Evidence has shown that the sarcoplasmic reticulum (SR) of cardiac cells releases Ca not only during excitation-contraction coupling but also during diastole, albeit at a much lower rate. This diastolic SR Ca release (leak) has also been implicated in the generation of spontaneous depolarization in latent atrial pacemaker cells of the cat right atrium. In the present work, we sought to measure Ca transients in pacemaker and nonpacemaker cells of the cat using the fluorescent Ca indicator indo 1. Atrial latent pacemaker cells develop a slow Ca transient when rested in the presence of both Na- and Ca-free solution and thapsigargin [used to inhibit Na/Ca exchange and SR Ca adenosinetriphosphatase (Ca-ATPase), respectively]. This increase in cytosolic Ca concentration ([Ca]j) is probably caused by the rate of SR Ca leak exceeding the capacity of the remaining Ca transport systems (e.g., sarcolemmal Ca-ATPase and mitochondria! Ca uptake). However, neither cat sinoatrial (SA) node cells nor myocytes from cat atrium or ventricle exhibited a similar increase in [Ca]j during the same protocol. This indicates that SR Ca leak in these cells occurred at a rate low enough to be within the capacity of the slow Ca transporters, as observed previously in rabbit ventricular myocytes. When atrial and ventricular myocytes were stimulated at higher frequencies, sufficient to markedly increase diastolic and systolic [Ca]; and approach Ca overload (and spontaneous activity), they responded to inhibition of SR Ca-ATPase and Na/Ca exchange with a slow Ca transient similar to that normally observed in atrial latent pacemaker cells. Furthermore, the SR Ca depletion by thapsigargin did not affect spontaneous activity of SA node cells, but it prevented or slowed pacemaker activity in the atrial latent pacemaker cells. These findings suggest that enhanced diastolic SR Ca efflux contributes significantly to the generation of spontaneous activity in atrial subsidiary pacemakers under normal conditions and in Ca-overloaded myocytes but not in S A node cells. sinoatrial node cells; sarcoplasmic reticulum calcium release; thapsigargin; calcium transients; automaticity Copyright © 1997 the American Physiological Society.422H886H892Bassani, R.A., Bassani, J.W.M., Bers, D.M., Mitochondrial and sarcolemmal Ca 2+ transport reduce Ca2 during caffeine contractures in rabbit cardiac myocytes (1992) J. Physiol. (Lond.), 453, pp. 591-608Bassani, J.W.M., Bassani, R.A., Bers, D.M., Relaxation in rabbit and rat cardiac cells: Species-dependent differences in cellular mechanisms (1994) J. Physiol. (Lond.), 476, pp. 279-293Bassani, R.A., Bers, D.M., Na-Ca exchange is required for rest-decay but not for rest-potentiation of twitches in rabbit and rat ventricular myocytes (1994) J. Mol. Cell. 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