MECHANISMS AND INHIBITION OF SICKLE CELL DEHYDRATION

镰状细胞脱水的机制和抑制

• 批准号: 6584666
• 负责人: Clinton H Joiner
• 金额: $ 22.86万
• 依托单位: CINCINNATI CHILDRENS HOSP MED CTR
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2003-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6584666
• 关键词: bone marrow; calcium channel blockers; cell age; cell growth regulation; cell morphology; cell water; clinical research; dipyridamole; drug screening /evaluation; erythrocyte membrane; erythrocytes; flow cytometry; growth factor; hematopoiesis; hemoglobin Ss; human subject; hydroxyurea; ion transport; membrane transport proteins; respiratory oxygenation; reticulocytes; sickle cell anemia; sickling inhibitor; tissue /cell culture

Dehydration of sickle red blood cells (SS RBC) contributes to pathology because sickling is highly dependent on Hb S concentration. Accordingly, inhibiting SS dehydration represents a potential therapeutic strategy. Three transport pathways mediate the cation loss which leads to dehydration: >1. Deoxygenation-induced (DI) fluxes of Na+, K+, and Ca++, 2. A Ca++-dependent K+ channel activated by DI Ca++ influx; 3. The KCI co- transporter (KCC). This project focuses on determining the therapeutic potential of several pharmacological inhibitors the DI pathway, identifying the membrane constituent which mediates the DI flux, and determining the basis for abnormal KCC activity in S RBC. In Specific Aim 1, the potency of dipyridamole and Ca channel antagonists as inhibitors of the DI pathway 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. To identify the membrane constituent associated with inhibition of DI fluxes, Specific Aim 2 employs covalent binding to SS RBC of another inhibitor of the DA pathway, 3H2-DIDS (4,4'-diisothiocyano- 2,2'-dihydrostilbene disulfonate). In Specific Aim 3, the hypothesis that increased KCC activity of SS RBC results either from a direct perturbation by Hb S or as a consequence of erythropoietic stress will be tested by comparing KCC in AA and SS reticulocytes with tightly defined age ranges. The rate and volume set point of KCC activated by a variety of stimuli will be measured by density shift assays using stractan gradients, and three subsets of reticulocytes identified by thiazole orange fluorescence using flow cytometry. RBC progenitors (CFUe) will be cultured from peripheral blood BFUe in vitro under defined conditions, and KCC activity of cultured AA and SS erythroblasts and reticulocytes compared. The direct effects of hematopoietic stimulation by growth factors on KCC expression will be examined in this culture system. The effects of hematopoietic stimulation by growth factors on KCC expression will be examined in this culture system. The effect of hydroxyurea will also be tested and the results integrated with measurements of KCC activity and hydration state of reticulocytes of patients taking HU (Project 4). The appearance of KCC activity during erythroid differentiation will be determined by density shift assay in SS and AA RBC precursors in bone marrow. These studies will elucidate the molecular mechanisms os SS RBC dehydration and will lay the groundwork for clinical trials aimed at preventing cellular dehydration of SS RBC in vivo by pharmacological inhibition of these cation transport pathways.

镰状红细胞 (SS RBC) 脱水导致病理 因为镰状化高度依赖于 Hb S 浓度。因此， 抑制 SS 脱水是一种潜在的治疗策略。 三种运输途径介导阳离子损失，从而导致 脱水：>1。 Na+、K+ 和 Ca++ 的脱氧诱导 (DI) 通量， 2. DI Ca++ 流入激活 Ca++ 依赖性 K+ 通道； 3. KCI 联合 运输商（KCC）。该项目的重点是确定治疗方法 DI途径的几种药理学抑制剂的潜力， 识别介导 DI 通量的膜成分，以及 确定 S RBC 中异常 KCC 活性的基础。特定目标 1、双嘧达莫和Ca通道拮抗剂作为抑制剂的效力 将确定 DI 途径，测量 Na、K 和 45Ca 的 DI 通量 并抑制体外培养的 SS RBC 密度变化 氧/脱氧循环。鉴定与相关的膜成分 抑制 DI 通量，Specific Aim 2 采用与 SS RBC 的共价结合 DA 途径的另一种抑制剂 3H2-DIDS（4,4'-二异硫氰基- 2,2'-二氢芪二磺酸盐）。在具体目标 3 中，假设 SS RBC 的 KCC 活性增加是由直接扰动导致的 Hb S 或红细胞生成应激的结果将通过以下方式进行测试 比较具有严格定义的年龄范围的 AA 和 SS 网织红细胞中的 KCC。 各种刺激激活 KCC 的速率和音量设定点 将使用 stractan 梯度通过密度位移测定进行测量，并且 噻唑橙荧光鉴定网织红细胞的三个亚群 使用流式细胞术。红细胞祖细胞 (CFUe) 将从 特定条件下的体外外周血 BFUe 和 KCC 活性 比较培养的 AA 和 SS 成红细胞和网织红细胞。直接的 生长因子刺激造血对KCC表达的影响 将在该文化系统中进行检查。对造血的影响 在此将检查生长因子对 KCC 表达的刺激 文化体系。羟基脲的效果也将得到测试， 结果与 KCC 活性和水合状态的测量相结合 服用 HU 的患者的网织红细胞（项目 4）。 KCC外观 红细胞分化期间的活性将由密度决定 骨髓中 SS 和 AA 红细胞前体的位移测定。这些研究将 阐明 SS RBC 脱水的分子机制并奠定基础 旨在预防细胞脱水的临床试验的基础 SS RBC 在体内通过药理学抑制这些阳离子转运 途径。