SR calcium depletion following reversal of the Na+/Ca2+-exchanger in rat ventricular myocytes

We previously reported that cytosolic calcium transiently increases after reversal of the sarcolemmal Na+/Ca2+ exchanger. Calcium released from sarcoplasmic reticulum (SR) constituted the major part of this cytosolic transient. The aim of this study was to test whether reversal of the Na+/Ca2+-exchanger affects SR calcium content, and whether altered SR calcium content is associated with direct triggering of SR calcium release or calcium release secondary to SR calcium overload. To this purpose we studied the change of SR calcium content after reversal of the Na+/Ca2+-exchanger and the dependence on the magnitude of change of its free energy (Delta G(exch)) in isolated rat ventricular myocytes. The Na+/Ca2+-exchanger was reversed by abrupt reduction of extracellular sodium ([Na+](o)). The magnitude of change of Delta G(exch) was varied with [Na+](o). Cytosolic free calcium ([Ca2+](i)) was measured with indo-1 and SR calcium content was estimated from the increase of [Ca2+](i) after rapid cooling (RC). SR function was manipulated either by blockade of the SR Ca2+-ATPase with thapsigargin or by blockade of SR calcium release channels with tetracaine. Reversal of the Na+/Ca2+-exchanger caused a transient increase of [Ca2+](i) of about: 180 s duration with a time to peak of about 30 s. During the first 30 s rapid small amplitude cytosolic calcium fluctuations were superimposed on this transient. The magnitude of the response of [Ca2+](i) to RC, during the course of the cytosolic [Ca2+](i) transient, also transiently increased from 174 in control myocytes to 480 nmol/l at the time of the peak value. After correction of [Ca2+](i) data for the fraction of mitochondrially compartmentalized indo-1 and mitochondrial calcium, total calcium released from SR after RC was calculated with the use of literature data on cytosolic calcium buffer capacity. Contrary to the measured RC-dependent increase of measured [Ca2+](i), after reversal of the Na+/Ca2+-exchanger, calculated total calcium released from SR transiently decreased. The extent of SR calcium depletion after reversal of the Na+/Ca2+-exchanger increased with the magnitude of change of Delta G(exch). Restitution of [Na+](o) 30 s after reversal of the Na+/Ca2+-exchanger, greatly accelerated both recovery of [Ca2+](i) and SR calcium content. Pretreatment of myocytes with thapsigargin caused almost entire depletion of SR and substantial reduction of the cytosolic transient of [Ca2+](i) following reversal of the Na+/Ca2+-exchanger. Application of tetracaine hardly affected SR calcium content, but caused an increase of the SR calcium content following reversal of the Na+/Ca2+-exchanger, while the cytosolic transient increase of [Ca2+](i) was substantially reduced. We conclude that reversal of the Na+/Ca2+-exchanger directly triggers SR calcium release and decreases SR calcium content in a Delta G(exch) dependent manner. (C) 2000 Academic Press.