marker of cardiovascular risk in humans. These studies are designed to allow us to initiate nitrite infusions in normal human subjects and those with a variety of ischemic (including sickle cell anemia) diseases. We have shown that the maximum production of NO from nitrite occurs near the p50 of hemoglobin and is dependent on the allosteric conformation of hemoglobin. We have also developed methods to measure nitrite levels precisely in human blood and have found that most of blood nitrite is contained in the red cells. In the recent reporting periods, we have measured the uptake of nitrite into the human red cell as a function of oxygen tension, pH, temperature and other variables. Such uptake appears to be an important factor controlling the overall rates of conversion of nitrite to NO intravascularly. Further, we have shown that dehydroascorbic acid in red cells can catalyze the oxidation of iron-nitrosylhemoglobin (NbNO) to release NO and that this NO is then converted to nitrite but not nitrate intracellularly. This reaction may be the major mechanism for the formation of red cell nitrite, which we believe is one of the major storage sites for bioactive NO in the body, and has led us to develop a model of the interaction of the ascorbic acid/dehyroascorbic acid and the NO/nitrite cycles inside the erythrocyte. We have also examined the hypothesis that in hypoxia cell-free hemoglobin could serve as an additional endocrine source of NO. In this study, we constructed a multicellular model to characterize the amount of NO delivered by the reaction of nitrite with both intraerythrocytic and cell-free hemoglobin and find that levels of NO production by this mechanism may be quite significant, comparable to production by the vascular wall itself. We have also examined in detail the reaction mechanism of nitrite with oxy- and deoxy-hemoglobin and find that at conditions likely to obtain physiologically and pharmacologically-especially with regard to the ratio of nitrite ions to hemoglobin molecules-the formation of free radicals (including that of the ferryl-form of hemoglobin) is not likely to occur to a major extent. This is important because the formation of free radicals had been of concern with regard to the development of nitrite (or NO) pharmacology. We believe that the above studies should contribute to our understanding of the role of the human erythrocyte in modulating NO bioactivity, especially via a nitrite intermediate, and also facilitate the development of nitrite as a useful drug for cardiovascular pathology. In recent work we have been investigating changes in NO-related species during red blood cell storage to ascertain whether these contribute to the complications of blood, especially red cell, transfusion know as the "storage lesion." We find, as expected from our previous work that nitrite levels fall rapidly after venisection but then, surprisingly level off at about 1/4 of the initial value for up to 42 days. We find no evidence of other relevant NO changes and are now investigating whether the nitrite changes contribute to red cell-induced pathology and, equally importantly, the mechanism of control and stabilization of red cell nitrite levels. In the last year we have shown that at physiological nitrite concentrations we can generate enough NO to inhibit platelet aggregation; we are now working on the physiological and pharmacological implications of these results, which appear to involve interactions of nitrite with circulating red blood cells and may contribute to physiological and pathophysiological modulation of platelet reactivity in the circulation. We have also shown that the levels of nitrite in platelets during storage in vitro drop slightly and if this is due to NO formation may contribute to keeping the platelets functional for transfusion. In recent months we have been able to show that we can measure the interaction of red cells, nitrite and blood clotting by thrombelastometry, which measures more steps in clotting than platelet aggregation or surface markers alone. Using this new instrument we may be in a position to expand our work on NO production by red cells to clinical evaluation of blood clotting in various physiological and disease states. This work is closely related to that described in our report on potential nitrite therapeutics.