Antigen and Thapsigargin Promote Influx of Ca2+ in Rat Basophilic RBL-2H3 Cells by Ostensibly Similar Mechanisms That Allow Filling of Inositol 1,4,5-Trisphosphate-Sensitive and Mitochondrial Ca2+ Stores

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Departmental Papers (Dental)
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Dentistry
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Ali, H.
Maeyama, K.
Sagi-Eisenberg, R.
Beaven, M. A.
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In single, Fura 2-loaded RBL-2H3 cells, antigen and thapsigargin depleted 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+). These increases were dependent on the presence of external Ca2+ and, presumably, on influx of Ca2+ across the cell membrane. Both stimulants enhanced intracellular accumulation of 45Ca2+ through ostensibly similar mechanisms because accumulation was blocked to similar extents by various multivalent cations or by depolarization with K+. Because thapsigargin blocked reuptake of Ca2+ into inositol 1,4,5-trisphosphate sensitive stores, uptake occurred independently of the refilling of these stores. Uptake was dependent instead on sequestration of 45Ca2+ in a pool of high capacity that was insensitive to thapsigargin, caffeine, GTP and inositol 1,4,5-trisphosphate but sensitive to ionomycin and mitochondrial inhibitors. The existence of an inositol 1,4,5-trisphosphate-insensitive pool was also apparent in permeabilized cells; at 0.1 μM Ca2+, uptake of 45Ca2+ was largely confined (> 80%) to the inositol 1,4,5-trisphosphate-sensitive pool, but at 2 μM Ca2+ uptake was largely (> 60%) into the inositol 1,4,5-trisphosphate-insensitive pool. Provision of mitochondrial inhibitors along with thapsigargin to block uptake into both pools, did not impair the thapsigargin-induced increase in Ca2+ or influx of Ca2+, as indicated by changes in Fura 2 fluorescence, but did block the intracellular accumulation of 45Ca2+. The studies illustrate the utility of simultaneous measurements of Ca2+ and 45Ca2+ uptake for a full accounting of Ca2+ homoeostasis as exemplified by the ability to distinguish between influx and mitochondrial uptake of Ca2+.

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1994
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Biochemical Journal
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