1. A method has been developed for calculating the flux of Ca2+ across the sarcoplasmic reticulum (SR) during excitation-contraction coupling in mammalian heart cells. F(SR) will symbolize the net rate of movement of Ca2+, per litre of accessible cytoplasm, into or out of the sarcoplasmic reticulum. F(SR) has the units M S-1. 2. A theory of the cytoplasmic [Ca2+]i transient in mammalian heart cells is presented in which the [Ca2+]i transient results from the various cellular processes that tend to increase or decrease cytoplasmic [Ca2+]i. According to the theory, F(SR) can be calculated if all cellular processes that contribute to the [Ca2+]i transient (other than Ca2+ fluxes across the SR) are either eliminated or are known quantitatively. 3. To obtain the measurements required to apply this theory, [Ca2+]i transients and membrane currents were recorded in guinea-pig single ventricular myocytes subjected to whole-cell voltage clamp and internal perfusion. [Ca2+]i transients were recorded through the use of the Ca2+ indicator, Fura-2 (pentapotassium salt). 4. Ca2+ fluxes through the sodium-calcium exchanger were eliminated in all experiments, by perfusing the cells, internally and externally, with Na+-free solutions. Ca2+ flux through the sarcolemmal L-type Ca2+ channel was measured as the verapamil-sensitive current. Influx of Ca2+ through all other voltage-dependent pathways was found to be negligible for the calculation of F(SR) over the time course of a single [Ca2+]i transient. 5. In the combined absence of Ca2+ current, Na+-Ca2+ exchange and fluxes across the SR (10 mM-caffeine), the net rate of removal of Ca2+ from the cytoplasm, which includes presumed contributions from sarcolemmal Ca2+-ATPase and mitochondrial Ca2+ transport, was found to be a negligible quantity in the calculation of F(SR), over the time course of a single [Ca2+]i transient. 6. Calculation of F(SR) requires that the Ca2+-binding capacity of cytoplasm be known. [Ca2+]i transients recorded during measurable total Ca2+ influx into the cytoplasm (verapamil-sensitive current in the absence of fluxes across the SR) were compared with theoretical Ca2+ transients computed on the assumption that the entering Ca2+ could bind only to intracellular ligands (values for ligands taken from literature) and to Fura-2 (30-mu-M). The slope of the regression line relating calculated total change in [Ca2+]i to the measured total Ca2+ influx was 0.99, not different from the perfect theoretical slope of 1.0 (correlation coefficient, 0.81; standard deviation of slope, 0.14; n = 7). 7. The results above indicate that, in the absence of Na+, the [Ca2+]i transient can be considered to be determined exclusively by the Ca2+ current, the flux across the SR, and intracellular Ca2+-binding ligands. According to this theory, F(SR) can be calculated from measurements of [Ca2+]i transients, Ca2+ currents and assumed characteristics of Ca2+-binding ligands. 8. Upon depolarization, Ca2+ is released from the SR, and F(SR) has a biphasic time course, with an early peak of net release of Ca2+ followed by a prolonged and slow phase of net uptake of Ca2+. The maximum F(SR) that was obtained in eight cells ranged between 2.7 and 9.5 mM s-1. 9. Upon repolarization from very positive potentials (e.g. +60 mV), Ca2+ was also released from the SR and F(SR) had a similar time course to that on depolarization. 10. The unidirectional efflux of Ca2+ from the SR, symbolized F(SR,rel) was calculated utilizing assumed characteristics of the Ca2+ pump of the SR. The value of F(SR,rel) was not affected by repolarization from voltage-clamp pulses greater than 150 ms in duration. 11. The peak amplitude of F(SR) elicited by depolarization was dependent on the pattern of prior stimulation. Peak F(SR) during a rested-state contraction was less than 10% of that during maximal contractions. 12. F(I)(Ca)), the net rate of movement of Ca2+ per litre of accessible cytoplasm across Ca2+ channels in the surface membrane, was 7% of F(SR) during the largest [Ca2+]i transients observed.
