MECHANISMS AND INHIBITION OF SICKLE CELL DEHYDRATION

镰状细胞脱水的机制和抑制

• 批准号: 6456256
• 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: 2001
• 资助国家: 美国
• 起止时间: 2001-04-01 至 2002-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6456256
• 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.

镰状红细胞脱水（SS RBC）有助于病理 因为病很高度取决于HB的浓度。因此， 抑制SS脱水代表了一种潜在的治疗策略。 三个传输途径介导导致阳离子损失 脱水：> 1。 Na+，K+和Ca ++的脱氧诱导（DI）通量， 2。ACa ++ - 依赖性K+通道被DI CA ++流入激活； 3。KCI共同 转运蛋白（KCC）。该项目着重于确定治疗性 DI途径的几种药理抑制剂的潜力， 识别介导DI通量的膜成分，并 确定S RBC中KCC活性异常的基础。在特定目标中 1，二吡啶胺和Ca通道拮抗剂的效力作为抑制剂 将确定DI途径，测量Na，K和45CA的DI通量 并抑制SS RBC的密度变化在体外孵育 氧/脱氧周期。确定与之相关的膜成分 抑制DI通量，特定目标2采用与SS RBC共价结合 在DA途径的另一种抑制剂中，3H2-DIDS（4,4'-diisothiocyano- 2,2'-dihydrostilbene二硫酸盐）。在特定的目标3中，假设 SS RBC的KCC活性增加了直接扰动的结果 由HB S或由于促红细胞增压的结果将通过 比较AA和SS网状细胞中的KCC的年龄范围紧密。 各种刺激激活KCC的速率和体积设定点 将通过使用Stractan梯度的密度转移测定法进行测量， 由噻唑橙荧光鉴定的三个子集的网状细胞 使用流式细胞仪。 RBC祖细胞（CFUE）将从 在定义的条件下，体外外周血BFUE和KCC活性 比较了培养的AA和SS红细胞和网状细胞。直接 生长因子造血刺激对KCC表达的影响 将在此文化系统中进行检查。造血的作用 在此中将检查生长因子对KCC表达的刺激 培养系统。羟基脲的作用也将被测试，并 与KCC活性和水合状态的测量相结合的结果 服用HU的患者的网状细胞（项目4）。 KCC的出现 红细胞分化过程中的活性将由密度确定 在骨髓中的SS和AA RBC前体中的移位测定。这些研究会 阐明分子机制OS SS RBC脱水，并将放置 旨在防止细胞脱水的临床试验的基础。 SS RBC在体内通过药理抑制这些阳离子运输 途径。