Intravesicular calcium release mediates the motion and exocytosis of secretory organelles: a study with adrenal chromaffin cells.
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.
Unidad de Farmacología, Facultad de Medicina, Universidad de La Laguna Tenerife, E-38071 La Laguna, Spain.
Marcial
M
Camacho
José D
JD
Machado
Javier
J
Alvarez
Ricardo
R
Borges
D000313:Adrenal Medulla / Q000166:cytology / Q000502:physiology*; D000818:Animals; D002118:Calcium / Q000502:physiology*; D002417:Cattle; D002465:Cell Movement / Q000187:drug effects / Q000502:physiology*; D019439:Chromaffin Cells / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D003594:Cytoplasmic Granules / Q000187:drug effects / Q000502:physiology / Q000648:ultrastructure; D003600:Cytosol / Q000187:drug effects / Q000502:physiology; D006801:Humans; D018942:Macrolides / Q000494:pharmacology; D016716:PC12 Cells; D051381:Rats; D014162:Transfection
D018942:Macrolides; C040929:bafilomycin A1; D002118:Calcium