IN VIVO STUDIES OF CELLULAR MAGNESIUM

虽然镁是最丰富的二价金属离子，在大多数 细胞内游离镁离子浓度的调节 (mg-I)，以及该离子水平的变化可能在 对多种毒素的致病机制尚不清楚。在……里面 为了解决这个问题，我们以前设计和合成了 核磁共振和荧光指示剂，可以加载到细胞中，并 对游离镁离子水平敏感。在过去的一年里，我们有 将这些指标应用于调节MG-1的机制的研究。在……里面 为了了解MG-I的改变可能如何参与细胞 损伤，重要的是要了解正常的生理调节 Mg-I。我们改变了细胞所在的培养液的离子成分 被灌流，并获得了间接影响的证据 细胞内钙、镁离子相互竞争结合 网站。为了了解调节MG-I的机制，我们 检测了改变胞浆pH的影响。养殖胚胎雏鸡 心脏细胞培养在盖玻片上，并装载有荧光 镁-I指示剂FURAPTRA或pH指示剂BCECF。体内的pH值发生了变化 通过将细胞暴露在10 mM NH4CL中。当中性NH3跨过 膜，细胞内pH瞬间上升到7.9左右；这是 伴随着镁离子浓度下降0.18 mm。几分钟后，PH值 恢复到7.25，mg-1恢复到对照水平。在移除时 胞外NH4Cl，胞内pH降至6.5，而镁离子升高 只差0.06毫米。原子吸收法测细胞总镁 光谱并没有因为这些操作而改变。尽管 钙离子在53微米处的离解常数远大于 基础细胞内钙水平，细胞内钙的变化可 这是使用该指示剂测定镁离子的一个重大限制。在……里面 为了克服这一缺点，我们付出了巨大的努力 用于合成具有更好的选择性的指示剂 镁/钙。

Although magnesium is the most abundant divalent metal ion found in most cells, the regulation of the cytosolic free magnesium ion concentration (Mg-i), and the role which alterations in the level of this ion may play in mediating the pathology of a variety, of toxins, is relatively unknown. In order to attack this problem, we have previously designed and synthesized both NMR and fluorescent indicators which can be loaded into cells and are sensitive to the free magnesium ion level. Over the past year we have applied these indicators to the study of mechanisms which regulate Mg-1. In order to understand how alterations in Mg-i might be involved in cell injury, it is important to understand the normal physiological regulation of Mg-i. We varied the ionic composition of the medium in which the cells were perfused and obtained evidence for an indirect effect reflecting competition between intracellular calcium and magnesium ions for binding sites. To understand the mechanisms involved in modulating Mg-i, we examined the effect of altering cytosolic pH. Cultured embryonic chick heart cells were grown on coverslips and loaded with either the fluorescent Mg-i indicator FURAPTRA, or the pH indicator BCECF. Internal pH was altered by exposing the cells, to 10 mM NH4CL . When neutral NH3 crossed the membrane, the intracellular pH rose transiently to about 7.9; this was accompanied by a 0.18 mM decrease in Mg-i. After a few minutes, pH recovered to 7.25 and Mg-1 returned to control levels. Upon removal of extracellular NH4Cl, intracellular pH fell to 6.5, whereas Mg-i increased by only 0.06 mM. Total cell magnesium measured by atomic absorption spectroscopy was not altered, by these manipulations. Although the dissociation constant of calcium ions from FURAPTRA of 53 uM is well above basal cytosolic calcium levels, variations in intracellular calcium can be a significant limitation on Mg-i determinations using this indicator. In order to overcome this shortcoming, we have devoted a significant effort toward the synthesis of indicators with improved selectivity for magnesium/calcium.