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

一种用荧光指示剂定量肌肉细胞内游离钙浓度的新方法。

基本信息

批准号：
05670055
负责人：
KONISHI Masato
金额：
$ 1.34万
依托单位：
The Jikei University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

项目摘要

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.

为了测量静止时骨骼肌纤维中的细胞内Ca^<2+>浓度([Ca^<2+>]_i)，将与葡聚糖缀合的Ca^<2+>指示剂fura-2(fura葡聚糖，MW -10,000)注射到青蛙的单抽动纤维中，并且指示剂在细胞质中的Ca^<2+>依赖性荧光为在 17°C 下测量。在用 β-七叶皂苷处理以透化细胞膜的肌纤维中进行指示剂荧光（以 [Ca^<2+>]_i 计）的校准。用5μMβ-七叶皂苷处理30-35分钟后，细胞膜可渗透小分子(例如Ca 2+ 、ATP)，而10kDa呋喃右旋糖酐仅缓慢地漏出细胞。细胞质蛋白的主要部分 (14-80 kDa) 保留在 β-七叶皂苷处理的细胞中，因为通过银染 SDS-PAGE 在细胞外溶液样品中仅检测到痕量蛋白质。因此可以通过将贝辛溶液中的Ca 2+ 浓度改变至不同水平来估计细胞中指示剂荧光的校准参数(在存在细胞蛋白质的情况下)。当沐浴溶液的[Ca 2+ ]变为pCa＞9至更高水平(pCa7-4)时，呋喃葡聚糖的Ca 2+ 依赖性荧光在几分钟内变为新的稳定水平。该指示剂在细胞中估计的Ca^2+(K_D)解离常数为1.0μM，比体外获得的解离常数(0.52μM)高两倍。估计 K_D 的第二种方法是对完整肌纤维中电刺激后指示剂荧光变化的动力学分析，得出的估计值为 2.1 μM。因此在细胞内部估计的 K_D 值 (1.0-2.0 μM) 比体外获得的值 (0.52 μM) 高两到四倍。根据纤维中估计的参数，我们得出结论，青蛙骨骼肌纤维中的静息[Ca^2+]_i可能在55-155nM的范围内。