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: 6162239
• 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/6162239
• 关键词: anomer; biomaterial development /preparation; calcium indicator; calcium metabolism; cations; chemical synthesis; cytoplasm; deoxyglucose; fluorine; glucose analog; intracellular transport; ion transport; laboratory rabbit; magnesium; membrane transport proteins; nuclear magnetic resonance spectroscopy; sarcoplasmic reticulum; uracil analog

Summary of Work: Cellular cations play fundamental roles in 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, which have improved relaxation characteristics, leading to greater sensitivity/unit time, and also additional fluorine nuclei, also resulting in improved sensitivity. Progress on the development of more magnesium selective inhibitors has been slow. A major difficulty in the development of fluorescent chelators is the considerable effort necessary to synthesize them. We have been exploring the use of new methodology to join together, in one step, the fluorophore and the chelator moiety. We have prepared a number of APTRA (o-aminophenol triacetate) derivatives with novel fluorescent groups and are now evaluating their properties. 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 2'-fluoro-5-methyl-b-L-arabinofuranosyl uracil (L-FMAU), a drug which has been shown to inhibit the growth of Epstein- Barr virus under in vitro conditions. Although the transport of L-FMAU was significantly inhibited (60-70 %) by nitrobenzylthioinosine (NBTI) and dipyridamole, these inhibitors did not achieve complete blockage of L-FMAU uptake. The uptake of L-FMAU was neither reduced in the presence of uracil, a competitor for transport via the nucleobase transporter, nor inhibited by the presence of up to 1000 muM papaverine. Upon inhibition of the nucleoside transporter by NBTI, L-FMAU influx rate increased linearly with its concentration, indicating a non-saturable transport mechanism. The residual transport of L-FMAU (30-40 %) measured in the presence of NBTI was found to be sensitive to butanol which affects membrane fluidity and would be expected to enhance non-facilitated membrane diffusion to the uptake of L-FMAU.

工作总结：细胞阳离子在激素合成中发挥重要作用 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， 改进的松弛特性，导致更高的灵敏度/单位 时间，以及另外的氟核，也导致改善的 灵敏度 高镁选择性的合成方法研究进展 抑制剂进展缓慢。 发展中的一个主要困难是 荧光螯合剂是合成 他们 我们一直在探索使用新的方法来加入 在一个步骤中，将荧光团和螯合剂部分结合在一起。 我们有 制备了许多APTRA（邻氨基苯酚三乙酸酯）衍生物， 新型荧光基团，目前正在评估它们的性质。 最近 氟化化合物的迁移实验， 细胞内/细胞外移位差异现象， 观察到，已集中在2 '-氟-5-甲基-b-L-阿拉伯呋喃糖基尿嘧啶 （L-FMAU），一种已被证明能抑制爱泼斯坦生长的药物， 体外条件下的巴尔病毒。虽然L-FMAU的运输 硝基苄硫代肌苷（NBTI）可显著抑制（60- 70%） 和潘生丁，这些抑制剂没有达到完全阻断 L-FMAU摄取。L-FMAU的摄取既不减少， 尿嘧啶，通过核碱基转运蛋白运输的竞争对手，也 被高达1000 μ M罂粟碱的存在抑制。 抑制后 核苷转运体的NBTI，L-FMAU内流率增加 与其浓度呈线性关系，表明非饱和运输 机制L-FMAU的残留转运（30- 40%）在 发现NBTI的存在对丁醇敏感，丁醇影响 膜流动性，预计将提高非易化 膜扩散对L-FMAU摄取的影响。