ANTIGEN AND THAPSIGARGIN PROMOTE INFLUX OF CA2-2H3 CELLS BY OSTENSIBLY SIMILAR MECHANISMS THAT ALLOW FILLING OF INOSITOL 1,4,5-TRISPHOSPHATE-SENSITIVE AND MITOCHONDRIAL CA2+ STORES( IN RAT BASOPHILIC RBL)

In single, Fura 2-loaded RBL-2H3 cells, antigen and thapsigargin deple ted the same intracellular pool of Ca2+ in the absence of external Ca2 +, provision of external Ca2+ induced immediate increases in levels of free Ca2+ ([Ca2+](i)). These increases were dependent on the presence of external Ca2+ and, presumably, on influx of Ca2+ across the cell m embrane. Both stimulants enhanced intracellular accumulation of Ca-45( 2+) through ostensibly similar mechanisms because accumulation was blo cked to similar extents by various multivalent cations or by depolariz ation with K+. Because thapsigargin blocked reuptake of Ca2+ into inos itol 1,4,5-trisphosphate sensitive stores, uptake occurred independent ly of the refilling of these stores. Uptake was dependent instead on s equestration of Ca-45(2+) in a pool of high capacity that was insensit ive to thapsigargin, caffeine, GTP and inositol 1,4,5-trisphosphate bu t sensitive to ionomycin and mitochondrial inhibitors. The existence o f an inositol 1,4,5-trisphosphate-insensitive pool was also apparent i n permeabilized cells; at 0.1 mu M [Ca2+](i), uptake of Ca-45(2+) was largely confined (> 80 %) to the inositol 1,4,5-trisphosphate-sensitiv e pool, but at 2 mu M [Ca2+](i) uptake was largely (> 60 %) into the i nositol 1,4,5-trisphosphate-insensitive pool. Provision of mitochondri al inhibitors along with thapsigargin to block uptake into both pools, did not impair the thapsigargin-induced increase in [Ca2+](i) or infl ux of Ca2+, as indicated by changes in Fura 2 fluorescence, but did bl ock the intracellular accumulation of Ca-45(2+). The studies illustrat e the utility of simultaneous measurements of [Ca2+](i) and Ca-45(2+) uptake for a full accounting of Ca2+ homoeostasis as exemplified by th e ability to distinguish between influx and mitochondrial uptake of Ca 2+.