Variation in myoplasmic Ca2+ concentration during contraction and relaxation studied by the indicator fluo-3 in frog...

1. The fluorescent dye fluo-3, in its permeant acetoxymethyl form, was used to monitor calcium transients during twitch and tetanus of single fibres isolated from the anterior tibialis muscle of Rana temporaria (2-5 degrees C). 2. Fluo-3 was loaded into the muscle fibre by diffusion. Under the experimental conditions used, approximately 45% of maximal fluorescence was reached during a 1 s fused isometric tetanus. Fluo-3 had no detectable effect on the mechanical response of the fibre. 3. The free calcium concentration in the myoplasm, [Ca2+]i, and its variation with time, was calculated from the fluorescence signal by accounting for the on- and off-rate constants for the binding of calcium to the dye. The time course of the calcium transient during twitch and tetanus determined in this way agreed well with previous measurements based on fast-reacting calcium-sensitive dyes. 4. [Ca2+]i declined steeply during the initial phase of force relaxation in both twitch and tetanus, but exhibited a secondary rise that closely coincided with the pseudoexponential fall of tension after the shoulder in the tetanus myogram. The rate of decay of [Ca2+]i during relaxation and the rate of decline of force both became progressively reduced by repetitive stimulation. 5. Stretch and shortening ramps performed during the plateau of an isometric tetanus had no detectable effect upon the calcium transient during the movement. By contrast, shortening and stretch imposed during the linear phase of relaxation both led to an increase of [Ca2+]i and to a steepening of the relaxation phase. 6. The results strongly suggest that the non-uniform length changes that are known to occur along a muscle fibre during relaxation enhance the release of calcium from the contractile system. The calcium mobilized in this way probably accounts for the transitory increase of [Ca2+]i that is observed during the latter part of force relaxation.