Measurement of sarcoplasmic reticulum calcium content in skinned mammalian cardiac muscle.

We developed an optical system for the measurement of the Ca2+ content of sarcoplasmic reticulum (SR) in saponin-treated ventricular muscles of ferrets. After the SR was loaded with Ca2+ by activating the Ca2+ pump of SR, caffeine (50 mM) was applied to release the accumulated Ca2+ from the SR into the bathing solution containing the fluorescent Ca2+ indicator, Fluo-3. As Fluo-3, at high concentrations (approximately 200 microM), predominantly binds most of the Ca2+ released from the SR, the Fluo-3 fluorescence change upon Ca2+ binding gave an estimate of the amount of accumulated Ca2+ in SR before caffeine application. The maximal Ca2+ content of SR, thus estimated, was about 370 mumol/l cytoplasm. The amount of Ca2+ loaded in SR showed bell-shaped dependence on the free Ca2+ concentration ([Ca2+]) of the loading solution, reflecting Ca(2+)-induced Ca2+ release at high [Ca2+] (> or = 1 microM). Mg2+ and H+ decreased the rate of Ca2+ uptake by SR. The present system provides a relatively direct means of measurement of the Ca2+ content of SR, and allows examination of the effects of various interventions on SR Ca2+ uptake, bypassing the large influence of intracellular Ca2+ buffer sites.