Csernoch L, Bernengo J C, Szentesi P, Jacquemond V
Department of Physiology, University Medical School of Debrecen, H-4012 Debrecen, Hungary.
Biophys J. 1998 Aug;75(2):957-67. doi: 10.1016/S0006-3495(98)77584-8.
Measurements of intracellular free magnesium concentration ([Mg2+]i) were performed on enzymatically isolated skeletal muscle fibers from mice, using the fluorescent ratiometric indicator mag-indo-1. An original procedure was developed to calibrate the dye response within the fibers: fibers were first permeabilized with saponin in the presence of a given extracellular magnesium concentration and were then embedded in silicone grease. The dye was then pressure microinjected into the saponin-permeabilized silicone-embedded fibers, and fluorescence was measured. The results show that for all tested [Mg2+], the value of the measured fluorescence ratio was higher than that found in aqueous solutions. Furthermore, the apparent binding curve that could be fit to the in vivo ratio data was shifted toward higher [Mg2+] by a factor of approximately 2. Using the in vivo calibration parameters, the mean resting [Mg2+]i was found to be 1.53 +/- 0.16 mM (n = 7). In an attempt to gain insight into the myoplasmic magnesium buffering capacity, we measured, together with mag-indo-1 fluorescence, the current elicited by the application of carbamylcholine (CCh) to the endplate of isolated fibers, in the presence of a high extracellular magnesium concentration. The results show that, under these conditions, a change in [Mg2+]i displaying a time course and amplitude qualitatively consistent with the CCh-induced inward current can be measured.
使用荧光比率指示剂mag-indo-1，对从小鼠酶解分离的骨骼肌纤维进行细胞内游离镁浓度（[Mg2+]i）的测量。开发了一种原始程序来校准纤维内的染料响应：首先在给定的细胞外镁浓度存在下，用皂素使纤维通透，然后将其包埋在硅脂中。然后将染料通过压力显微注射到经皂素通透且包埋在硅脂中的纤维中，并测量荧光。结果表明，对于所有测试的[Mg2+]，测量的荧光比率值高于在水溶液中发现的值。此外，能够拟合体内比率数据的表观结合曲线向更高的[Mg2+]移动了约2倍。使用体内校准参数，发现平均静息[Mg2+]i为1.53±0.16 mM（n = 7）。为了深入了解肌浆镁缓冲能力，在高细胞外镁浓度存在的情况下，我们在测量mag-indo-1荧光的同时，测量了向分离纤维的终板施加氨甲酰胆碱（CCh）所引发的电流。结果表明，在这些条件下，可以测量到[Mg2+]i的变化，其时间进程和幅度在定性上与CCh诱导的内向电流一致。
