CONTROL OF MAXIMUM SARCOPLASMIC-RETICULUM CA LOAD IN INTACT FERRET VENTRICULAR MYOCYTES - EFFECTS OF THAPSIGARGIN AND ISOPROTERENOL

In steady state, the Ca content of the sarcoplasmic reticulum (SR) of cardiac myocytes is determined by a balance among influx and efflux pa thways. The SR Ca content may be limited mainly by the ATP-supplied ch emical potential that is inherent in the gradient between SR and cytos ol. That is, forward Ca pumping from cytosol to SR may be opposed by e nergetically conservative reverse pumping dependent on intra-SR free [ Ca]. On the other hand, SR Ca loading may be limited by dissipative pa thways (pump slippage and/or pump-independent leak). To assess how SR Ca content is limited, we loaded voltage-clamped ferret ventricular my ocytes cumulatively with known amounts of Ca via L-type Ca channels (I -Ca), using Na-free solutions to prevent Na/Ca exchange. We then measu red the maximal resulting caffeine-released SR Ca content under contro l conditions, as well as when SR Ca pumping was accelerated by isoprot erenol (1 mu M) or slowed by thapsigargin (0.2-0.4 mu M) Under control conditions, SR Ca content reached a limit of 137 mu mol . liter cytos ol(-1) (nonmitochondrial volume) when measured by integrating caffeine -induced Na/Ca exchange currents (integral I(NaCaX)dt) and of 119 mu m ol . liter cytosol(-1) when measured using fluorescence signals depend ent on changes in cytosolic free Ca ([Ca](i)). When Ca-ATPase pumping rate was slowed 39% by thapsigargin, the maximal SR Ca content decreas ed by 5 (integral I(NaCaX)dt method) or 23% (fluorescence method); whe n pumping rate was increased 74% by isoproterenol, SR Ca content incre ased by 10% (fluorescence method) or 20% (integral I(NaCaX)dt method). The relative stability of the SR Ca load suggests that dissipative lo sses have only a minor influence in setting the SR Ca content. Indeed, it appears that the SR Ca pump in intact cells can generate a [Ca] gr adient approaching the thermodynamic limit.