Effects of adenosine diphosphate on Ca2+ fluxes and Ca2+ accumulation of sarcoplasmic reticulum

Ca2+ transport by sarcoplasmic reticulum vesicles was examined by incubating sarcoplasmic reticulum vesicles (0.15 mg/ml) at 37 degrees C in, either normal medium that contained 0.15 M sucrose, 0.1 M KCl, 60 microM CaCl2, 2.5 mM ATP and 30 mM Tes at pH 6.8, or a modified medium for elimination of ADP formed from ATP hydrolysis by including, in addition, 3.6 mM phosphocreatine and 33 U/ml of creatine phosphokinase. In normal medium, Ca2+ uptake of sarcoplasmic reticulum vesicles reached a plateau of about 100 nmol/mg. In modified medium, after this phase of Ca2+ uptake, a second phase of Ca2+ accumulation was initiated and reached a plateau of about 300 nmol/mg. The second phase of Ca2+ accumulation was accompanied by phosphate uptake and could be inhibited by ADP. Since, under these experimental conditions, there was no significant difference of the rates of ATP hydrolysis in normal medium and modified medium, extra Ca2+ uptake in modified medium but not in normal medium could not be explained by different phosphate accumulation in the two media. Unidirectional Ca2+ influx of sarcoplasmic reticulum near steady state of Ca2+ uptake was measured by pulse labeling with 45Ca2+. The Ca2+ efflux rate was then determined by subtracting the net uptake from the influx rate. At the first plateau of Ca2+ uptake in normal medium, Ca2+ influx was balanced by Ca2+ efflux with an exchange rate of 240 nmol/mg per min. This exchange rate was maintained relatively constant at the plateau phase. In modified medium, the Ca2+ exchange rate at the first plateau of Ca2+ uptake was about half of that in normal medium. When the second phase of Ca2+ uptake was initiated, both the influx and efflux rates started to increase and reached a similar exchange rate as observed in normal medium. Also, during the second phase of Ca2+ uptake, the difference between the influx and efflux rates continued to increase until the second plateau phase was approached. In conditions where the formation of ADP and inorganic phosphate was minimized by using a low concentration of sarcoplasmic (7.5 micrograms/ml) and/or using acetyl phosphate instead of ATP, the second phase of Ca2+ uptake was also observed. These data suggest that the Ca2+ load attained by sarcoplasmic reticulum vesicles during active transport is modulated by ADP accumulated from ATP hydrolysis. ADP probably exerts its effect by facilitating Ca2+ efflux, which subsequently stimulates Ca2+ exchange.