Processes that remove calcium from the cytoplasm during excitation-contraction coupling in intact rat heart cells

1. The processes that remove Ca2+ rapidly from the cytoplasm were studied in isolated rat ventricular myocytes subjected to whole-cell voltage clamp and internal perfusion with the Ca2+ indicator, indo-1. Na(+)-Ca2+ exchange was eliminated in most experiments by removing Na+ both internally and externally. 2. When the Ca(2+)-pumping ATPase of the sarcoplasmic reticulum (SR) was inhibited with cyclopiazonic acid and ryanodine interfered with the release of Ca2+ from the SR, [Ca2+]i transients rose slowly and declined extremely slowly. We concluded that transport of Ca2+ by mitochondria and the surface membrane Ca(2+)-pumping ATPase would be negligible over the time course of a single [Ca2+]i transient. 3. The influence of cytoplasmic Ca2+ ligands was characterized by internal perfusion with high concentrations of diffusible Ca2+ ligands (indo-1) or by superfusion with the membrane-permeant Ca2+ ligand, BAPTA AM. As the concentration of indo-1 in the cell increased from < 0.1 mM to at least 0.5 mM, the time constant of the decline of [Ca2+]i increased from about 0.15 s to nearly 3 s. 4. Calcium bound to endogenous Ca2+ ligands during depolarizing clamp pulses was characterized quantitatively as the difference between the total Ca2+ entering the cell via L-type Ca2+ channels and [Ca2+]i, in experiments in which SR function had been abolished. As total calcium increased during the entry of Ca2+, total calcium was found to agree reasonably well with that predicted by assuming that Ca2+ could bind to endogenous intracellular Ca2+ ligands and to indo-1. 5. The results indicate that, in the absence of Na+, the major factors determining the removal of cytoplasmic free Ca2+ are the Ca(2+)-pumping ATPase of the SR and the binding of Ca2+ to endogenous and exogenous Ca2+ ligands. 6. Several hypothetical 'Ca2+ removal functions' were fitted to the declining phase of [Ca2+]i transients. The best fit was one in which the flux of Ca2+ through the SR Ca(2+)-pumping ATPase was described by a Michaelis-Menten-type equation. The decline of the [Ca2+]i transient was thus described by a linear, first-order differential equation having terms giving the rate of Ca2+ transport by the SR Ca(2+)-pumping ATPase (Vmax and KM), the rates of complexation of Ca2+ with the various Ca2+ ligands (L), and a leak of Ca2+ into the cytoplasm from the SR (FSR,leak).(ABSTRACT TRUNCATED AT 400 WORDS)