Regulation of calcium influx across the plasma membrane of the human T-leukemic cell line, JURKAT: dependence on a rise in cytosolic free calcium can be dissociated from formation of inositol phosphates.
A rise in the cytosolic free Ca2+ concentration due to both mobilization of Ca2+ from internal stores and influx of extracellular Ca2+ across the plasma membrane through 'second messenger-operated Ca2+ channels' is one of the first transmembrane signals detected following activation of CD2 or CD3 receptors on T-cells. In this study, we have further elucidated the regulation of these channels in the human T-leukemic cell line, JURKAT. Stimulation with either OKT3 or PHA induced a prompt influx of Ca2+ as assessed by MN2+ quenching of intracellular fura-2 fluorescence. When cytosolic free Ca2+ transient was partially buffered by loading the cells with BAPTA, neither agonist could induce Ca2+ entry into the cells as depicted by the lack of quenching of the fluorescence signal by Mn2+. This is in good agreement with our previous data on agonist-induced 45Ca2+ influx demonstrating that a rise in cytosolic free Ca2+ due to agonist-induced mobilization of Ca2+ from intracellular stores, could, directly or indirectly via the inositol cycle, initiate Ca2+ influx in these cells. Further support of this idea comes from the data demonstrating that agonist-induced mobilization of Ca2+ precedes the influx of Ca2+ across the plasma membrane. The present findings show that agonist-stimulation significantly increased the levels of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 after only 5 s, indicating that one or both of these substances could play a role in the regulation of Ca2+ influx. However, when agonist-induced Mn2+ influx was totally abolished, by partially buffering the cytosolic free Ca2+ rise, the formation of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 was not affected. Consequently, the dependence of an initial rise in cytosolic free Ca2+ for the subsequent regulation of Ca2+ influx across the plasma membrane, can be dissociated from the formation of both Ins(1,4,5)P3 and Ins(1,3,4,5)P4.
Ng
J
J
Department of Immunology, Karolinska Institute, Stockholm, Sweden.
Gustavsson
J
J
Jondal
M
M
Andersson
T
T
D000634:Aminoquinolines / Q000494:pharmacology; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001572:Benzofurans; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000378:metabolism*; D004533:Egtazic Acid / Q000494:pharmacology / Q000031:analogs & derivatives; D005456:Fluorescent Dyes; D016257:Fura-2; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D007295:Inositol Phosphates / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D015458:Leukemia, T-Cell / Q000378:metabolism*; D008345:Manganese; D010835:Phytohemagglutinins / Q000494:pharmacology; D015290:Second Messenger Systems; D013050:Spectrometry, Fluorescence; D014407:Tumor Cells, Cultured
D000634:Aminoquinolines; D000911:Antibodies, Monoclonal; D001572:Benzofurans; D015220:Calcium Channels; D005456:Fluorescent Dyes; D007295:Inositol Phosphates; D010835:Phytohemagglutinins; C081655:inositol-1,3,4,5-tetrakisphosphate; D008345:Manganese; D004533:Egtazic Acid; D015544:Inositol 1,4,5-Trisphosphate; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; C035739:Quin2; D002118:Calcium; D016257:Fura-2