TYRPHOSTIN A9 INHIBITS CALCIUM RELEASE-DEPENDENT PHOSPHORYLATIONS ANDCALCIUM-ENTRY VIA CALCIUM RELEASE-ACTIVATED CHANNEL IN JURKAT T-CELLS

The mechanism by which calcium-depleted intracellular stores may trigg er an external calcium influx through a calcium release-activated chan nel was investigated by analyzing the effects of several protein tyros ine kinase inhibitors on calcium movements in Jurkat T cells. Tyrphost in A9, an inhibitor of the kinase activity of the platelet-derived gro wth factor (PDGF) receptor, dramatically impaired the sustained elevat ion of cytosolic calcium concentration, induced by either CD3 mAbs, th apsigargin, ionomycin at low (10(-7) M) concentration, or passive depl etion of intracellular stores; other tested tyrphostins, lavendustin, genistein, and compound 5 lacked significant effect. Tyrphostin A9, ad ded during the plateau phase, was able to return cytosolic calcium to resting concentration, Likewise, it abrogated manganese entry in cells stimulated by CD3 or thapsigargin, measured by the quenching of the f luorescence of Indo-1. However, it did not measurably modify kinetics of intracellular calcium releases monitored in the absence of extracel lular calcium, nor did it reverse the inhibition of phosphatidylserine that occurs as a consequence of emptying intracellular stores. Analys es of tyrosine phosphorylations demonstrated that A9 inhibited the pho sphorylation of proteins, which occurred every time that internal calc ium stores were depleted. These phosphorylations were not impaired by chelation of external Ca2+, nor by La3+ that inhibits calcium release- induced calcium entry. We concluded that their inhibition was not a co nsequence, but may be a cause, of the blockade of calcium release-acti vated channel by tyrphostin A9.