DEVELOPMENT OF INTRACELLULAR INDICATORS AND ION TRANSPORT STUDIES

细胞内指示剂和离子传输研究的发展

• 批准号: 2574384
• 负责人: R E LONDON
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/2574384
• 关键词: anomer; biomaterial development /preparation; calcium indicator; calcium metabolism; cations; chemical synthesis; cytoplasm; deoxyglucose; fluorescent dye /probe; fluorine; glucose analog; intracellular transport; ion transport; laboratory rabbit; magnesium; membrane transport proteins; nuclear magnetic resonance spectroscopy; sarcoplasmic reticulum; uracil analog

Cellular cations play fundamental roles in the mediation of hormonal signaling, and are also involved in the mediation of cell injury. The development of intracellular indicators for cytosolic cations and other parameters of interest has had a major impact on the field of cell biology. Our goals in this area have included the development of more sensitive intracellular calcium indicators, and more selective intracellular magnesium indicators. During the last year, several improved fluorinated BAPTA derivatives were developed for the measurement of intracellular calcium. These have improved relaxation characteristics, leading to greater sensitivity/unit time, and also additional fluorine nuclei, resulting in improved sensitivity. One of the most useful niches which physiological NMR studies can fill involves the analysis of sub-cellular ion distributions. The realization of this objective requires the development of fast exchange NMR indicators which will effectively load into both the cytosol and other subcellular compartments. During the past year, we demonstrated that the tetrafluoroBAPTA (TFBAPTA) NMR calcium indicator previously developed by our group can be effectively loaded into both the cytosol and the sarcoplasmic reticulum of perfused rabbit hearts. Subsequent physiological studies involving chemical agents which selectively reduce SR calcium uptake (cyclopiezonic acid) or promote calcium release (caffeine) have allowed assignment of these resonances to the two pools. Gating experiments have allowed measurement of the SR calcium pool as a function of the cardiac cycle. Work has continued on the preparation of a new series of Mg2+ selective indicators, and although progress has been made in the preparation of these molecules, useful indicators have not yet been prepared. We anticipate evaluation of further derivatives in the next year. Recent experiments on the transport of fluorinated compounds which are based on the intra/extracellular shift difference phenomenon which we initially observed, have focused on the drug 2'-fluoro-5-methyl-beta-L- arabinofuranosyl uracil (L-FMAU), a fluorinated L-nucleoside which has been shown to inhibit the growth of Epstein-Barr virus under in vitro conditions. These studies show that the uptake of this analog occurs by both the nucleoside transporter mediated as well as by a non-facilitated diffusion pathway. Equilibrium exchange studies of glucosyl fluoride in human erythrocytes show that the beta-anomer is transported more rapidly than the ` anomer, in contrast with 2-, 3-, 4-,. and 6-fluorodeoxy glucose analogs. It was proposed that this reversal reflects a reduced interaction of the glucosyl fluoride with the externally oriented carrier.

细胞阳离子在激素调节中起着重要作用。 信号传导，并且还参与细胞损伤的介导。 的 细胞溶质阳离子和其它离子的细胞内指示剂的开发 感兴趣的参数对细胞生物学领域产生了重大影响。 生物学 我们在这一领域的目标包括发展更多的 敏感的细胞内钙指示剂， 细胞内镁指示剂。 去年，几个 开发了改进的氟化BAPTA衍生物用于测量 细胞内钙离子 这些改善了放松 特性，导致更高的灵敏度/单位时间，并且 额外的氟核，导致灵敏度提高。 之一 生理核磁共振研究所能填补的最有用的领域包括 亚细胞离子分布分析。 实现这一 目标要求开发快速交换NMR指示剂， 将有效地装载到细胞质和其他亚细胞中， 隔间 在过去一年，我们证明， BAPTA（TFBAPTA）核磁共振钙指示剂，之前由 我们的基团可以被有效地装载到细胞质和 灌注兔心脏的肌浆网。 后续 生理学研究涉及选择性降低 SR钙摄取（环吡酮酸）或促进钙释放 （咖啡因）已经允许将这些共振分配给两个池。 门控实验已经允许测量SR钙池， 心动周期的功能。 继续进行了编写 一系列新的Mg 2+选择性指示剂，虽然进展已经 在制备这些分子时，有用的指示剂还没有 还没有准备好。 我们预计， 未来一年 最近关于含氟化合物迁移的实验， 基于细胞内/细胞外移位差异现象， 最初观察到的，集中在药物2 '-氟-5-甲基-β-L- 阿拉伯呋喃糖基尿嘧啶（L-FMAU），一种氟化L-核苷， 在体外条件下， 条件 这些研究表明，这种类似物的摄取发生在 核苷转运蛋白介导的以及非易化的 扩散途径 氟葡萄糖在水溶液中的平衡交换研究 人红细胞显示β-端基异构体的转运更快， 与2-，3-，4-，.和6-氟脱氧 葡萄糖类似物。 有人提出，这一逆转反映了一种减少的 葡萄糖基氟与外部取向的 载体