DEVELOPMENT OF INTRACELLULAR INDICATORS AND ION TRANSPORT STUDIES

众所周知，离子可以作为细胞内的信使发挥作用。 因此，离子传输的变化并不令人惊讶。 经常会导致细胞功能障碍。例如，许多癌基因产品 改变离子传输。这个项目的目标有两个：(1)发展 用于测量细胞内离子浓度的新指示器， (2)离子输运及其调控的研究； 伴随致病状态的微扰。以前我们有过 开发了新的核磁共振活性指示剂，用于细胞内钙、镁和 氯化物，以及细胞体积。这些核磁共振指标通常是 (虽然不一定)氟化分子，因为氟-19是100% 丰富，对核磁共振检测高度敏感，由于细胞 基本上没有任何背景氟核磁共振信号。两大 过去一年的项目有：(1)发展新高 解离常数(Kd)细胞内钙离子指示剂“TFBAPTA”， 和(2)几种“NEAP”衍生物的合成和鉴定，我们的 新近开发的pH敏感指示剂。其中一位校长 细胞内钙离子指示剂在灌流液中应用的局限性 心脏一直是收缩能力的显著扰动，其结果 由于细胞内钙离子螯合剂的引入。这就是 我们开发新指标TFBAPTA的主要动机是 具有相当高的钙离子离解常数，并且 因此，基本上不会让人感到不安。尽管不适合于 基础钙离子浓度的测定，该指示剂是有用的 用于测量伴随着诸如缺血等扰动的增加 或某些化学制剂的管理。我们还制作了 对pH指示剂NEAP进行了几次进一步修改，以降低 并添加一个内部基准信号，以便于 量化。我们还在继续研究运输机制。 以远低于其电化学性质的方式调节胞内游离镁 均衡。

It is well recognized that ions can function as intracellular messengers. It is not surprising, therefore, that alterations in ion transport frequently cause cellular dysfunction. For example, many oncogene products alter ion transport. The goal of this project is twofold: (1) development of new indicators for the measurement of intracellular ion concentrations, and (2) investigation of ion transport and its regulation, and the perturbations which accompany pathogenic states. Previously we have developed new NMR-active indicators for cytosolic calcium, magnesium, and chloride, and for cell volume. These NMR indicators are generally (although not necessarily) fluorinated molecules since fluorine-19 is 100% abundant, highly sensitive for NMR detection, and since cells are essentially free of any background fluorine NMR signals. Two major projects over the past year were: (1) development of a new, high dissociation constant (KD) intracellular calcium ion indicator, "TFBAPTA", and (2) synthesis and evaluation of several derivatives of "NEAP", our recently developed pH sensitive indicator. One of the principal limitations on the use of cytosolic calcium ion indicators in the perfused heart has been the significant perturbation of contractility which results from the introduction of intracellular calcium ion chelators. This was the primary motivation for our development of a new indicator, TFBAPTA, which has a considerably higher calcium ion dissociation constant and is therefore essentially non-perturbing. Although not suited for the determination of basal calcium ion concentrations, this indicator is useful for measuring the increases which accompany perturbations such as ischemia or the administration of certain chemical agents. We have also made several further modifications of the pH indicator NEAP in order to reduce leakage and to add an internal reference signal in order to facilitate quantitation. We are also continuing studies on the transport mechanisms that regulate cytosolic free magnesium well below its electrochemical equilibrium.