STUDIES OF SICKLING MECHANISMS AND RED CELL MEMBRANES

该项目涉及镰状细胞的分子和细胞病理生理学 红细胞疾病和相关的红细胞疾病，主要关注 循环中致密、脱水的SS细胞的起源，并强调 与其他红细胞疾病和细胞生理学相关的基本问题：I. 镰状化是如何作用于SS网织红细胞（网织红细胞）异质性， 不同的成熟红细胞亚群 二. 的本质是什么 镰状化诱导的渗透性途径，以及它如何产生观察到的 离子运输和含量异常？ 三. 如何形成， SS中脱氧血红蛋白S聚合物的分解和可能的结构变异 细胞直接改变细胞体积、离子分布和新陈代谢？ 为此目的进行的研究将：I.进一步发展我们的假设， 最密集的SS细胞的网状起源：使用我们新的 非稳态红细胞和retic模型来预测条件， 分离具有不同输运特性的SS反射体;识别其 转运不均一性，Ca 2 =和Mg 2 =代谢，以及我们新发现的 Ca 2 =-敏感的Cl渗透性;并评估“应力抑制剂”在 脱水过程，以及在血管闭塞中致密细胞的变化 镰刀危机; II. 研究镰状化诱导的渗透性途径 （“Psickle”）和SS中离子、pH和体积异常的机制 视网膜和老年细胞;表征我们新发现的肝素效应， 在不存在Ca 2+的情况下放大的Psickle-Na/K，用于运输， 渗漏的超微结构研究;荧光成像单细胞 含有Ca 2+螯合剂的溶液，以定位Ca 2+泄漏和Ca 2+的分布 正常和镰状细胞中的Pca和稳态[Ca 2 +];测量pO 2 并且需要聚合物级分来透化不同密度的S细胞， 测试致密的SS细胞可能被渗透的假设， 循环时间;由内而外囊泡中K：C1共转运试验 从视网膜和成熟的红细胞，如果它可以被激活暴露于 体外Hb S或C;检测我们新发现的肌苷增加是否 SS细胞中的一磷酸盐反映了他们暴露于[Ca 2 +];研究 使用电子顺磁共振技术在SS级分中形成metHb和血红素 共振 三. 应用新的精确方法来（i）估计 （ii）测试聚合物中的Hb浓度Cp; 影响聚合物溶解度的细胞因子（C）;（iii）估计 将Hbsa，F和C掺入聚合物中（作为杂化物，四聚物， 和T或R构象），并掺入低和中等MW 聚合物缔合物水隔室（PWC，衍生自 Cp），其不包括可溶性大分子。 我们可以：预测 聚合的渗透效应作为细胞MCHC的函数，并测试 直接预测;通过测量Cp和非S Hb掺入，研究 聚合物超微结构，并评估糖酵解作用 在密集的SS细胞中由于聚合而引起的酶-底物再分布。

This project concerns the molecular and cellular pathophysiology of sickle cell disease and related red cell disorders, with a major focus on the origin of dense, dehydrated SS cells in the circulation, and emphasis on basic issues relevant to other red cell disorders and cell physiology: I. How does sickling act on SS reticulocyte (retic) heterogeneity to generate different mature red cell subpopulations? II. What is the nature of the sickling-induced permeability pathway and how does it produce the observed abnormalities of ion transport and content? III. How does the formation, breakdown and possible structural variations of deoxy-Hb S polymers in SS cells directly alter cell volume, ion distribution, and metabolism? Studies to these ends will: I. Develop further our hypothesis of a direct retic origin of most dense SS cells: use simulations of our new non-steady-state red cell and retic models to predict conditions to separate SS retics with different transport properties; identify their transport heterogeneities, Ca2= and Mg2= metabolism, and our newly found Ca2=-sensitive Cl permeability; and assess the role of "stress retics" in the dehydration process, and in dense cell variations in vasoocclusive sickle crises; II. Study the sickling-induced permeability pathway ("Psickle") and the mechanisms of ion, pH and volume abnormalities in SS retics and older cells; characterize our newly found heparin effect of a magnified Psickle-Na/K in the absence of Ca2+, for transport and ultrastructural studies of the leak; fluorescence-image single cells containing Ca2+ chelators, to locate the Ca2+ leaks and the distribution of Pca and steady state [Ca2+] in normal and sickle cells; measure what pO2's and polymer fractions are needed to permeabilize different density S cells, testing the hypothesis that dense SS cells may be permeabilized most of the time in the circulation; test for K:C1 cotransport in inside-out vesicles from retics and mature red cells, and if it can be activated by exposure to Hb S or C in vitro; test whether our newly found increased inosine monophosphate in SS cells reflects their exposure to [Ca2+]; and study metHb and hemichrome formation in SS fractions using electron paramagnetic resonance. III. Apply new accurate methods to (i) estimate the concentration of Hb in the polymer, Cp; (ii) test whether Cp changes with cell factors affecting polymer solubility (C); (iii) estimate the incorporation of Hbs a, F and C into the polymer (as hybrids, tetramers, and in T or R conformations), and incorporation of low and intermediate MW substances into the polymer-associates water compartment (PWC, derived from Cp) which excludes soluble macromolecules. We can then: predict the osmotic effects of polymerization as a function of cell MCHC, and test the predictions directly; with Cp and non-S Hb incorporation measured, study polymer ultrastructure by electronmicroscopy, and assess glycolytic effects of enzyme-substrate redistribution due to polymerization in dense SS cells.