Fast calcium removal during single twitches in amphibian skeletal muscle fibres

Fluorescence signals from the calcium sensitive dyes Fluo-3 or Rhod-2 were obtained simultaneously with isometric tension in single fibres isolated from the anterior tibialis muscle of Leptodactylus insularis (20-22 degrees C). Fluo-3 fluorescence signals were transformed into [Ca2+](i) transients as previously described. Most of the decay phase of single twitch transient is well fitted by a single exponential (tau of about 10 ms), followed by a slower declining component lasting tens of milliseconds. During short periods, 10 to 20 s, of low frequency stimulation, between 0.2 and 5 Hz, the basal [Ca2+](i) increased slowly from 0.1 to about 0.4 mu M, with only minor changes in the exponentially decaying phase. In fibres poisoned with thapsigargin or cyclopiazonic acid (1-2 mu M) the tau of decay of fluorescence or Ca2+ transients of single twitches was very similar to that observed in non-poisoned fibres. Nevertheless, in poisoned fibres challenged with repetitive stimulation, the tau of Ca2+ transients decay increased from about 10 ms to > 40 ms, while the basal [Ca2+](i) increased from 0.1 to 2 mu M. Short rest periods (about 5 min) could reverse these effects, indicating that they were not a direct consequence of SR Ca2+-ATPase inhibition. The correlation coefficient between tau of decay and basal [Ca2+](i) was > 0.8 (P < 0.0001). Qualitatively similar results were obtained measuring Rhod-2 fluorescence signals. A lumped, two-compartment model could account for these results. Loading the fibres with EGTA-AM, diminished the effects of prolonged stimulation observed in poisoned fibres. Moreover, we show that the Na+ - Ca2+ exchange mechanism does not participate appreciably in fast Ca2+ removal.