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。我们现在重点研究HB S与红细胞膜相互作用改变细胞功能的机制，从而在临床上导致广泛的微血管闭塞和溶血性贫血。我们的近期目标是：1.研究镰刀样诱导的离子通透途径(S)的功能性质和可能的分子性质，该途径是由脱氧Hb S聚合物与SS细胞的细胞膜表面相互作用而产生的。P-镰刀介导的钙内流是SS细胞脱水的关键步骤。我们解决了几个关于P-镰刀的基本问题：对于不同的阳离子，是否存在单一的、选择性较差的渗透途径或不同的途径？不同SS细胞间P-镰刀的程度有何不同？它与pO2和[Ca~(2+)]_0有何不同？单程电导是高还是低，是恒定的还是可变的？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镰刀单位数还是它们的单位电导？识别刺激或抑制P-镰刀的药物可能指向所涉及的膜成分和其分子性质的线索。2.探讨我们发现的高Na+、低K+、低密度、阳离子渗漏的SS和正常红细胞的产生机制(S)，并检验假设(I)这些细胞中的许多细胞来自致密的不可逆镰状细胞(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K池；以及(Iii)它们是镰状细胞和可能是正常红细胞细胞死亡的主要最终途径。我们的假设是，循环SS和变异红细胞的体积和密度(或细胞Hb浓度)的显著异质性是网织红细胞转运系统异质性的结果，Hb-膜相互作用的直接和间接影响。这些研究使用了我们最新可用的流式细胞仪技术(改进的H*3)，在实验设计中，细胞之间的转运蛋白分布是用它们的体积变化率来表示的。我们集成的RBC和网织红细胞模型将用于测试网织红细胞容量控制(以及SS细胞中的去控制)的替代机制。我们的目标是详细地描述SS和正常的RETICS/RBC的运输异质性，特别强调RETICS对细胞脱水的贡献。IV.对多种转基因镰状细胞病小鼠模型的Hb聚合性和红细胞离子转运功能进行表征，以确定最适合检测镰状细胞病的各种病理生理机制和治疗方法的模型。一旦确定了特征，这些将服务于上述三个目标的调查。

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.