SICKLING MECHANISMS AND RED CELL MEMBRANES

描述：(改编自调查人员摘要)长期目标 是对分子和分子的透彻理解 镰刀病的细胞病理生理学。当前的分子焦点 属于血红蛋白聚合过程和细胞焦点 是关于镰状红细胞膜转运的异常。特定目标 1将检查网织红细胞离子转运的异质性和生成 致密的镰状细胞，这些研究分为三类。研究 对网织红细胞中离子传输的研究探索了标志着 循环中Hb浓度、体积和离子含量的不均一性 红细胞在很大程度上是由早期阳离子渗透作用决定的。 网织红细胞或具有多种表达的早期前体细胞 离子转运体。使用示踪剂通量方法的组合，一种新的 高精度渗透裂解法和一种新的流式细胞仪 技术，申请者将鉴定和分离镰状网织红细胞 具有不同运输特性的亚群，包括应激 网织红细胞，并鉴定它们的主要离子转运蛋白 不同的成熟阶段，测试假设这些 差异会影响快速脱水的易感性。作为这项工作的一部分 在这项工作中，他将进一步定义K：CL-CO-的pH敏感性 转运蛋白及其抑制剂对其pH激活成分的影响。 其次，他将跟进他的新奇观察，即镁疗法 可通过检查体内的体外相关性来授予好处 研究正在进行中。这些研究将包括体内(大鼠肠系膜 镁对镰状红细胞血管扩张作用的检测 微血管血流行为。第三，流式细胞仪系统将 被用来检验这样一个假设，即早秋的一个决定因素 血管闭塞危象中的致密细胞包括释放减少的 新的应激性网织红细胞进入循环。第二个具体目标 将研究镰刀引起的红细胞通透性的本质 和网织红细胞，重点是区分钙的作用- 钠通透性和钙诱导钾通道的诱导抑制 在脱水过程中的激活。在这些实验中，他将 使用肝素作为这一途径的标志物，他将尝试定义 肝素刺激镰刀样渗漏的机制 网织红细胞。他将检验阳离子泄漏不是 定位于针状体，他将尝试使用体外大鼠 肠系膜微循环切变对钙的影响 正常和镰刀状红细胞的通透性。第三个具体目标是 继续研究HBS聚合物的结构，重点是 它的形成和分解对细胞内的影响。这一目标将 更准确地采用调查员开发的新方法 测量聚合物浓度和聚合物水的大小 车厢。他将测试这些参数的可变性，如 受非S血红蛋白的影响，并检测其分布 PWC中的细胞质低相对分子质量物质，预期形成聚合物 来影响细胞新陈代谢。申请人的新方法也将是 用来进一步定义聚合物的分子间相互作用 通过测试重组突变体。第二个重点领域将是 用EPR分析镰状细胞半色性的产生 孵化，检验这一假说是由氧合血红蛋白引起的 S不稳定，而不是聚合。此外，震级 镰刀状诱导的网织红细胞和视盘细胞的内吞作用 试验研究钙蓄积的发育机制 囊泡，重点是确定至少其中的一些 都是由Retic衍生的细胞器。

DESCRIPTION: (Adapted from investigator's abstract) The long-term goal of this project is a thorough understanding of the molecular and cellular pathophysiology of sickle disease. The current molecular focus pertains to the hemoglobin polymerization process and the cellular focus is on the membrane transport abnormalities of sickle RBC. Specific Aim 1 will examine reticulocyte ion transport heterogeneity and generation of dense sickle cells, studies that fall into three categories. Studies of ion transport in reticulocytes explore the hypothesis that marked heterogeneity of Hb concentration, volume, and ion content of circulating RBC is largely determined by early cation permeabilization of reticulocytes or earlier precursors with a diversity of expression of ion transporters. Using a combination of tracer flux methods, a novel high-precision osmotic lysis method, and a new flow cytometric technology, the applicant will identify and separate sickle reticulocyte subpopulations with different transport properties, including stress reticulocytes, and characterize their major ion transporters at different stages of maturity, testing the hypothesis that these differences affect susceptibility to rapid dehydration. As part of this work, he will further define the pH sensitivity of the K:Cl co- transporter and the effect of inhibitors on its pH-activated component. Secondly, he will follow up his novel observation that magnesium therapy may confer benefit by examining in vitro correlates of the in vivo studies ongoing. These studies will include in vivo (rat mesenteric system) examination for vasodilatory effects of magnesium on sickle RBC microvascular flow behavior. Thirdly, the flow cytometry system will be used to test the hypothesis that a determinant of the early fall in dense cells during vaso-occlusive crisis comprises decreased release of new stress reticulocytes into the circulation. The second Specific Aim will examine the nature of sickling-induced permeability in red cells and reticulocytes, with emphasis on distinguishing the roles of calcium- induced inhibition of sodium permeability and calcium-induced K channel activation in the dehydration process. In these experiments, he will use heparin as a marker of this pathway, and he will attempt to define the mechanism of heparin stimulation of sickling-induced leak in reticulocytes. He will test the hypothesis that cation leak is not localized to spicules and he will attempt to use the ex vivo rat mesocecum to test the effect of microcirculatory shear on calcium permeability of normal and sickle RBC. The third Specific Aim will continue studies of the structure of the HbS polymer, with emphasis on the intracellular effects of its formation and breakdown. This aim will employ a new method developed by the investigator more accurately measuring polymer concentration and the size of the polymer water compartment. He will test the variability of these parameters as influenced by non-S hemoglobins, and examine the distribution of cytoplasmic lower MW substances in the PWC, expecting polymer formation to influence cell metabolism. The applicant's new method also will be used to further define the intermolecular interactions of the polymer by testing recombinant mutants. The second area of emphasis will be to use EPR assays of hemichrome generation in sickle cells during incubation, testing the hypothesis that this results from oxyhemoglobin S instability rather than polymerization. Additionally, the magnitude of sickling-induced endocytosis in reticulocytes and discocytes will be tested to examine mechanisms of development of calcium-accumulating vesicles, with an emphasis on determining whether at least some of these are retic-derived organelles.