Quantitative Measurement of Intracellular Ca Transient by the Simultaneous Application of Furaー2 and Aequorin

同时应用 Fura-2 和水母发光蛋白定量测量细胞内 Ca 瞬态

• 批准号: 01570049
• 负责人: KURIHARA Satoshi
• 金额: $ 1.15万
• 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: 1989
• 资助国家: 日本
• 起止时间: 1989 至 1990
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-01570049/
• 关键词: Skeletal muscle; Ca transient; Ca indicator; Intracellular Ca; Fura-2; Aequorin; furaー2; 蛍光色素; fura-2

Furaー2 with high affinity for Ca^<2+> and aequorin were injected into single skeletal muscle fibers, and intracellular Ca concentration ([Ca^<2+>]_i) during and after contraction was measured using the in vitro calibration curves of both indicators. [Ca^<2+>]_i at resting state became negative value if the ratio (340/380 nm of excitation wave length) of fura-2 fluorescence was converted to [Ca^<2+>]_i. [Ca^<2+>]_i during twitch and tetanus (50 Hz, 1 sec) measured with fura-2 was 0.5 and 1.1 muM respectively. At high stimulation frequency (1/1s - 1/0.3s), an increase in the resting ratio signal of fura-2 was observed. In aequorin, [Ca^<2+>]_i during twitch and tetanus was 5 and 7 muM. Fura-2 ratio signal did not return to the control level even 30 sec after twitch and a higher level of fura-2 ratio signal persisted 90 sec after tetanus. Aequorin was not sensitive to detect a small increase in resting [Ca^<2+>]_i after twitch and tetanus. A small value of [Ca^<2+>]_i measured with fura-2 might be due to (1) binding of fura-2 to intracellular soluble proteins, (2) inner filter effect of fura-2 and (3) intrinsic absorbency of the fiber at a shorter wave length. However, fura-2 is advantageous to qualitatively detect a small change in resting [Ca^<2+>]_i but is not suitable for measuring Ca transients of the skeletal muscle fibers. The present result also suggests that Ca^<2+> released from the sarcoplasmic reticulum (SR) takes much longer time to return to the SR than that reported.

将对Ca^2+和水母发光蛋白具有高亲和力的Furaー2注射到单根骨骼肌纤维中，利用这两种指标的体外校准曲线测量收缩期间和收缩后的细胞内Ca浓度（[Ca^2+]_i）。如果将fura-2荧光的比率(激发波长340/380nm)换算成[Ca^2+]_i，则静息状态下的[Ca^2+]_i变为负值。用fura-2测量的抽搐和破伤风(50Hz，1秒)期间的[Ca 2+ ]_i分别为0.5和1.1μM。在高刺激频率（1/1s - 1/0.3s）下，观察到 fura-2 的静息比信号增加。在水母发光蛋白中，抽搐和破伤风期间的[Ca 2+ ]_i 为5和7μM。即使在抽搐后 30 秒，Fura-2 比率信号也没有恢复到控制水平，并且破伤风后 90 秒，Fura-2 比率信号仍保持较高水平。水母发光蛋白对于检测抽搐和破伤风后静息[Ca^2+]_i 的小幅增加不敏感。使用fura-2测量的[Ca^2+]_i值较小可能是由于(1)fura-2与细胞内可溶性蛋白质的结合，(2)fura-2的内部过滤效应和(3)纤维在较短波长下的固有吸收性。然而，fura-2有利于定性检测静息[Ca^2+]_i的微小变化，但不适合测量骨骼肌纤维的Ca瞬变。目前的结果还表明，从肌浆网(SR)释放的Ca^2+返回到SR所需的时间比报道的要长得多。

项目成果

O.Okazaki: "Modulation of Ca^<2+> transients and contractile proーperties by betaーadrenoceptor stimulation in ferret ventricular muscles" Journal of Physiology. 423. 221-240 (1990)

O.Okazaki：“雪貂心室肌肉中β-肾上腺素受体刺激对Ca^2+瞬变和收缩特性的调节”生理学杂志423. 221-240 (1990)。

Kurihara.S.: "Application of aequorin and fura-2 to single skeletal muscle fibres for monitoring intracellular Ca transients and tension" Journal of Muscle Research and Cell Motility. 10. 262 (1989)

Kurihara.S.：“应用水母发光蛋白和 fura-2 到单骨骼肌纤维来监测细胞内 Ca 瞬变和张力”《肌肉研究和细胞运动杂志》。

M.Watanabe: "Binding of murine monoclonal antibodies to the active and inactive configurations of aequorin" FEBS Letters. 246. 73-77 (1989)

M.Watanabe：“鼠单克隆抗体与水母发光蛋白的活性和非活性构型的结合”FEBS Letters。

N.Suda: "Intracellular calcium signals mesured with furaー2 and aequorin in frog skeletal muscle fibers." Japanese journal of physiology.

N.Suda：“用青蛙骨骼肌纤维中的 fura-2 和水母发光蛋白测量细胞内钙信号。”

M. Watanabe: "Binding of murine monoclonal antibodies to the active and inactive configurations of aequorin" FEBS Letters. 246. 73-77 (1989)

M. Watanabe：“鼠单克隆抗体与水母发光蛋白的活性和非活性构型的结合”FEBS Letters。