Akerman K E, Heinonen E
Biochim Biophys Acta. 1983 Jul 13;732(1):117-21. doi: 10.1016/0005-2736(83)90193-1.
If arsenazo III is present during homogenization of brain this metallochromic indicator is entrapped within subsequently isolated synaptosomes. A large proportion of the entrapped indicator is released upon addition of digitonin to disrupt the synaptosomal plasma membrane. A similar proportion of [3H]sucrose is also trapped within synaptosomes if present in the homogenization medium, suggesting that homogenization causes a transient opening of the nerve ending as it is chopped off from the axon. Addition of the ionophore A23187 or depolarization of the plasma membrane by adding veratridine, gramicidin or increasing external K+ changes the absorbance of the entrapped dye, with peaks of absorbance around 600 and 650 nm, typical of the arsenazo III-Ca2+ complex. The response to veratridine is inhibited by the Ca2+-channel antagonist, verapamil, while that of A23187 is unaffected. The present method provides a sensitive technique for measurements of changes in cytosolic calcium ion concentrations within nerve endings.
如果在脑匀浆过程中存在偶氮胂III，这种金属显色指示剂就会被困在随后分离出的突触小体中。加入洋地黄皂苷破坏突触小体质膜后，大部分被困的指示剂会被释放出来。如果在匀浆介质中存在[3H]蔗糖，类似比例的[3H]蔗糖也会被困在突触小体中，这表明匀浆过程会导致神经末梢在从轴突切断时出现短暂的开放。添加离子载体A23187或通过添加藜芦碱、短杆菌肽或增加细胞外钾使质膜去极化，会改变被困染料的吸光度，吸光度峰值在600和650纳米左右，这是偶氮胂III - Ca2+复合物的典型特征。Ca2+通道拮抗剂维拉帕米可抑制对藜芦碱的反应，而对A23187的反应则不受影响。本方法为测量神经末梢内细胞溶质钙离子浓度的变化提供了一种灵敏的技术。
