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J. Gen. Physiol.,
Volume 111, Number 4, April 1, 1998 491-504
From the Department of Physiology, Loyola University Chicago, Stritch School of Medicine, Maywood, Illinois 60153
In steady state, the Ca content of the sarcoplasmic reticulum (SR) of cardiac myocytes is determined
by a balance among influx and efflux pathways. The SR Ca content may be limited mainly by the ATP-supplied
chemical potential that is inherent in the gradient between SR and cytosol. That is, forward Ca pumping from cytosol to SR may be opposed by energetically conservative reverse pumping dependent on intra-SR free [Ca]. On
the other hand, SR Ca loading may be limited by dissipative pathways (pump slippage and/or pump-independent leak). To assess how SR Ca content is limited, we loaded voltage-clamped ferret ventricular myocytes cumulatively
with known amounts of Ca via L-type Ca channels (ICa), using Na-free solutions to prevent Na/Ca exchange. We
then measured the maximal resulting caffeine-released SR Ca content under control conditions, as well as when
SR Ca pumping was accelerated by isoproterenol (1 µM) or slowed by thapsigargin (0.2-0.4 µM). Under control
conditions, SR Ca content reached a limit of 137 µmol·liter cytosol
1 (nonmitochondrial volume) when measured
by integrating caffeine-induced Na/Ca exchange currents (
INaCaXdt) and of 119 µmol·liter cytosol1 when measured using fluorescence signals dependent on changes in cytosolic free Ca ([Ca]i). When Ca-ATPase pumping rate
was slowed 39% by thapsigargin, the maximal SR Ca content decreased by 5 (INaCaXdt method) or 23% (fluorescence method); when pumping rate was increased 74% by isoproterenol, SR Ca content increased by 10% (fluorescence method) or 20% (INaCaXdt method). The relative stability of the SR Ca load suggests that dissipative
losses 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] gradient approaching the thermodynamic limit.
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