OXIDATION AND DEFORMATION IN SICKLE RBC DEHYDRATION

镰状红细胞脱水中的氧化和变形

• 批准号: 3353266
• 负责人: ROBERT P HEBBEL
• 金额: $ 7.94万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-09-30 至 1989-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3353266
• 关键词: autooxidation; biophysics; body water dehydration; cell water; erythrocytes; free radicals; human tissue; lipid peroxides; membrane permeability; molecular pathology; sickle cell anemia; thiols

The as yet undefined membrane defect leading to sickle RBC K+ loss and dehydration is arguably the most important defect of the sickle RBC membrane. The proposed studies will examine a specific hypothesis regarding the etiology of K+ leak in sickle RBC cells and they will provide an accurate biophysical description of the K+ leak pathway(s) in these cells. Specifically, I hypothesize that autoxidative membrane damage induces a defect which makes the sickle RBC membrane abnormally susceptible to the potentially adverse effects of cellular deformation accompanying deoxygenation. This could explain the apparently reversible nature of sickling-induced abnormalities of cation homeostasis. Four approaches will be used. (1) RBC from different sickle patients and from density-subpopulations of single patients will be examined to see if the dehydration abnormality (as defined by size of the abnormally dense subpopulation and by monovalent cation content) correlates with various "footprints" of sickle RBC oxidation (radical generation, membrane-bound heme, thiol oxidation, lipid peroxidation). (2) It will be determined whether in vitro oxidative perturbation accurately models the K+ leak of unmanipulated sickle RBC. Oxidative insults will include intracellular generation of superoxide, thiol oxidation without lipid peroxidation and lipid peroxidation without thiol oxidation. (3) It will be determined whether minimal oxidative insult (which itself does not cause K+ leak in the absence of deformation) predisposes RBC to K+ leak during minimal deformation (which itself does not cause K+ leak in the absence of oxidation). In these studies deformation will be induced by quantitative application of shear stress in a concentric cylinder viscometer. (4) Finally, the K+ leak pathway of normal, sickle and minimally-oxidized normal RBC will be compared under oxygenated and deoxygenated conditions, and during and after deformation. Under these conditions, K+ leak will be described in terms of actual pore size and pore number per cell (using differential seiving of radio-probes), the possible role of calcium, the ion selectivity sequence of the K+ leak pore, and its flux kinetics and activation energy.

尚未明确的膜缺陷导致镰状红细胞K+损失， 脱水是镰状红细胞最重要的缺陷 膜的 拟议的研究将检验一个特定的假设 关于镰状红细胞中K+渗漏的病因，他们将提供 这些细胞中K+渗漏途径的准确生物物理描述 细胞 具体来说，我假设自氧化性膜损伤 会导致镰状红细胞膜的缺陷， 细胞变形的潜在不利影响， 脱氧 这可以解释明显可逆的性质， 镰状化引起的阳离子稳态异常。 四种方法将 被利用 (1)来自不同镰状患者和来自 将检查单个患者的密度亚群，以查看 脱水异常（如由异常致密的 亚群和单价阳离子含量）与各种 镰状红细胞氧化的“足迹”（自由基生成，膜结合 血红素、硫醇氧化、脂质过氧化）。 (2)将确定 体外氧化扰动是否准确地模拟了 未经处理的镰状红细胞。 氧化损伤将包括细胞内 产生超氧化物，硫醇氧化，无脂质过氧化， 脂质过氧化而无硫醇氧化。 (3)将确定 是否存在最小的氧化损伤（其本身不会导致K+泄漏， 不存在变形）使RBC在最小的 变形（其本身在不存在 氧化）。 在这些研究中，变形将通过定量的 剪切应力在同心圆筒粘度计中的应用。 （四） 最后，正常、镰状和最小氧化的K+渗漏途径 将在充氧和脱氧条件下比较正常RBC， 以及在变形期间和变形之后。 在这些条件下，K+泄漏将 根据每个单元的实际孔尺寸和孔数量（使用 差异seiving的无线电探针），钙的可能作用， K+渗漏孔的离子选择性序列及其通量动力学， 活化能