Ca exchange under non-perfusion-limited conditions in rat ventricular cells: identification of subcellular...

Freshly prepared ventricular myocytes from rat hearts, aliquots of which were tested for sarcolemmal integrity by La exposure, were labeled at high 45Ca specific activity. Isotope was subsequently washed out at a perfusion rate of 2.8 ml/s with washout solution sampled each 1 s. No initial unrecorded period of washout was imposed. Four compartments were distinguishable: 1) a "rapid" compartment (RC) containing 2.6 mmol Ca/kg dry wt of La-displaceable Ca, half time (t1/2) less than 1 s; 2) an "intermediate" compartment(s) (IC) containing 2.1 mmol, t1/2 = 3 and 19 s; 3) a "slow" compartment (SC) containing 1.6 mmol, t1/2 = 3.6 min; 4) an "inexchangeable" compartment that demonstrated no 45Ca uptake after 60-min labeling containing 1.2 mmol. Introduction of 10 mM caffeine as a probe for sarcoplasmic reticulum (SR) content at various times during the washouts caused an increased release of 45Ca. The net increased 45Ca release plotted as a function of time at which caffeine was introduced produced a biexponential curve with t1/2s of 2 and 22 s, very similar to the t1/2s of the IC. Ryanodine (1 microM) significantly reduced the caffeine-induced 45Ca release, confirming the SR locus of the IC. Cells were perfused with 10 mM NaH2PO4 to specifically increase mitochondrial 45Ca labeling. Subsequent removal of PO4 at various times during washouts produced large increases in effluent 45Ca. A plot of the net peak release of 45Ca vs. time of PO4 removal was monoexponential with t1/2 = 3.3 min, very similar to the SC t1/2. The large La-accessible RC remains unlocalized, but the rapidity of its exchange places it in the sarcolemma and/or at sites in rapid equilibrium with the sarcolemma.