Localization of calcium changes in stimulated rat mast cells

We studied intracellular free, bound, and sequestered calcium in rat mast cells after various stimulations. The use of a fluorescent probe combined with digitized imaging on individual living cells demonstrated transient increases of free Ca2+ in the micromolar range. The use of histochemical techniques (K pyroantimonate and anhydrous fixation), together with X-ray microanalysis, energy electron-loss spectroscopy, and electron spectroscopic imaging, revealed large amounts of stored calcium within the cells (in the millimolar range). Chelation experiments and stimulations enabled us to identify at least two pools of bound calcium which exhibited different dynamic behaviors. Stimulation in the presence of EGTA did not modify calcium from granules, granule membranes, and heterochromatin, whereas it decreased calcium from other cell compartments. Stimulation triggered variations in the amount of bound calcium but they did not parallel free calcium movements. Hence, whereas free calcium is implicated in exocytosis, bound calcium may be involved in altogether different cell functions.