NITRIC OXIDE DIFFUSION AND REACTION WITH ERTHROCYTES

该项目的长远目标是研究一氧化氮（NO）在血液中的扩散和反应，特别是它与红细胞（rbc）的相互作用。据推测，红细胞具有通过调节膜对NO的渗透性来调节NO消耗率的特定机制。具体而言，将实现以下目标。特异性目的1：红细胞的NO消耗是否受跨膜扩散调节？特异性目的2：红细胞内是否有特异性分子参与NO猝灭的调控？具体目标3：红细胞对NO消耗的调节如何影响血管调节？前两个目标将通过使用竞争性实验和专门设计的差分膜生物反应器来测量NO-RBC反应速率来解决。动力学模型将用于分析数据。为了回答这些问题，生物物理（EPR和荧光）和生化（酶、代谢物和脂质的表征）技术将应用于红细胞、红细胞鬼和合成脂质体。最后一个目的是利用分离的猪冠状动脉微血管作为生物测定来确定NO猝灭及其调控的功能作用。上述提出的假设与目前认为NO消耗不受调节以及RBC膜对NO“完全可渗透”的理解有很大的不同。除了对基础生理学的贡献外，拟议的工作还直接影响临床医学的多个方面，包括NO吸入疗法和血液代用品的设计。此外，所提出的机制可能有助于几种疾病的病理，如原发性和肺动脉高压、与糖尿病相关的外周血管疾病、镰状细胞性贫血和其他遗传性红细胞疾病。在这些情况下，红细胞对红细胞膜消耗的改变对于临床干预的发展和理解NO在生理和病理条件下发挥的复杂作用至关重要。

The broad, long-term goal of this project is to investigate the diffusion and reaction of nitric oxide (NO) in blood, particularly its interaction with red blood cells (RBCs). It is hypothesized that RBCs possess specific mechanisms that regulate the NO consumption rate through modulation of membrane permeability to NO. Specifically the following aims will be pursued. Specific Aim 1: Is NO consumption by RBC regulated by transmembrane diffusion? Specific Aim 2: Do any specific intra- erythrocytic molecules participate in the regulation of NO quenching? Specific Aim 3: How does the regulation of NO consumption by RBCs affect vessel regulation? The first two aims will be addressed by use of a competitive experiment and a differential membrane bioreactor specifically designed to measure the NO-RBC reaction rate. Kinetic models will be used to analyze the data. Biophysical (EPR and fluorescence) and biochemical (characterization of enzymes, metabolites, and lipids) techniques will be applied to RBCs, RBC ghosts, and synthetic liposomes in order to answer these questions. The last aim will be addressed using isolated porcine coronary microvessels as a bio-assay to determine the functional role of NO quenching and its regulation. The hypotheses proposed above are a significant departure from the current understanding that NO consumption is not regulated and that the RBC membrane is "completely permeable" to NO. In addition to its contribution to fundamental physiology, the proposed work directly impacts multiple aspects of clinical medicine, including NO inhalation therapy and the design of blood substitutes. Furthermore, the proposed mechanism might contribute to the pathology of several diseases, such as essential and pulmonary hypertension, peripheral vascular disease associated with diabetes mellitus, sickle cell anemia, and other hereditary RBC disorders. In these situations, altered RBC membrane consumption by RBCs is essential to the development of clinical intervention and understanding of the complex roles that NO plays under physiological and pathological conditions.