Zhao M, Hollingworth S, Baylor S M
Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia 19104-6085, USA.
Biophys J. 1997 Jun;72(6):2736-47. doi: 10.1016/S0006-3495(97)78916-1.
The AM loading of a number of different fluorescent Ca2+ indicators was compared in intact single fibers of frog muscle. Among the 13 indicators studied, loading rates (the average increase in the fiber concentration of indicator per first 60 min of loading) varied approximately 100-fold, from approximately 3 microM/h to >300 microM/h (16 degrees C). Loading rates were strongly dependent on the molecular weight of the AM compounds, with the rate increasing steeply as molecular weight decreased below approximately 850. Properties of delta F/F (the Ca2(+)-related fluorescence signal observed with fiber stimulation) were also measured in AM-loaded fibers and compared with those previously reported for fibers microinjected with indicator. In general, the time course of delta F/F was very similar with AM-loading and microinjection; however, the amplitude of delta F/F was usually smaller with AM-loading. There was a strong correlation between the rate of indicator loading and the value of the parameter f (the ratio of the amplitude of delta F/F in AM-loaded versus microinjected fibers). For indicators with small loading rates (<10 microM/h, N = 5), f values were generally small (< or =0.4, N = 4); whereas with large loading rates (>100 microM/h, N = 4), f values were large (> or =0.8, N = 4). This suggests that, with any AM indicator, a small concentration may associate nonspecifically with the fiber (either the indicator is incompletely de-esterified or, if completely de-esterified, not located in the myoplasmic compartment). If the loaded concentration is small, the nonspecific indicator will present a significant source of error in the estimation of [Ca2+]i.
在青蛙肌肉的完整单纤维中比较了多种不同荧光Ca2+指示剂的AM加载情况。在所研究的13种指示剂中，加载速率（加载最初60分钟内指示剂在纤维中的浓度平均增加量）变化约100倍，从约3 microM/h到>300 microM/h（16摄氏度）。加载速率强烈依赖于AM化合物的分子量，当分子量降至约850以下时，速率急剧增加。还在AM加载的纤维中测量了δF/F（纤维刺激时观察到的与Ca2+相关的荧光信号）的特性，并与先前报道的显微注射指示剂的纤维的特性进行了比较。一般来说，AM加载和显微注射时δF/F的时间进程非常相似；然而，AM加载时δF/F的幅度通常较小。指示剂加载速率与参数f的值（AM加载纤维与显微注射纤维中δF/F幅度之比）之间存在很强的相关性。对于加载速率小的指示剂（<10 microM/h，N = 5），f值通常较小（<或=0.4，N = 4）；而加载速率大时（>100 microM/h，N = 4），f值较大（>或=0.8，N = 4）。这表明，对于任何AM指示剂，低浓度可能会非特异性地与纤维结合（要么指示剂脱酯不完全，要么即使完全脱酯，也不在肌质区室中）。如果加载浓度较低，非特异性指示剂将在估计[Ca2+]i时产生显著的误差来源。
