SICKLING MECHANISMS AND RED CELL MEMBRANES

主要的长期目标是彻底了解镰状细胞（SS）病的分子和细胞病理生理学。我们现在关注Hb S与红细胞膜相互作用改变细胞功能的机制，导致临床上广泛的微血管闭塞和溶血性贫血。我们的直接目标是：1 .表征镰状细胞诱导的离子渗透途径（“p -镰状”）的功能特性和可能的分子性质，“p -镰状”是由脱氧血红蛋白s聚合物与SS细胞的细胞质膜表面相互作用产生的。p -镰状蛋白介导的Ca2+内流是SS细胞脱水的关键步骤。我们解决了关于p型镰状细胞的几个基本问题：是否存在单一的低选择性渗透途径或不同阳离子的不同途径？SS细胞中p -镰状的程度有何不同？它是如何随pO2和[Ca2+]0变化的？单通道电导是高还是低，恒定还是可变？平均p -镰刀值是否反映了数万或数千个聚合物膜接触？随机事件是每个细胞的p -镰状单位数还是它们的单位电导？识别刺激或抑制p -镰状蛋白的药物可能指向涉及的膜成分和其分子性质的线索。2。为了研究我们发现的高na +、低k +、低密度、阳离子泄漏的SS和正常红细胞的产生机制，并验证假设(i)这些细胞中的许多来自致密不可逆镰状细胞（ISCs）；（ii）它们构成了我们在低密度SS细胞中发现的非常快速周转的钾库；（iii）它们代表镰状细胞和正常红细胞死亡的主要最终途径。3。为了验证我们的假设，即循环SS和变异红细胞之间体积和密度（或细胞Hb浓度）的显著异质性是由于网状红细胞运输系统的异质性，以及Hb-膜相互作用的直接和间接影响。这些研究采用我们最新的流式细胞术技术（改进的H*3）进行实验设计，其中细胞间的转运体分布以其体积变化率表示。我们的综合红细胞和网织红细胞模型将用于测试网织红细胞体积控制（和在SS细胞中去控制）的替代机制。我们的目标是详细描述SS和正常红细胞/红细胞的运输异质性，特别强调红细胞对细胞脱水的贡献。IV.表征各种转基因镰状细胞小鼠模型的hb聚合特性和红细胞离子转运功能，以确定最适合测试镰状细胞病各种病理生理机制和治疗策略的模型。一旦确定特征，这些将服务于上述三个目标范围内的调查。

The main long-term goal is a thorough understanding of the molecular and cellular pathophysiology of sickle cell (SS) disease. We now focus on the mechanisms by which Hb S interacts with the RBC membranes to alter cell functions, leading clinically to widespread microvascular occlusion and hemolytic anemia. Our immediate aims are: I. To characterize the functional properties and possible molecular nature of the sickling-induced ion permeability pathway(s), "P-sickle", generated by interaction of deoxy-Hb S polymers with the cytosolic membrane surface of SS cells. P-sickle -mediated Ca2+ influx is a critical step in SS cell dehydration. We address several fundamental questions about P-sickle: is there a single poorly selective permeability pathway or different pathways for the different cations? How does the extent of P-sickle vary among SS cells? How does it vary with pO2 and [Ca2+]0? Is the single pathway conductance high or low, constant or variable? Does a mean P-sickle value reflect tens or thousands of polymer-membrane contacts? Is the stochastic event the number of P-sickle units per cell or their unit conductance? Identifying agents that stimulate or inhibit P-sickle may point to membrane components involved and clues to its molecular nature. II. To investigate the mechanism(s) of generation of the high-Na+, low-K+, low-density, cation-leaky SS and normal RBCs we discovered, and test the hypotheses (i) that many of these cells are derived from dense irreversibly sickled cells (ISCs); (ii) that they comprise the very rapid-turnover K pool we found among low density SS cells; and (iii) that they represent a major final pathway of cell death for sickle cells, and perhaps for normal RBCs. III. To pursue our hypothesis that the marked heterogeneity of volume and density (or cell Hb concentration) among circulating SS and variant RBCs results from heterogeneity of transport systems in the reticulocytes, with the direct and indirect effects of Hb-membrane interactions. These studies employ our newly available flow cytometric technology (modified H*3) in an experimental design in which the transporter distribution among cells is expressed in their rates of volume change. Our integrated RBC and reticulocyte models will be used to test alternative mechanisms of reticulocyte volume control (and decontrol in SS cells). We aim to characterize in detail the transport heterogeneity of SS and normal retics/RBCs, with particular emphasis on the contribution of retics to cell dehydration. IV. To characterize the Hb-polymerization properties and RBC ion-transport functions of a variety of transgenic sickle mouse models to identify those most suitable to test various pathophysiological mechanisms and therapeutic maneuvers in sickle cell disease. Once characterized, these will serve investigations within the above three aims.