IN VIVO STUDIES OF CELLULAR MAGNESIUM

尽管镁是在大多数生物体中发现的最丰富的二价金属离子， 细胞内游离镁离子浓度的调节 （Mg-1），以及该离子水平的改变可能在 介导多种毒素的病理学是相对未知的。在 为了解决这个问题，我们以前设计和合成了 NMR和荧光指示剂都可以装载到细胞中， 对游离镁离子水平敏感。在过去的一年里， 将这些指标应用于调节Mg-1的机制的研究。在 为了了解Mg-i的改变如何参与细胞凋亡， 受伤后，重要的是要了解正常的生理调节 关于MG我们改变了培养基的离子组成， 并获得了间接影响的证据， 细胞内钙镁离子竞争结合 网站.为了理解参与调节Mg-1的机制，我们 研究了改变细胞溶质pH值的影响。培养的鸡胚 心脏细胞在盖玻片上生长， Mg-1指示剂FURAPTRA或pH指示剂BCECF。内部pH值改变 通过将细胞暴露于10 mM NH4CL。当中性NH3穿过 膜，细胞内pH值瞬时上升至约7.9;这是 伴随Mg2+降低0.18 mM。几分钟后，pH 恢复至7.25，Mg-1恢复至对照水平。一旦移除 胞外NH_4Cl，胞内pH下降至6.5，而Mg~（-1）增加 通过原子吸收测量的总细胞镁 通过这些操作，光谱学没有改变。虽然 FURAPTRA的钙离子解离常数为53 μ M，远高于 基础细胞溶质钙水平，细胞内钙的变化， 使用该指示剂对Mg-1测定的显著限制。在 为了克服这一缺点，我们付出了巨大的努力， 以合成具有更高选择性的指标， 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.