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

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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.