INHIBITION OF ION TRANSPORT IN DEOXYGENATED SICKLE CELLS

脱氧镰状细胞中离子传输的抑制

• 批准号: 2771557
• 负责人: Clinton H Joiner
• 金额: $ 22.75万
• 依托单位: CINCINNATI CHILDRENS HOSP MED CTR
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-09-30 至 2000-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2771557
• 关键词: body water dehydration; calcium channel blockers; cell osmotic pressure; erythrocytes; erythroid stem cell; hemoglobin Ss; human subject; ion transport; membrane channels; membrane permeability; oxygen tension; oxygen transport; sickle cell anemia; sickle cell crisis; sickling inhibitor; transport inhibitor

Deoxygenation of red blood cells containing Hemoglobin S(SS RBC) increases membrane permeability to Na, K, and Ca, which perturbs a number of ion transport mechanisms and leads to cellular dehydration. This potentiates sicking because of its high dependence on Hb S concentration, so that inhibiting SS RBC dehydration is a potential therapeutic strategy i sickle cell disease. DIDS (4,4'-diisothiocyano-2,2'-disulfostibene), dipyridamole, and Ca channel blockers inhibit deoxygenation-induced (DI) cation fluxes associated with sickling. The goal of this project is to develop the therapeutic potential of these drugs for inhibiting cellular dehydration in sickle cell patients and to exploit their inhibitory properties to elucidate the molecular and biophysical nature of the DK pathway and its role in the perturbation of cell volume regulation during erythroid differentiation. In Specific Aim 1, the inhibitory potency of dipyridamole and Ca channel blockers will be determined, measuring DI fluxes of Na, K and 45Ca and inhibition of density changes in SS RBC incubated in vitro during oxy/deoxy cycles. Specific Aim 2 employs covalent 3H2-DIDS binding to SS RBC to identify the membrane constituent associated with inhibition of DI fluxes. Specific Aim 2 will test the dual hypotheses that the DI pathway is localized to membrane spicules formed during sickling and represents the activity of a nonselective cation channel. Tracer cation fluxes will be measured in isolated membrane spicules (IMS) and IMS incorporated into artificial lipid bilayers will be analyzed electrophysiologically using single channel techniques. Vesicle fluxes and single channel activities will be tested for physiological and pharmacological properties which characterize DI fluxes in intact SS RBC. In Specific Aim 4, normal and SS RBC progenitors (BFU-e/CFU-e) will be cultured in vitro under defined conditions, and density distribution of erythroblasts and reticulocytes determined on stractan gradients. These experiments will test the hypothesis that the presence of Hb S +/-sickling perturbs volume regulation prior to the reticulocyte stage and alters the expression of volume regulatory transporters. This system will permit testing of transport inhibitors for effects on the hydration state of erythroblasts and early reticulocytes and will shed light on which transport systems are involved in reticulocyte dehydration. These studies will lay the groundwork for clinical trials aimed at preventing cellular dehydration of SS RBC in vivo by pharmacological inhibition of the deoxygenation-induced cation transport pathway.

含有血红蛋白 S (SS RBC) 的红细胞脱氧作用增加 膜对 Na、K 和 Ca 的渗透性，会扰乱许多离子 运输机制并导致细胞脱水。 这增强了 生病是因为它高度依赖 Hb S 浓度，因此 抑制 SS RBC 脱水是一种潜在的治疗策略 细胞疾病。 DIDS（4,4'-二异硫氰基-2,2'-二硫代苯）， 双嘧达莫和 Ca 通道阻滞剂抑制脱氧诱导 (DI) 与镰状化相关的阳离子通量。 该项目的目标是 开发这些药物抑制细胞的治疗潜力 镰状细胞病患者脱水并利用其抑制作用 阐明 DK 的分子和生物物理性质的特性 途径及其在细胞体积调节扰动中的作用 红细胞分化。 在具体目标 1 中，抑制效力 将测定双嘧达莫和 Ca 通道阻滞剂，测量 DI Na、K 和 45Ca 的通量以及对 SS RBC 密度变化的抑制 在氧/脱氧循环期间在体外孵育。 具体目标 2 采用 共价 3H2-DIDS 与 SS RBC 结合以识别膜成分 与 DI 通量的抑制有关。 具体目标2将测试双重 假设 DI 途径定位于形成的膜针 在镰状化过程中，代表非选择性阳离子的活性 渠道。 示踪剂阳离子通量将在隔离膜中测量 骨针 (IMS) 和 IMS 纳入人工脂质双层将 使用单通道技术进行电生理学分析。 囊泡 将测试通量和单通道活动的生理和 表征完整 SS RBC 中 DI 通量的药理学特性。 在具体目标 4 中，正常和 SS RBC 祖细胞 (BFU-e/CFU-e) 将被 在规定的条件下进行体外培养，并进行密度分布 在 stractan 梯度上测定红细胞和网织红细胞。 这些 实验将检验 Hb S +/- sickling 的存在这一假设 扰乱网织红细胞阶段之前的容量调节并改变 体积调节转运蛋白的表达。 该系统将允许 测试运输抑制剂对水合状态的影响 成红细胞和早期网织红细胞，并将揭示哪些 运输系统参与网织红细胞脱水。 这些研究 将为旨在预防细胞的临床试验奠定基础 通过药物抑制 SS RBC 体内脱水 脱氧诱导的阳离子转运途径。