IMAGING OF TOTAL INTRACELLULAR CALCIUM AND CALCIUM INFLUX AND EFFLUX IN INDIVIDUAL RESTING AND STIMULATED TUMOR MAST-CELLS USING ION MICROSCOPY

Ion microscopy was employed to investigate intracellular total calcium concentrations and calcium influx, and efflux in resting and antigen- stimulated tumor mast cells (RBL-2H3 cells). The nucleus, a perinuclea r region which included the Golgi apparatus (Golgi region), and the re maining cytoplasm were spatially resolved with the Cameca IMS-3f ion m icroscope in cryogenically prepared cells. In resting cells the nucleu s contained about 0.60 mM, the Golgi region about 1.2 mM, and the rema ining cytoplasm about 1.0 mM total calcium. Antigen stimulation of rat basophilic leukemia cells resulted in a significant loading of calciu m in all three cellular compartments. Antigen stimulation in the absen ce of extracellular calcium resulted in a significant loss of total ca lcium from all three intracellular compartments. Influx and efflux of calcium were measured simultaneously in resting and stimulated cells b y using stable Ca-44 in the extracellular solution, and by imaging mas s 40 to determine the native intracellular calcium (Ca-40) and mass 44 to localize the Ca-44 that entered the cell from extracellular soluti on. After a 10-min incubation, 0.240 fmol of the total calcium per cel l had been replaced with Ca-44, which amounts to about 33% of the tota l cell calcium. If antigen was present during this incubation there wa s an additional loss of 0.229 fmol of Ca-40 and an added gain of 0.476 fmol of Ca-44 per cell, which corresponds to a net increase in total intracellular calcium of 0.247 fmol.