Measurement of SR Ca2+ content in the presence of caffeine in permeabilised rat cardiac trabeculae

This study was designed to measure the Ca2+ content of rat cardiac sarcopla smic reticulum (SR) after equilibration with normal diastolic levels of Ca2 + (100 nM), in the absence and presence of caffeine. Measurements of [Ca2+] based on Fura-2 fluorescence were made from a limited bath volume (230 nl) containing individual saponin-permeabilised rat cardiac trabeculae. Inject ion of caffeine (5-40 mM) into this volume caused an initial release of Ca2 + from the SR, but within 30 s the SR was able to re-accumulate a significa nt proportion of the Ca2+. Ca2+ re-accumulation into the SR could be pre ve nted by removal of ATP to inhibit the SR Ca2+ pump. Incubation of the prepa ration in an ATP-containing solution containing caffeine (5-40 mM) and 100 nM Ca2+ indicated that the SR's ability to retain Ca2+ depends inversely on the dose of caffeine. The relative Ca2+ content of the SR after preincubat ion with caffeine was 86.7+/-3.5% at a caffeine concentration of 5 mM, 62.5 +/-5.1% at 10 mM caffeine, 37.8+/-8.1% at 20 mM caffeine and 7.1+/-1.9% at 40 mM caffeine. Measurement of the SR Ca2+ release in the presence of diffe rent BAPTA concentrations was used to calculate (1) the Ca2+-binding capaci ty of the preparation (equivalent to 245+/-10 mu M BAPTA) and (2) the Ca2content of the SR accessed by caffeine after equilibration with 100 nM Ca2 (186+/-11 mu mol/l cell vol ume or 5.6 mmol/l SR volume).