Temporal separation of vesicle release from vesicle fusion during exocytosis

During exocytosis, vesicles in secretory cells fuse with the cellular membrane and release their contents in a Ca2+-dependent process. Release occurs initially through a fusion pore, and its rate is limited by the dissociation of the matrix-associated contents. To determine whether this dissociation is promoted by osmotic forces, we have examined the effects of elevated osmotic pressure on release and extrusion from vesicles at mast and chromaffin cells. The identity of the molecules released and the time course of extrusion were measured with fast scan cyclic voltammetry at carbon fiber microelectrodes. In external solutions of high osmolarity, release events following entry of divalent ions (Ba2+ or Ca2+) were less frequent. However, the vesicles appeared to be fused to the membrane without extruding their contents, since the maximal observed concentrations of events were less than 7% of those evoked in isotonic media. Such an isolated, intermediate fusion state, which we term "kiss-and-hold," was confirmed by immunohistochemistry at chromaffin cells. Transient exposure of cells in the kiss and hold state to isotonic solutions evoked massive release. These results demonstrate that an osmotic gradient across the fusion pore is an important driving force for exocytotic extrusion of granule contents from secretory cells following fusion pore formation.