Effects of staurosporine on the capacitative regulation of the state of the Ca reserves in activated Jurkat T lymphocytes |
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Authors: | Charaf E. Ahnadi Marcel D. Payet Gilles Dupuis |
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Affiliation: | aProgram Group of the Medical Research Council of Canada on Immuno-cardiovascular Interactions, Departments of Biochemistry, Faculty of Medicine, University of Sherbrooke, Sherbrooke, Quebec, Canada;bProgram Group of Medical Research Council of Canada on Immuno-cardiovascular Interactions, Departments of Physiology and Biophysics, Faculty of Medicine, University of Sherbrooke, Sherbrooke, Quebec, Canada |
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Abstract: | Staurosporine (Stp) is an inhibitor of protein kinase C (PKC) that has been used to address the role of this enzyme in a variety of cells. However, Stp can also inhibit protein tyrosine kinases (PTK). We have investigated the effects of Stp on the InsP3- (using mAb C305 directed against the β chain of the T cell receptor (TcR)/CD3 complex) and the thapsigargin (Tg)-dependent release and influx of Ca2+ in human (Jurkat) T cells. The addition of Stp (200 nM) during the sustained phase of the TcR-dependent Ca2+ response resulted in a rapid inhibition of the influx of Ca2+ that was not seen when Ca2+ mobilization was triggered by Tg (1 μM). When the cells were preincubated with Stp (200 nM), there was an inhibition of the mAb C305- but not the Tg-dependent Ca2+ response. The effect of Stp was not the result of the inhibition of PKC as shown by down-regulation of PKC and with the use of the specific PKC inhibitor bis-indolyl maleimide GF 109203X. The effect of Stp on the entry of Ca2+ in activated (mAb C305) Jurkat lymphocytes was dose-related and was not the result of a direct inhibition of plasma membrane Ca2+ channels based on an absence of effect on the Tg-dependent entry of Ca2+ and the use of Ca2+ channel blockers (econazole and Ni2+). These blockers terminated the influx of Ca2+ but the Tg-sensitive Ca2+ reserves were not refilled in marked contrast to the effect of Stp. Quantification of InsP3 revealed that the addition of Stp resulted in an approximate 40% reduction in mAb C305-activated Jurkat cells. The effects of Stp can be explained as follows. Stp decreases the mAb C305-induced production of InsP3 by inhibiting the TcR/CD3-dependent activation of PTK associated with the stimulation of phospholipase C-γ1. A decrease in [InsP3] without a return to baseline is sufficient to close the InsP3 Ca2+ channel, endoplasmic Ca2+ ATPases use the incoming Ca2+ to refill the Ca2+ pools and that terminates the capacitative entry of Ca2+. A simple kinetic model reproduced the experimental data. |
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Keywords: | Abbreviations: CD, cluster of differentiation [Ca2+]i or [Ca2+]c, cytosolic concentration of Ca2+ [Ca2+]i, Ca2+ concentration in the cellular reserves DAG, 1,2-sn-diacylglycerol DMSO, dimethyl-sulfoxide EGTA, ethyleneglycol-bis(β-aminoethyl ether) N,N N&prime ,N&prime -tetraacetic acid InsP3, D-myo-inositol-1,4,5-trisphosphate InsP3R, InsP3 receptor mAb, monoclonal antibody PBS, Dulbecco's phosphate-buffered saline PKC, protein kinase C PLC, phospholipase C PMCA, plasma membrane Ca2+ transport ATPase PTK, protein tyrosine kinase SERCA, sarco- and endoplasmic Ca2+ transport ATPase Stp, staurosporine Tg, thapsigargin TcR, T cell receptor TPA, 12-O-tetradecanoyl-13-O-acetyl phorbol Tris, tris(hydroxymethyl)aminomethane |
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