MEASUREMENT OF SARCOPLASMIC-RETICULUM CA2+ CONTENT AND SARCOLEMMAL CA2+ FLUXES IN ISOLATED RAT VENTRICULAR MYOCYTES DURING SPONTANEOUS CA2+RELEASE

1. Intracellular calcium concentration ([Ca2+](i)) and Na+-Ca2+ exchan ge currents were measured in calcium-overloaded voltage-clamped rat ve ntricular myocytes loaded with the Ca-2+-sensitive fluorescent indicat or indo-1. Sarcoplasmic reticulum (SR) Ca2+ content was measured from the integral of the caffeine-envoked current. In cells that had sponta neous SR Ca2+ release in 1 mM external Ca2+ concentration ([Ca2+](0)), raising [Ca2+](0) increased the frequency of release with no effect o n SR Ca2+ content. In quiescent cells, increased [Ca2+](0) produced sp ontaneous Ca2+ release associated with increased SR Ca2+ content. Furt her increase of [Ca2+](0) had no effect on SR Ca2+ content. The amount of Ca2+ leaving the cell during each release was constant over a wide range of frequencies and [Ca2+](0) values. It appears there is a maxi mum level of SR Ca2+ content, perhaps because spontaneous Ca2+ release results when the content reaches a threshold. 2. From the relationshi p between [Ca2+](i) and Na+-Ca2+ exchange current during a caffeine re sponse, it is possible to estimate the changes in Na+-Ca2+ exchange cu rrent expected from a change of [Ca2+](i). The data show that the calc ium oscillations contribute a significant fraction of the total extra Ca2+ efflux induced by Increasing [Ca2+](0). Raising [Ca2+](0) decreas ed the rate of calcium removal from the cell as measured from the rate of decay of the caffeine response, suggesting that both inhibition of Ca2+ efflux and increased Ca2+ entry account for the Ca2+ overload at elevated [Ca2+](0). 3. Inhibiting spontaneous SR Ca2+ release increas es resting [Ca2+](i). The Ca2+ efflux is identical to that in the pres ence of release. It is concluded that spontaneous release of calcium, although potentially arrhythmogenic, is an effective way to activate C a2+ efflux in overloaded conditions and minimizes any increase of dias tolic tension.