DEVELOPMENT OF INTRACELLULAR INDICATORS AND ION TRANSPORT STUDIES

胞内离子指示剂极大地扩展了我们的 了解这些离子在正常和病理状态下的作用 条件。最近的努力集中在几个钙离子指示剂上。 具有相对较高的(几十微摩尔)离解常数。虽然 一般来说，如果Kd与离子相匹配，则测量是最准确的 在研究中，高Kd指标有几个优点 和潜在的应用。这些措施包括：(1)减少离子缓冲 瞬变；(2)适用于离子水平可能增加的情况 远远超出基础值；(3)在细胞器研究中的应用 其中钙离子水平可能显著高于胞浆中的 水平。例如，胞质钙瞬变的缓冲 用5FBAPTA负载灌流的大鼠心脏的结果，目前的核磁共振 选择的指示器，显著改变收缩能力。该指示器 我们合成和评价的TFBAPTA提供了一个有吸引力的 解决上述问题。这个指示器有两种氟 共振，其中之一不会改变钙的络合作用，以及 第二个以10ppm的速度移动。在使用的典型场强下， 钙敏感共振在化学位移上快速交换 比例。快速交换指标的一个重要优点是 TFBAPTA是，如果指示器加载到不同的蜂窝隔间 对于不同的钙离子浓度，将有单独的共振 观察到的。一种类似的高KD荧光指示剂Fura-F也 是人工合成的。Fura-F的钙离子Kd为20fM，并表现出 类似于Fura-2的钙离子络合时的激发转移。在……里面 除了指标制定工作外，我们还启动了研究 探讨~-受体激动剂对大鼠MGI和镁外排的影响 镁离子指示剂灌流大鼠心脏和S49淋巴瘤细胞 之前作为该计划的一部分开发的。初步研究表明 MGI没有变化，但添加了 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.