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

H. Ali, K. Maeyama, R. Sagi-Eisenberg, M. A. Beaven

Research output: Contribution to journalArticlepeer-review

Abstract

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+](i)). 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+](i), uptake of 45Ca2+ was largely confined (> 80%) to the inositol 1,4,5-trisphosphate-sensitive pool, but at 2 μM [Ca2+](i) 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+](i) 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+](i) and 45Ca2+ uptake for a full accounting of Ca2+ homoeostasis as exemplified by the ability to distinguish between influx and mitochondrial uptake of Ca2+.

Original languageEnglish
Pages (from-to)431-440
Number of pages10
JournalBiochemical Journal
Volume304
Issue number2
DOIs
StatePublished - 1994
Externally publishedYes

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