Fluorescence signals from the calcium sensitive dyes Fluo-3 or Rhod-2 were
obtained simultaneously with isometric tension in single fibres isolated fr
om the anterior tibialis muscle of Leptodactylus insularis (20-22 degrees C
). Fluo-3 fluorescence signals were transformed into [Ca2+](i) transients a
s 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 perio
ds, 10 to 20 s, of low frequency stimulation, between 0.2 and 5 Hz, the bas
al [Ca2+](i) increased slowly from 0.1 to about 0.4 mu M, with only minor c
hanges in the exponentially decaying phase. In fibres poisoned with thapsig
argin 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 repetiti
ve 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 the
y were not a direct consequence of SR Ca2+-ATPase inhibition. The correlati
on coefficient between tau of decay and basal [Ca2+](i) was > 0.8 (P < 0.00
01). Qualitatively similar results were obtained measuring Rhod-2 fluoresce
nce signals. A lumped, two-compartment model could account for these result
s. Loading the fibres with EGTA-AM, diminished the effects of prolonged sti
mulation observed in poisoned fibres. Moreover, we show that the Na+ - Ca2 exchange mechanism does not participate appreciably in fast Ca2+ removal.