ESTIMATE OF NET CALCIUM FLUXES AND SARCOPLASMIC-RETICULUM CALCIUM CONTENT DURING SYSTOLE IN RAT VENTRICULAR MYOCYTES

1. The experiments were performed on voltage-clamped cells in which in tracellular calcium concentration ([Ca2+](i)) was measured with the fl uorescent indicator indo-1 (acetoxymethyl ester (AM) loading). When ce lls were stimulated with a short (100 ms) depolarizing pulse, followin g a rest, the magnitude of the first systolic calcium transient was gr eater 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 u sed then the systolic calcium transient was either unaffected or incre ased in magnitude following a rest. During constant stimulation, if th e length of the pulse is decreased, then the magnitude of the calcium transient decreased reversibly over several beats. 2. The calcium entr y into the cell was measured from the integral of the inward calcium c urrent and the efflux from the Na+-Ca2+ exchange current on repolariza tion. During the negative staircase the calcium current was approximat ely constant whilst the Na+-Ca2+ exchange current decayed in parallel with the systolic calcium transient. A net loss of calcium from the ce ll can be calculated from the extra Na+-Ca2+ exchange current followin g the initial pulses. 3. The application of caffeine produces a transi ent increase of both [Ca2+](i) and an inward Na+-Ca2+ exchange current . The integral of this current can be used to estimate the caffeine-re leasable calcium content of the sarcoplasmic reticulum (SR), which dec reases following stimulation with short compared to long pulses. This difference in SR calcium content is quantitatively similar to that est imated from the sarcolemmal currents. 4. At a given membrane potential , the relationship between [Ca2+](i) and current during the caffeine e xposure can be used to estimate the Na+-Ca2+ exchange flux from the me asured [Ca2+](i) and thence the Na+-Ca2+ exchange flux during depolari zation. 5. For a long depolarizing pulse the extrusion of calcium from the cell on Na+-Ca2+ exchange is comparable to the entry on the calci um 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 t hat 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 sa rcolemmal fluxes.