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.
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 microM [Ca2+]i, uptake of 45Ca2+ was largely confined (> 80%) to the inositol 1,4,5-trisphosphate-sensitive pool, but at 2 microM [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+.
Ali
H
H
Laboratory of Chemical Pharmacology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892-1760.
Maeyama
K
K
Sagi-Eisenberg
R
R
Beaven
M A
MA
D000818:Animals; D000941:Antigens / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D002132:Calcium Radioisotopes; D002412:Cations; D016257:Fura-2; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology*; D007700:Kinetics; D008407:Mast Cells / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D011188:Potassium / Q000494:pharmacology; D051381:Rats; D013050:Spectrometry, Fluorescence; D013729:Terpenes / Q000494:pharmacology*; D019284:Thapsigargin; D014407:Tumor Cells, Cultured
D000941:Antigens; D002132:Calcium Radioisotopes; D002412:Cations; D013729:Terpenes; D019284:Thapsigargin; D015544:Inositol 1,4,5-Trisphosphate; D011188:Potassium; D002118:Calcium; D016257:Fura-2