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

测量细胞内Ca^<2+>浓度静息状态下骨骼肌纤维中的[Ca^<2+>] _i，Ca^<2+>指示剂fura-2与葡聚糖结合（呋喃葡聚糖，MW约10，000）注射到青蛙的单个抽搐纤维中，并在17 ℃下测量细胞质中指示剂的Ca^<2+>依赖性荧光。（以[Ca^<2+>] _i计）在用β-七叶皂苷处理的肌纤维中进行，以透化细胞膜。在用5 μ M β-七叶皂苷处理30-35分钟后，细胞膜对小分子（例如，Ca^<2+>，ATP），而10 kDa的呋喃右旋糖酐仅缓慢地从细胞中漏出。细胞质蛋白质的主要部分（14-80 kDa）保留在β-七叶皂苷处理的细胞中，因为通过SDS-PAGE和银染色在细胞外溶液样品中仅检测到痕量的蛋白质。因此，可以通过改变培养液中Ca^2+的浓度来估计细胞中指示剂荧光的校准参数（在细胞蛋白存在的情况下）。当浴槽溶液中的[Ca^2+]改变为pCa>9到更高的水平（pCa 7 -4）时，呋喃葡聚糖的Ca^2+依赖性荧光在几分钟内改变到一个新的稳定水平。该指示剂对Ca^2+的解离常数（K_D）在细胞中估计为1.0 μ M，比体外获得的值（0.52 μ M）高两倍。第二种估算K_D的方法是对完整肌纤维进行电刺激后指示剂荧光变化的动力学分析，其估算值为2.1 μ M。由此估算的细胞内部的K_D值（1.0-2.0 μ M）比体外获得的K_D值（0.52 μ M）高2 - 4倍。从估计的纤维中的参数，我们得出结论，青蛙骨骼肌纤维中的静息[Ca^<2+>]_i可能在55-155 nM的范围内。

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.