Biochimica et Biophysica Acta 818 (1985) 373-385 373 Elsevier BBA 72714 Calcium oxalate and calcium phosphate capacities of cardiac sarcoplasmic reticulum Joseph J. Feher and Grayson B. Lipford Department of Physiology and Biophysics, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA 23298 (U.S.A.) (Received May 21st, 1985) Key words: Ca 2 + transport; Sarcoplasmic reticulum; Ryanodine; (Canine heart) Both oxalate-supported and phosphate-supported calcium uptake by canine cardiac sarcoplasmic reticulum initially increase linearly with time but fall to a steady-state level within 20 min. The departure from linearity could be due to a decrease in influx or to an increase in efflux of calcium. Because Ca2+-ATPase activity is linear, a decrease in the influx of calcium is an unlikely cause of the non-linear calcium uptake curves. A possible cause of an increase in calcium efflux is rupture of the vesicles. This hypothesis was tested by investigating the amount of calcium which could be released upon addition of 5 mM EGTA. The amount of rapidly releasable calcium was zero until a threshold calcium uptake of about 4-6 pmol calcium oxalate or calcium phosphate per mg was reached. After that point the rapidly releasable calcium continued to increase with calcium oxalate to reach more than 23 #mol/mg, but stayed constant at about 0.7 p mol/mg for calcium phosphate. The rapidly releasable calcium was attributed to calcium oxalate or calcium phosphate crystals externalized by vesicle rupture. The differences in the amounts of rapidly releasable calcium were attributed to different kinetics of calcium phosphate and calcium oxalate dissolution. Addition of ryanodine caused a marked increase in the threshold for rapidly releasable calcium oxalate. Transmission electron micrographs showed that vesicles can become filled with calcium oxalate crystals, but the vesicles were heterogeneous with respect to their size and their sensitivity to ryanodine. These observations support the hypothesis that calcium oxalate and calcium phosphate capacities are limited by vesicle rupture and that ryanodine increases the capacity by closing a calcium channel in a subpopulation of vesicles that otherwise would not accumulate calcium. Introduction Calcium-precipitating anions have long been used in studies of calcium transport by sarcop- lasmic reticulum in order to augment the amount of calcium accumulated [1,2]. One current hy- pothesis for this augmentation of calcium uptake is that the oxalate enters the vesicle after being protonated by the CaE+-ATPase [3] and that the Abbreviation: EGTA, ethylene glycol bis(fl-aminoethyl ether)- N, N'-tetraacetic acid. transported calcium increases in concentration un- til calcium oxalate precipitation occurs [4]. Since the early addition of ionophore X537A or A23187 completely release the accumulated calcium [3,5], apparently it is not immediately precipitated as calcium oxalate. After further incubation, calcium oxalate crystallization begins, and these early crystals seed crystallization in the intravesicular space [5,20]. During the linear phase of calcium uptake, the Ca2+-ATPase pumps calcium inward against a calcium gradient and the transported calcium either precipitates as calcium oxalate or 0005-2736/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)