On the role of intravesicular calcium in the motion and exocytosis of secretory organelles

Abstract

Secretory vesicles of sympathetic neurons and chromaffin granules maintain a pH gradient towards the cytosol (5.5 vs. 7.2) promoted by the V-ATPase activity. This gradient of pH is mainly responsible for the accumulation of amines. The secretory vesicles contain large amounts of total Ca2+, but the free intragranular [Ca2+], the mechanisms for Ca2+ uptake and release from the granules and their physiological relevance regarding exocytosis are still matters of debate.We have recently shown that disruption of the pH gradient of secretory vesicles slowed down exocytosis. Fluorimetric measurements, using the dye Oregon green BAPTA-2, showed that the V-ATPase inhibitor bafilomycin A1 directly released Ca2+ from freshly isolated vesicles. Accordingly, vesicle alkalinization released Ca2+ from the granules to the cytosol, measured with fura-2 in intact chromaffin cells. Using TIRFM in cells over-expressing the EGFP-labeled synaptobrevin (VAMP2-EGFP) protein, we have then shown that the Ca2+ released from the vesicles to the cytosol in the presence of bafilomycin, dramatically increased the granule motion of chromaffin- or PC12-derived granules, and triggered exocytosis (measured by amperometry).We conclude that the gradient of pH of secretory vesicles might be involved in the homeostatic regulation of the local cytosolic Ca2+ around the vesicles and in two of the major functions of secretory cells, vesicle motion and exocytosis.1