OXIDATION AND DEFORMATION IN SICKLE RBC DEHYDRATION

尚未明确的膜缺陷导致镰刀状RBC K丢失和 脱水可以说是镰状红细胞最重要的缺陷 薄膜。拟议的研究将检验一个具体的假设。 关于镰状红细胞K泄漏的原因，他们将提供 K渗漏途径的准确生物物理描述(S) 细胞。具体地说，我假设自氧化性膜损伤 引起一种缺陷，使镰刀状红细胞膜异常敏感 细胞变形的潜在不利影响 去氧作用。这可以解释显然可逆的性质 镰刀致阳离子动态平衡异常。四种方法将 被利用。(1)来自不同镰刀患者和来自不同来源的RBC 密度-单身患者的亚群将被检查，以确定是否 脱水异常(由异常密度的大小定义 亚群和单价阳离子含量)与各种 镰刀状红细胞氧化的“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 体外氧化微扰是否能准确模拟钾的渗漏 未经处理的镰刀状红细胞。氧化侮辱将包括细胞内 产生超氧化物，无脂质过氧化的硫醇氧化和 没有硫醇氧化的脂质过氧化。(3)将确定 是否最小程度的氧化损伤(本身不会导致钾泄漏 没有变形)使红细胞在极小的时间内容易发生钾泄漏 变形(这本身不会导致K泄漏 氧化)。在这些研究中，变形将由定量的 剪切应力在同心圆筒粘度计中的应用(4) 最后，正常、镰刀状和微氧化的钾泄漏途径 正常的红细胞将在充氧和脱氧条件下进行比较， 以及变形过程中和变形后。在这种情况下，K泄漏将被 根据实际孔径和每个单元的孔数进行描述(使用 无线电探头的差异接收)，钙的可能作用， K漏孔的离子选择性序列及其通量动力学和 活化能。

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.