DEVELOPMENT OF INTRACELLULAR INDICATORS AND ION TRANSPORT STUDIES

细胞阳离子在荷尔蒙的调节中起着重要作用 信号转导，也参与细胞损伤的调节。这个 胞内阳离子和其他离子指示剂的研究进展 感兴趣的参数对细胞的领域产生了重大影响 生物学。我们在这一领域的目标包括开发更多 灵敏的细胞内钙指示剂，更具选择性 细胞内镁指示剂。在过去的一年里，几个 改进的氟化BAPTA衍生物被开发出来用于测定 细胞内的钙离子。这些都改善了放松 特性，导致更高的灵敏度/单位时间，还 额外的氟核，从而提高了灵敏度。其中之一 生理学核磁共振研究可以填补的最有用的利基领域包括 亚细胞离子分布的分析。这一点的实现 目标要求开发快速交换核磁共振指示剂 将有效地负载到胞浆和其他亚细胞中 车厢。在过去的一年里，我们证明了 四氟BAPTA(TFBAPTA)核磁共振钙指示剂 我们的组可以有效地负载到细胞质和 兔心脏灌流后的肌浆网。后续 涉及化学试剂的生理学研究选择性地降低 SR钙摄取(环哌酸)或促进钙释放 (咖啡因)允许将这些共振分配给两个池。 门控实验使测量SR钙池成为一种 心动周期的功能。筹备工作仍在继续 一系列新的镁离子选择性指标，尽管取得了进展 在制备这些分子时，有用的指示剂还没有 但已经做好了准备。我们预计将在年对进一步的衍生品进行评估 第二年。 最近关于含氟化合物的传输的实验是 基于我们发现的细胞内/细胞外移位差异现象 初步观察，已集中在药物2‘-氟-5-甲基-β-L- 阿糖尿嘧啶(L-FMAU)，一种含氟的L核苷 已被证明在体外条件下抑制EB病毒的生长 条件。这些研究表明，这种类似物的吸收是通过 核苷转运体既有介导性的也有非促进性的 扩散途径。葡萄糖氟化物在水溶液中的平衡交换研究 人类红细胞显示，β-异构体的运输速度更快 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 葡萄糖氟化物与外向型分子间的相互作用 承运人。

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.