Berlin J R, Konishi M
Bockus Research Institute, Graduate Hospital, Philadelphia, Pennsylvania 19146.
Biophys J. 1993 Oct;65(4):1632-47. doi: 10.1016/S0006-3495(93)81211-6.
Intracellular calcium ion ([Ca2+]i) transients were measured in voltage-clamped rat cardiac myocytes with fura-2 or furaptra to quantitate rapid changes in [Ca2+]i. Patch electrode solutions contained the K+ salt of fura-2 (50 microM) or furaptra (300 microM). With identical experimental conditions, peak amplitude of stimulated [Ca2+]i transients in furaptra-loaded myocytes was 4- to 6-fold greater than that in fura-2-loaded cells. To determine the reason for this discrepancy, intracellular fura-2 Ca2+ buffering, kinetics of Ca2+ binding, and optical properties were examined. Decreasing cellular fura-2 concentration by lowering electrode fura-2 concentration 5-fold, decreased the difference between the amplitudes of [Ca2+]i transients in fura-2 and furaptra-loaded myocytes by twofold. Thus, fura-2 buffers [Ca2+]i under these conditions; however, Ca2+ buffering is not the only factor that explains the different amplitudes of the [Ca2+]i transients measured with these indicators. From the temporal comparison of the [Ca2+]i transients measured with fura-2 and furaptra, the apparent reverse rate constant for Ca2+ binding of fura-2 was at least 65s-1, much faster than previously reported in skeletal muscle fibers. These binding kinetics do not explain the difference in the size of the [Ca2+]i transients reported by fura-2 and furaptra. Parameters for fura-2 calibration, Rmin, Rmax, and beta, were obtained in salt solutions (in vitro) and in myocytes exposed to the Ca2+ ionophore, 4-Br A23187, in EGTA-buffered solutions (in situ). Calibration of fura-2 fluorescence signals with these in situ parameters yielded [Ca2+]i transients whose peak amplitude was 50-100% larger than those calculated with in vitro parameters. Thus, in vitro calibration of fura-2 fluorescence significantly underestimates the amplitude of the [Ca2+]i transient. These data suggest that the difference in amplitude of [Ca2+]i transients in fura-2 and furaptra-loaded myocytes is due, in part, to Ca2+ buffering by fura-2 and use of in vitro calibration parameters.
在使用fura-2或furaptra的电压钳制大鼠心肌细胞中测量细胞内钙离子（[Ca2+]i）瞬变，以定量[Ca2+]i的快速变化。膜片钳电极溶液含有fura-2（50微摩尔）或furaptra（300微摩尔）的钾盐。在相同的实验条件下，furaptra负载的心肌细胞中刺激的[Ca2+]i瞬变的峰值幅度比fura-2负载的细胞大4至6倍。为了确定这种差异的原因，研究了细胞内fura-2的钙缓冲、钙结合动力学和光学性质。通过将电极fura-2浓度降低五倍来降低细胞内fura-2浓度，使fura-2和furaptra负载的心肌细胞中[Ca2+]i瞬变幅度之间的差异降低了两倍。因此，在这些条件下fura-2缓冲[Ca2+]i；然而，钙缓冲并不是解释用这些指示剂测量的[Ca2+]i瞬变不同幅度的唯一因素。从用fura-2和furaptra测量的[Ca2+]i瞬变的时间比较来看，fura-2的钙结合表观反向速率常数至少为65s-1，比先前在骨骼肌纤维中报道的要快得多。这些结合动力学并不能解释fura-2和furaptra报告的[Ca2+]i瞬变大小的差异。fura-2校准的参数Rmin、Rmax和β是在盐溶液（体外）以及暴露于Ca2+离子载体4-Br A23187的心肌细胞中，在EGTA缓冲溶液（原位）中获得的。用这些原位参数对fura-2荧光信号进行校准，得到的[Ca2+]i瞬变的峰值幅度比用体外参数计算的幅度大50-100%。因此，fura-2荧光的体外校准显著低估了[Ca2+]i瞬变的幅度。这些数据表明，fura-2和furaptra负载的心肌细胞中[Ca2+]i瞬变幅度的差异部分归因于fura-2的钙缓冲作用以及体外校准参数的使用。
