DEVELOPMENT OF INTRACELLULAR INDICATORS AND ION TRANSPORT STUDIES

细胞阳离子在荷尔蒙中起着重要作用 信号转导，也参与细胞损伤的调节。这个 胞内阳离子指示剂的研究进展 其他令人感兴趣的参数对 细胞生物学。我们在这方面的目标包括发展 更灵敏的细胞内钙指示剂，以及更多 选择性细胞内镁指示剂。在过去一年中， 我们致力于开发一条新的一般路线 BAPTA相关荧光螯合剂的合成研究进展 [双(邻氨基苯氧基)乙烷-N，N，N？，N？-四乙酸]或 APTRA(邻氨基酚-N，N，O-三乙酸)，用于 测定细胞中的钙和镁离子水平。关键一步 是钯催化的螯合部分偶联到 荧光团。使用这种方法，可以更快地 合成和筛选具有多种潜力的指示器 荧光团。三氟化钠偶联 [4-(CF3SO3)C6H3NO2-2-(邻苯基)]与几种多芳烃 硼和锡的品种产量很高。进一步 2-苄氧基硝基苯环的精制(还原， 然后用BrCH2COOMe和 皂化)得到所需的APTRA螯合剂。《荧光报》 其中几个分子对镁的反应是 已评估。除了上述研究外，我们最近还 成功地开发了一种对镁的选择性比 上述APTRA分子。这些荧光灯 分子在镁的络合作用下表现出发射位移， 对钙离子相对不敏感。衍生品更多 适合装入细胞的材料目前正在准备中。在……里面 与探头研制工作并行的生理学研究 继续与墨菲·S博士合作使用核磁共振 NIEHS的小组和杜克大学的Steenbergen？S博士小组。 这些研究的目的是了解离子的变化 发生在压力时期，以及这些因素的作用 改变打法会造成不可逆转的伤害。在过去一年中， 对灌流的大鼠心脏进行的核磁共振研究表明 预适应条件下细胞内pH的差异 而非预适应心脏不是由于H+增加所致 在前者中挤出。含氟有机物的输运研究 核苷：2‘-氟-5-甲基-β-L-阿糖尿嘧啶 (L-民盟)，最近结束了。

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 past year, we have worked on the development of a new and general route towward the synthesis of fluorescent chelators related to BAPTA [bis(o-aminophenoxy)ethane-N,N,N?,N?-tetraacetic acid] or APTRA (o-aminphenol-N,N,O-triacetic acid), which are used for determining calcium and magnesium ion levels in cells. The key step is a palladium-catalyzed coupling of the chelating moiety to the fluorophore. Using this method, it becomes possible to more rapidly synthesize and screen indicators with a range of potential fluorophores. Coupling of triflate [4-(CF3SO3)C6H3NO2-2-(OBenzyl)] with several polyaromatic boron and tin species proceeds with excellent yields. Further elaboration of the 2-benzyloxy-nitrophenyl ring (reduction, followed by triple alkalyation with BrCH2COOMe and saponification) gives the desired APTRA chelators. The fluorescent response to magnesium of several of these molecules has been evaluated. In addition to the above studies, we have recently succeeded in developing a more magnesium selective chelator than the APTRA molecules referred to above. These fluorescent molecules exhibit an emission shift upon magnesium complexation, and are relatively insensitive to calcium ions. Derivatives more suitable for loading into cells are currently in preparation. In parallel with the probe development work, physiological studies utilizing NMR have continued in collaboration with Dr. Murphy?s group at NIEHS and Dr. Steenbergen?s group at Duke University. These studies have been aimed at understanding the ionic changes which occur during periods of stress, and the role that these changes play in causing irreversible injury. During the last year, NMR studies performed on perfused rat hearts indicated that differences in intracellular pH observed between pre-conditioned and non pre-conditioned hearts are not due to increased H+ extrusion in the former. Studies on the transport of the fluorinated nucleoside: 2'-fluoro-5-methyl-beta-L-arabinofuranosyl uracil (L-FMAU), were recently concluded.