NITRIC OXIDE DIFFUSION AND REACTION WITH ERTHROCYTES

该项目的广泛，长期目标是研究一氧化氮（NO）在血液中的扩散和反应，特别是其与红细胞（RBC）的相互作用。据推测，红细胞具有特定的机制，通过调节膜的渗透性NO调节NO的消耗率。具体来说，将追求以下目标。具体目标1：红细胞对NO的消耗是否受跨膜扩散的调节？目的2：红细胞内是否有特异性分子参与NO淬灭的调控？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用于测量NO-RBC反应速率的差分膜生物反应器来解决。动力学模型将用于分析数据。生物物理（EPR和荧光）和生物化学（酶，代谢物和脂质的表征）技术将应用于RBC，RBC血影和合成脂质体，以回答这些问题。最后一个目的将使用离体猪冠状动脉微血管作为生物测定来确定NO淬灭及其调节的功能作用。上述提出的假设是一个显着偏离目前的理解，即NO消耗不受监管，红细胞膜是“完全渗透”的NO。除了其对基础生理学的贡献，拟议的工作直接影响临床医学的多个方面，包括NO吸入治疗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学，如原发性肺动脉高压、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RBC疾病。在这些情况下，改变红细胞的红细胞膜消耗是必不可少的临床干预和了解的复杂作用，NO在生理和病理条件下发挥的发展。

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.