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: 3841114
• 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/3841114
• 关键词: acidity /alkalinity; analytical method; calcium indicator; chemical association; chemical synthesis; intracellular transport; ion transport; magnesium; pathologic process; reagent /indicator

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.

众所周知，离子可以作为细胞内信使发挥作用。 因此，离子转运的改变并不奇怪， 经常导致细胞功能障碍。 例如，许多癌基因产物 改变离子运输。 本项目的目标有两个：（1）发展 用于测量细胞内离子浓度的新指标， （2）离子转运及其调控的研究， 伴随致病状态的扰动。 以前我们有 开发了新的核磁共振活性指示剂，用于细胞溶质钙，镁， 氯化物和细胞体积。 这些NMR指示剂通常是 （虽然不一定）氟化分子，因为氟-19是100% 丰富，对NMR检测高度敏感，并且由于细胞是 基本上没有任何背景氟NMR信号。 两大 过去一年的项目是：（1）开发一个新的，高 解离常数（KD）细胞内钙离子指示剂，“TFBAPTA”， （2）几种“NEAP”衍生物的合成和评价， 最近开发的pH敏感指示剂。 的一个主要 在灌注的细胞中使用细胞溶质钙离子指示剂的局限性 心脏一直是收缩性的显着扰动， 从细胞内钙离子螯合剂的引入。 这是 我们制定新指标TFBAPTA的主要动机， 具有相当高的钙离子解离常数， 因此基本上是无扰动的。 虽然不适合 测定基础钙离子浓度，该指示剂是有用的 用于测量伴随扰动的增加 或使用某些化学药剂。 我们也取得了 pH指示剂NEAP的几个进一步的修改，以减少 并添加内部参考信号以便于 定量 我们也在继续研究运输机制 其调节细胞溶质游离镁远低于其电化学 均衡