Ca uptake by heart cells: I. Ca uptake by the sarcoplasmic reticulum of intact heart cells in suspension.

Electric field stimulation of adult rat heart cells suspended in medium with 0.2 mM Ca and isoproterenol caused 45Ca uptake at a rate (5.25 pmol/mg/beat) proportional to stimulation frequency. Uptake was strongly inhibited by verapamil or thapsigargin. 45Ca autoradiography showed that stimulation dependent verapamil sensitive uptake was associated with the rod shaped cells, while the uptake by round cells was unaffected by stimulation and was verapamil-insensitive. 45Ca efflux measurements revealed a caffeine-sensitive component of uptake which was abolished by thapsigargin, and a caffeine-insensitive component. Part of the latter was sensitive to thapsigargin but not to 30 s of stimulation; another part was sensitive to such stimulation but not to thapsigargin. With longer times of stimulation, the caffeine-insensitive pool increased in size, part of which appeared to be mitochondrial Ca uptake via a thapsigargin-sensitive pool. The caffeine-sensitive pool labelled quickly in stimulated cells and its size and rate of labelling was increased by stimulation frequency (3.87 pmol/mg/beat), while the caffeine-insensitive pool labelled more slowly and was relatively insensitive to stimulation (0.77 pmol/mg/beat). We conclude that essentially all of the SR Ca pool, as defined by its involvement in excitation-contraction coupling, is released by caffeine.

Ca uptake by heart cells: II. Most entering Ca appears to leave without mixing with the sarcoplasmic reticulum Ca pool.

The rate of verapamil-sensitive uptake of 45Ca by rat heart cells stimulated to beat in suspension with 0.2 mM Ca and isoproterenol was increased > 2-fold by cell loading with the chelator Quin-2. No effect of Quin-2 loading was observed on the rate of uptake of trace levels of 54Mn, present in addition to Ca, which was used as an index of Ca channel activity. Quin-2 loading also had little effect on the rate of 45Ca uptake by cells diluted into a high K/low Na medium, where Ca uptake was primarily by Na/Ca exchange. The fast chelator 1,2-bis(o-aminophenoxy)ethane-N,N,-N',N'-tetraacetic acid (BAPTA) was 3-fold more effective than the slow chelator EGTA at preventing Ca efflux. BAPTA loading also caused an increase in sarcoplasmic reticulum (SR) Ca content. These results suggest that chelator loading had little effect on the rate of Ca influx by Ca channels or by Na/Ca exchange, and that the increased rate of 45Ca uptake seen with Quin-2 loading was caused by an inhibition of Ca efflux, either directly by chelation or by increased Ca uptake by the SR or by other intracellular organelles. This further suggests that most of the Ca entering the cell without chelator leaves again within the same beat, and that this may result from Ca efflux from a kinetically limited Ca pool in or around the diad cleft.

Effect of ATP depletion on kinetics of Na/Ca exchange-mediated Ca influx in Na-loaded heart cells.

The kinetics of Ca influx by Na/Ca exchange into adult rat heart cells loaded with Na and depleted of ATP were investigated, to further elucidate how ATP regulates exchanger properties in the intact heart cell. We found an eight-fold reduction in Vmax for Ca uptake by ATP depletion, with no significant change in the K(m) for extracellular Ca or the K1 for inhibition by extracellular Na. Autoradiography of ATP depleted cells after45Ca uptake showed no gross heterogeneity of Ca uptake, as would occur if a small fraction of cells still retained some ATP. We conclude that the reduction in Vmax is a property of all of the ATP depleted cells. These kinetics are consistent with regulation of the putative calmodulin binding site on the exchanger. They are also consistent with sequestration of the exchanger at the intracellular face of the sarcolemma by intracellular Na dependent inactivation, a property of the exchanger observed in membrane patches to be relieved by ATP. However, no effect of heptalysine was observed on exchange activity in cells with ATP, nor was the restoration of exchange activity to ATP depleted cells by ATP resynthesis blocked by heptalysine. This suggests that in intact cells the activation of exchange activity by ATP is not caused by phosphatidylserine translocation to the cytosolic leaflet of the sarcolemma. Also, no effect of cytochalasin D was observed on exchanger activity or kinetics, in contrast with observations in Chinese hamster ovary (CHO) cells transfected with bovine cardiac Na/Ca exchanger by ATP, and these are differently expressed depending on the exchanger environment.