EFFECTS OF STAUROSPORINE ON THE CAPACITATIVE REGULATION OF THE STATE OF THE CA2-LYMPHOCYTES( RESERVES IN ACTIVATED JURKAT T)

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. H owever, Stp can also inhibit protein tyrosine kinases (PTK). We have i nvestigated the effects of Stp on the InsP(3)-(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 (J urkat)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 trigge red by Tg (1 mu M). When the cells were preincubated with Stp (200 nM) , there was an inhibition of the mAb C305- but not the Tg-dependent Ca 2+ 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 specif ic PKC inhibitor bis-indolyl maleimide GF 109203X. The effect of Stp o n the entry of Ca2+ in activated (mAb C305) Jurkat lymphocytes was dos e-related and was not the result of a direct inhibition of plasma memb rane Ca2+ channels based on an absence of effect on the Tg-dependent e ntry of Ca2+ and the use of Ca2+ channel blockers (econazole and Ni2+) . These blockers terminated the influx of Ca2+ but the Tg-sensitive Ca 2+ reserves were not refilled in marked contrast to the effect of Stp. Quantification of InsP(3) revealed that the addition of Stp resulted in an approximate 40% reduction in mAb C305-activated Jurkat cells. Th e effects of Stp can be explained as follows. Stp decreases the mAb C3 05-induced production of InsP(3) by inhibiting the TcR/CD3-dependent a ctivation of PTK associated with the stimulation of phospholipase C-ga mma(1). A decrease in [InsP(3)] without a return to baseline is suffic ient to close the InsP(3) Ca2+ channel, endoplasmic Ca2+ ATPases use t he incoming Ca2+ to refill the Ca2+ pools and that terminates the capa citative entry of Ca2+. A simple kinetic model reproduced the experime ntal data.