Intracellular calcium signals measured with fura-2 and aequorin in frog skeletal muscle fibers.

Intracellular Ca(2+)-related optical signals during and after contraction (twitch and tetanus) were measured in single frog skeletal muscle fibers with fura-2 and aequorin. In twitch response, the peaks of [Ca2+]i estimated from the in vitro calibrations of fura-2 and aequorin were significantly different (0.5 microM for fura-2 and 5 microM for aequorin). Even 30s after twitch response, the fura-2 fluorescence ratio (F340/F380) signal did not recover to the resting level before stimulation. When the stimulation frequency was increased, an increase in the resting fura-2 ratio signal became obvious and after the cessation of stimulation this increase gradually recovered to the level before stimulation. After tetanus (50 Hz for 1 s), a higher fura-2 ratio signal than that before stimulation was sustained longer than 90 s. These results indicate that the dissociation of the released Ca2+ from the intracellular Ca2+ binding sites probably takes longer time than that previously reported. The present results, therefore, demonstrate that fura-2 is advantageous for qualitative monitoring of a slight change in the resting level of [Ca2+]i, which is not easily detectable with aequorin. In addition, the problems encountered in quantitative estimation of [Ca2+]i with fura-2 are also discussed.