Intravesicular Calcium Release Mediates the Motion and Exocytosis of Secretory Organelles: A STUDY WITH ADRENAL CHROMAFFIN CELLS

Marcial Camacho,José D Machado,Javier Alvarez,Ricardo Borges

doi:10.1074/jbc.m800552200

Abstract

Secretory vesicles of sympathetic neurons and chromaffin granules maintain a pH gradient toward the cytosol (pH 5.5 versus 7.2) promoted by the V-ATPase activity. This gradient of pH is also responsible for the accumulation of amines and Ca2+ because their transporters use H+ as the counter ion. We have recently shown that alkalinization of secretory vesicles slowed down exocytosis, whereas acidification caused the opposite effect. In this paper, we measure the alkalinization of vesicular pH, caused by the V-ATPase inhibitor bafilomycin A1, by total internal reflection fluorescence microscopy in cells overexpressing the enhanced green fluorescent protein-labeled synaptobrevin (VAMP2-EGFP) protein. The disruption of the vesicular gradient of pH caused the leak of Ca2+, measured with fura-2. Fluorimetric measurements, using the dye Oregon green BAPTA-2, showed that bafilomycin directly released Ca2+ from freshly isolated vesicles. The Ca2+ released from vesicles to the cytosol 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 cytosolic Ca2+ and in two of the major functions of secretory cells, vesicle motion and exocytosis.

Highlights

Secretory granules from chromaffin cells are large dense core vesicles similar to vesicles present in many other neuroendocrine cells and in sympathetic neurons [1]
We have found that bafilomycin A1-mediated alkalinization causes the release of Ca2ϩ from granules to cytosol, and this effect is able to increase the lateral motion of chromaffin granules and to trigger exocytosis, even in Ca2ϩ-free medium
Even considering that secretory vesicles contain large amounts of Ca2ϩ [15] and that this cation is crucial for processes that take place “just across their membrane,” like vesicle movement or exocytosis, they have received little attention in terms of Ca2ϩ homeostasis

Summary

Introduction

Secretory granules from chromaffin cells are large dense core vesicles similar to vesicles present in many other neuroendocrine cells and in sympathetic neurons [1]. We have found that bafilomycin A1-mediated alkalinization causes the release of Ca2ϩ from granules to cytosol, and this effect is able to increase the lateral motion of chromaffin granules and to trigger exocytosis, even in Ca2ϩ-free medium.

Results

Conclusion