A23187 RELEASES BOUND RATHER THAN FREE CALCIUM FROM CALCIUM-LOADED LIPOSOMES

We have investigated sonication-, osmotic shock-, and A23187-induced c alcium and horseradish peroxidase release from phosphatidylcholine lip osomes, We report that sonication and osmotic shock both cause release of liposome-entrapped horseradish peroxidase activity, thus, complete ly releasing the entrapped volume into and mixing it with the external medium. However, we find that neither sonication nor osmotic shock re leases significant amounts of calcium from liposomes pre-loaded with c alcium, while A23187 causes release of significant amounts of calcium from similarly prepared liposomes. We also find that A23187 can releas e calcium from liposomes after release of entrapment volume by sonicat ion or osmotic shock. Alteration of the net charge of liposomes by sub stitution of phosphatidylcholine with phosphatidylserine or stearylami ne dramatically changes the amount of calcium associated with calcium- loaded liposomes; sonication or addition of A23187 to liposomes contai ning significant amounts of PS does not appear to release calcium boun d to these vesicles. These results suggest that a large fraction (>99% ) of the calcium associated with liposomes is bound to the membranes o f the liposomes and that the calcium released by A23187 is due to rele ase of bound rather than entrapped calcium. (C) 1994 Academic Press, I nc.