The experiments were performed on voltage-clamped cells in which intracellular calcium concentration ([Ca2+]i) was measured with the fluorescent indicator indo-1 (acetoxymethyl ester (AM) loading). When cells were stimulated with a short (100 ms) depolarizing pulse, following a rest, the magnitude of the first systolic calcium transient was greater than that in the steady state (rest potentiation) and decayed to its steady level over a few stimuli. If a longer pulse (800 ms) was used then the systolic calcium transient was either unaffected or increased in magnitude following a rest. During constant stimulation, if the length of the pulse is decreased, then the magnitude of the calcium transient decreased reversibly over several beats. 2. The calcium entry into the cell was measured from the integral of the inward calcium current and the efflux from the Na(+)-Ca2+ exchange current on repolarization. During the negative staircase the calcium current was approximately constant whilst the Na(+)-Ca2+ exchange current decayed in parallel with the systolic calcium transient. A net loss of calcium from the cell can be calculated from the extra Na(+)-Ca2+ exchange current following the initial pulses. 3. The application of caffeine produces a transient increase of both [Ca2+]i and an inward Na(+)-Ca2+ exchange current. The integral of this current can be used to estimate the caffeine-releasable calcium content of the sarcoplasmic reticulum (SR), which decreases following stimulation with short compared to long pulses. This difference in SR calcium content is quantitatively similar to that estimated from the sarcolemmal currents. 4. At a given membrane potential, the relationship between [Ca2+]i and current during the caffeine exposure can be used to estimate the Na(+)-Ca2+ exchange flux from the measured [Ca2+]i and thence the Na(+)-Ca2+ exchange flux during depolarization. 5. For a long depolarizing pulse the extrusion of calcium from the cell on Na(+)-Ca2+ exchange is comparable to the entry on the calcium current. In contrast, for short pulses the extrusion of calcium on the Na(+)-Ca2+ exchange immediately after the pulse is greater than the entry during the pulse on the calcium current. 6. These results show that rest potentiation can be correlated with changes in the amount of calcium stored in the SR and this, in turn, can be accounted for by sarcolemmal fluxes.
