title: Effects of staurosporine on the capacitative regulation of the state of the Ca2+ reserves in activated Jurkat T lymphocytes.
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 beta 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 microM). 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-gamma 1. 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.
authors.affiliation: Program Group of the Medical Research Council of Canada on Immuno-cardiovascular Interactions, Faculty of Medicine, University of Sherbrooke, Quebec, Canada.
authors.forename: C E
authors.initials: CE
authors.lastname: Ahnadi
authors.forename: M D
authors.initials: MD
authors.lastname: Payet
authors.forename: G
authors.initials: G
authors.lastname: Dupuis
mesh_terms: D000911:Antibodies, Monoclonal / Q000276:immunology; D001692:Biological Transport / Q000187:drug effects / Q000276:immunology; D002118:Calcium / Q000032:analysis / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000187:drug effects / Q000378:metabolism*; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000276:immunology; D002614:Chelating Agents / Q000494:pharmacology; D003198:Computer Simulation; D004464:Econazole / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D016257:Fura-2 / Q000494:pharmacology; D006801:Humans; D019169:Jurkat Cells / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D007700:Kinetics; D008213:Lymphocyte Activation; D018448:Models, Immunological; D009532:Nickel / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000276:immunology; D019311:Staurosporine / Q000494:pharmacology*; D013601:T-Lymphocytes / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D019284:Thapsigargin / Q000494:pharmacology
chemical_list: D000911:Antibodies, Monoclonal; D002121:Calcium Channel Blockers; D015220:Calcium Channels; D002614:Chelating Agents; D004791:Enzyme Inhibitors; D016693:Receptors, Antigen, T-Cell, alpha-beta; D004533:Egtazic Acid; D019284:Thapsigargin; C022838:nickel chloride; D004464:Econazole; D009532:Nickel; D011493:Protein Kinase C; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium; D016257:Fura-2