Red Blood Cell Function in Nitric Oxide Biotransport

描述（由申请人提供）：我们的长期目标是在生理学和病理生理学上量化微循环中一氧化氮（NO）的运输。在血管壁，内皮源性一氧化氮到达平滑肌，激活可溶性鸟苷酸环化酶（sGC）并调节血管张力。生物可利用一氧化氮的水平是通过其在微循环中的产生和消耗来维持的。尽管对一氧化氮从产生部位（内皮）转运到靶部位（平滑肌）的研究非常深入，但内皮来源的一氧化氮是否以及如何在血管腔内有效的一氧化氮清除剂红细胞（rbc）存在的情况下到达平滑肌仍然存在疑问。本研究将提供一氧化氮与红细胞相互作用中血流动力学和生化参数的功能关系。本研究的具体目的是：1)确定氧化作用在NO与红细胞相互作用中的作用；2)量化NO与游离Hb和RBC Hb相互作用中NO最终产物形成的差异。根据初步数据，我们将检验的假设是：(a) NO与氧化红细胞的相互作用比NO与脱氧红细胞的相互作用产生更高的亚硝酸盐形成速率，(b)氧化红细胞的总NO浓度高于脱氧红细胞的总NO浓度。(c)与游离血红蛋白溶液或含有少量游离血红蛋白的红细胞溶液相比，结合在红细胞膜上的血红蛋白（RBC-血红蛋白）会产生更高的总NO浓度，从而具有更高的NO生物利用度。为了验证这些假设，我们将使用一种新的生物反应器来模拟NO和红细胞在体内的相互作用。我们将比较一氧化氮与氧合红细胞和脱氧红细胞的相互作用产物、游离血红蛋白与红细胞和游离血红蛋白的存在。我们将使用化学发光法测量NO的反应产物，包括中间产物（s-亚硝基血红蛋白和亚硝基血红蛋白）和最终产物（亚硝酸盐和硝酸盐）。我们将使用NO电极测量气态和水溶液中的NO浓度。我们还将使用紫外/可见分光光度法来量化这些浓度。通过设计一个新的实验模型来研究NO和红细胞的生化相互作用，我们将进一步提高生物医学研究人员对这些分子相互作用的认识。这可能导致对镰状细胞性贫血、肺动脉高压、感染性休克和血液替代品等多种领域的治疗方法进行评估。公共卫生相关性：一氧化氮（NO）在许多生理功能中发挥重要作用，包括调节血管张力、神经传递、免疫反应和抑制血小板聚集。本研究将大大促进我们对NO微循环代谢的基本认识，并为生物医学研究人员研究NO与红细胞的生化相互作用提供新的实验模型。本研究还将量化游离Hb对NO代谢的影响。这一知识对于发展血红蛋白为基础的氧载体和理解慢性溶血在镰状细胞病和肺动脉高压中的作用至关重要。

DESCRIPTION (provided by applicant): Our long-term goal is to quantify nitric oxide (NO) transport in the microcirculation in physiology and pathophysiology. In vascular wall, endothelium-derived nitric oxide reaches smooth muscle where it can activate soluble guanylate cyclase (sGC) and modulate vascular tone. The level of bioavailable NO are maintained by its production and consumption in the microcirculation. Despite intense research on the transport of NO from the production site (endothelium) to the target site (smooth muscle), question remains on whether and how significant amount of endothelium-derived NO reaches smooth muscle in the presence of efficient NO scavenger red blood cells (RBCs) in vascular lumen. The proposed research will provide functional relationship between hemodynamic and biochemical parameters in NO and RBC interaction. The Specific Aims for the proposed research are 1) to determine the role of oxygenation on NO interaction with RBC and 2) to quantify the differences in NO end-products formation from NO interaction with free Hb and RBC Hb. Based on the preliminary data, the hypotheses we will test are that (a) NO interaction with oxygenated RBCs will result in higher nitrite formation rate as compared to nitrite formation rate of NO interaction with deoxygenated RBCs, (b) oxygenated RBCs will have higher total NO concentration as compared to total NO concentration of deoxygenated RBCs, and (c) hemoglobin bound in the RBC membrane (RBC-hemoglobin) will results in higher total NO concentration and thus will have higher NO bioavailability as compared to that of free hemoglobin solution or RBC solution containing small amount of free Hb. For testing these hypotheses, we will use a novel bioreactor that simulates in vivo interactions of NO and RBCs. We will compare interaction products of NO with oxygenated- and deoxygenated- RBCs, presence of free hemoglobin with RBCs, and free hemoglobin. We will measure the reaction products of NO including intermediate products (s-nitrosohemoglobin & nitrosyl-Hb) and end-products (nitrite & nitrate) using chemiluminescence method. We will measure the gaseous and aqueous NO concentration using an NO electrode. We will additionally use UV/Vis spectrophotometric spectrum to quantify these concentration. By designing a new experimental model for studying biochemical interactions of NO and RBCs, we will advance the knowledge of biomedical researchers on these molecular interactions. This may lead to evaluation of therapeutics in areas as diverse as sickle cell anemia, pulmonary hypertension, septic shock, and blood substitutes. PUBLIC HEALTH RELEVANCE: Nitric oxide (NO) plays important roles in numerous physiological functions including regulation of vascular tone, neurotransmission, immune response and inhibition of platelet aggregation. The proposed research will significantly advance our fundamental understanding of NO metabolism in the microcirculation and provide biomedical researchers a new experimental model for studying biochemical interactions of NO and RBCs. The proposed research will also quantify the effect of free Hb on the NO metabolism. This knowledge is critical to the development of hemoglobin based oxygen carrier and the understanding the effects of chronic hemolysis in sickle cell disease and pulmonary hypertension.