NITRIC OXIDE DIFFUSION AND REACTION WITH ERTHROCYTES

这个项目的长期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。假设红细胞具有通过调节膜对NO的通透性来调节NO消耗率的特定机制。具体而言，将实现以下目标。具体目标1：红细胞的NO消耗是否受跨膜扩散的调节？具体目标2：是否有特定的红细胞内分子参与NO猝灭的调节？具体目标3：红细胞对NO消费的管制如何影响船舶管制？前两个目标将通过竞争实验和专门为测量无红细胞反应速率而设计的差动式膜生物反应器来实现。将使用动力学模型来分析数据。生物物理(EPR和荧光)和生化(酶、代谢物和脂类的表征)技术将应用于红细胞、RBC重影和合成脂质体，以回答这些问题。最后一个目的将使用分离的猪冠状动脉微血管作为生物测定，以确定NO猝灭的功能作用及其调节。以上提出的假设大大背离了目前人们的理解，即NO的消耗不受监管，红细胞膜对NO“完全可渗透”。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吸入疗法和血液替代品的设计。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、糖尿病相关的外周血管疾病、镰状细胞贫血和其他遗传性红细胞疾病。在这种情况下，改变红细胞膜的消耗对于临床干预的发展和理解NO在生理和病理条件下所起的复杂作用是至关重要的。

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.