A23187 releases bound rather than free calcium from calcium-loaded liposomes

We have investigated sonication-, osmotic shock-, and A23187-induced calcium and horseradish peroxidase release from phosphatidylcholine liposomes. We report that sonication and osmotic shock both cause release of liposome-entrapped horseradish peroxidase activity, thus, completely releasing the entrapped volume into and mixing it with the external medium. However, we find that neither sonication nor osmotic shock releases significant amounts of calcium from liposomes pre-loaded with calcium, while A23187 causes release of significant amounts of calcium from similarly prepared liposomes. We also find that A23187 can release calcium from liposomes after release of entrapment volume by sonication or osmotic shock. Alteration of the net charge of liposomes by substitution of phosphatidylcholine with phosphatidylserine or stearylamine dramatically changes the amount of calcium associated with calcium-loaded liposomes; sonication or addition of A23187 to liposomes containing significant amounts of PS does not appear to release calcium bound to these vesicles. These results suggest that a large fraction (> 99%) of the calcium associated with liposomes is bound to the membranes of the liposomes and that the calcium released by A23187 is due to release of bound rather than entrapped calcium.