DEVELOPMENT OF INTRACELLULAR INDICATORS AND ION TRANSPORT STUDIES

胞质离子的细胞内指示剂极大地扩展了我们的 了解这些离子在正常和病理条件下的作用 条件 最近的努力集中在几个钙离子指标 具有相对高的（数十微摩尔）解离常数。 虽然 通常，如果KD与离子匹配，则测量是最准确的 在研究的浓度下，高KD指示剂具有几个优点 和潜在的应用。 这些措施包括：（1）减少离子缓冲 瞬态;（2）应用于离子水平可能增加的情况 远超过基础值;（3）应用于细胞器的研究， 其钙离子水平可能显著高于胞质 水平 例如，细胞溶质钙瞬变的缓冲， 结果从加载灌流大鼠心脏与5 FBAPTA，目前的NMR 选择的指标，显著改变收缩性。 指示器 TFBAPTA，我们已经合成和评估提供了一个有吸引力的 解决上述问题。 这个指示剂有两个氟 共振，其中一个在钙络合时不移动， 其中第二个偏移10 ppm。 在所使用的典型场强下， 钙敏感共振在化学位移上是快速交换的 规模 一个重要的优点是快速交换指标，如 TFBAPTA是如果指示剂加载到不同的细胞隔室， 在不同的钙离子浓度下， 观察 类似的高KD荧光指示剂fura-F也具有 被合成。 Fura-F具有20 fM的钙离子KD，并且表现出 与Fura-2类似，钙离子络合后的激发位移。 在 除了制订指标的工作外，我们亦已展开研究， 研究~-受体激动剂对Mgi和镁外流的影响， 使用镁离子指示剂灌注大鼠心脏和S49淋巴瘤细胞 以前作为这个项目的一部分。 初步研究表明 Mgi无变化，但在添加 异丙肾上腺素灌流大鼠心脏。

Intracellular indicators for cytosolic ions have greatly extended our understanding of the role of these ions under normal and pathological conditions. Recent efforts have focused on several calcium ion indicators with relatively high (tens of micromolar) dissociation constants. Although in general measurements are most accurate if the KD is matched to the ion concentration under study, high KD indicators have several advantages and potential applications. These include: (1) reduced buffering of ion transients; (2) application to situations where the ion level may increase well beyond the basal value; (3) application to studies of organelles in which the calcium ion levels may be considerably higher than the cytosolic level. For example, the buffering of cytosolic calcium transients which results from loading a perfused rat heart with 5FBAPTA, the current NMR indicator of choice, significantly alters contractility. The indicator TFBAPTA which we have synthesized and evaluated provides an attractive solution to the above problems. This indicator has two fluorine resonances, one of which does not shift upon calcium complexation, and the second of which shifts by 10 ppm. At typical field strengths used, the calcium sensitive resonance is in fast exchange on the chemical shift scale. One important advantage of a fast exchange indicator such as TFBAPTA is that if the indicator loads into different cellular compartments with different calcium ion concentrations, separate resonances will be observed. An analogous high KD fluorescent indicator, fura-F has also been synthesized. Fura-F has a calcium ion KD of 20fM, and exhibits an excitation shift upon calcium ion complexation similar to fura-2. In addition to the indicator development work, we have also initiated studies to investigate the effect of ~-agonists on Mgi and magnesium efflux from perfused rat heart and S49 lymphoma cells using magnesium ion indicators previously developed as part of this program. Preliminary studies show no changes in Mgi, but a significant magnesium efflux upon addition of isoproterenol to perfused rat heart.