Direct measurement of free Ca in organelles of gastric epithelial cells

When loaded as the acetoxymethyl ester (AM) derivative, the fluorescent probe mag-fura 2 accumulates in both the cytoplasm and the subcellular compartments. The relatively high dissociation constant of this dye for Ca (53 microM) permits the measurement of changes in the free concentration of this ion in a variety of organelles where Ca concentration ([Ca]) is high. To characterize Ca stores in gastric cells, we used digitonin to permeabilize cells within isolated rabbit gastric glands loaded with mag-fura 2-AM. This allowed cytosolic dye to leak out, leaving only compartmentalized dye behind. It appears that mag-fura 2 monitors [Ca] changes in several ATP-dependent Ca-sequestering pools; an inositol 1,4,5-trisphosphate (IP3)-releasable and thapsigargin-sensitive store (which probably includes the endoplasmic reticulum), a pool that is released by the mitochondrial inhibitors oligomycin+azide, valinomycin, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone, and ruthenium red (and therefore likely represents mitochondria), and a residual pool that was resistant to release by mitochondrial inhibitors and thapsigargin. Ca sequestration into all pools was sensitive to changes in [ATP], indicating that treatments that reduce cellular [ATP] will cause certain organelles to lose their Ca to the cytoplasm. Caffeine and ryanodine, which mobilize Ca from internal stores in many cell types, induced Ca sequestration into an IP3-insensitive store of gastric cells, and caffeine caused a reduction in cytoplasmic [Ca] (as measured with fura 2). We also show that the quantitation of free [Ca] in a given pool is complicated by a nonlinearity in the relationship between the mag-fura 2 ratio and [Ca]. This effect is likely a consequence of monitoring the fluorescence from multiple pools simultaneously. However, this limitation does not detract from the ability of this method to yield important qualitative information about the nature and number of Ca stores within single gastric cells.