A Novel Method to Quantify Intracellular Free Calcium Concentration in Muscle Cells with a Fluorescent Indicator.

为了测定静息状态下骨骼肌纤维内Ca^<2+>浓度（[Ca^<2+>] _i），将Ca^<2+>指示剂fura-2 （fura dextran, MW -10,000）偶联于右旋糖酐，注射到蛙的单肌纤维中，在17 C下测定该指示剂在细胞质中的Ca^<2+>依赖性荧光。对经β -叶escin处理的肌纤维进行了指示荧光（以[Ca^<2+>] _i表示）校准，以通透细胞膜。5mum β -escin处理30- 35min后，细胞膜可渗透小分子（如Ca^<2+>， ATP），而10kda fura dexran只是缓慢地泄漏出细胞。β -果皮素处理的细胞中保留了主要的细胞质蛋白（14-80 kDa），因为通过SDS-PAGE银染色在细胞外溶液样品中只检测到微量的蛋白质。因此，可以通过改变溶液中Ca^<2+>的浓度到不同的水平来估计细胞中指示荧光的校准参数（在细胞蛋白存在的情况下）。当洗浴液中的[Ca^<2+>]变为更高水平的pCa>9 （pCa7-4）时，呋喃葡聚糖的Ca^<2+>依赖性荧光在几分钟内变化到一个新的稳定水平。该指示剂在细胞内对Ca^<2+>的解离常数（K_D）的估计为1.0 muM，比在体外得到的解离常数（0.52 muM）提高了2倍。第二种估计K_D的方法是对完整肌肉纤维电刺激后指示荧光变化的动力学分析，估计值为2.1 muM。因此，在细胞内部估计的K_D值（1.0-2.0 muM）比在体外获得的K_D值（0.52 muM）高2 - 4倍。从纤维中估计的参数，我们得出青蛙骨骼肌纤维的静息[Ca^<2+>]_i可能在55-155 nM范围内。

To measure intracellular Ca^<2+> concentration ( [Ca^<2+>] _i) in skeletal muscle fibers at rest, the Ca^<2+> indicator, fura-2, conjugated to dextran (fura dextran, MW -10,000) was injected into single twitch fibers of frogs, and the indicator's Ca^<2+> -dependent fluorescence in the cytoplasm was measured at 17ﾟC.Calibration of the indicator fluorescence (in terms of [Ca^<2+>] _i) was carried out in the muscle fibers treated with beta-escin to permeabilize the cell membrane. After the treatment with 5 muM beta-escin for 30-35 min, the cell membrane was permeable to small molecules (e.g., Ca^<2+>, ATP) , while the 10 kDa fura dexran only slowly leaked out of the cell. The major fraction of cytoplasmic proteins (14-80 kDa) was retained in the beta-escin-treated cells, as only a trace amount of proteins was detected in the exracellular solution samples by SDS-PAGE with silver staining. It was thus possible to estimate the calibration parameters of the indicator fluorescence in the cell (in the presence of cellular proteins) by changing the Ca^<2+> concentration in the bething solution to various levels. When [Ca^<2+>] of the bathing solution was changed to pCa>9 to higher levels (pCa7-4), the Ca^<2+> -dependent fluorescence of fura dextran changed to a new steady level within a few minutes. The indicator's dissociation constant for Ca^<2+> (K_D) estimated in the cell was 1.0 muM,which is two-fold higher than that obtained in vitro (0.52 muM). The second method to estimate the K_D, the kinetic analysis of the indicator fluorescence change following electrical stimulation in intact muscle fibers, gave an estimated value of 2.1 muM.The K_D values thus estimated in the cell interior (1.0-2.0 muM) is two- to four-fold higher than that obtained in vitro (0.52 muM). From the parameters estimated in the fibers, we conclude that the resting [Ca^<2+>]_i in frog skeletal muscle fibers is likely in the range of 55-155 nM.