INHIBITION OF BRADYKININ-INDUCED AND THAPSIGARGIN-INDUCED CA(2+) ENTRY BY TYROSINE KINASE INHIBITORS

We examined the involvement of tyrosine kinase activity in the bradyki nin (BK)-mediated signal transduction process. Immunoblots with anti-p hosphotyrosine antibodies following BK stimulation of human fibroblast s showed tyrosine phosphorylation of specific proteins that could be i nhibited by the tyrosine kinase inhibitors genistein and tyrphostin. I mage analysis data from individual cells stimulated by BK in the prese nce of genistein and tyrphostin showed that these inhibitors block the plateau phase but not the rapid transient phase of the BK-induced cal cium response. That the loss of the plateau phase was due to blockage of calcium entry rather than stimulation of calcium pump activity was confirmed by examining the influx of Ba2+ following BK stimulation. Th e Ca2+ imaging results were confirmed by Ca-45(2+) uptake measurements and extended to another tyrosine kinase inhibitor (methyl 2,5-dihydro xycinnamate), which was found to interfere with the fura-2 signal and therefore could not be used in imaging experiments. The fact that thre e structurally distinct inhibitors of tyrosine kinase activity inhibit ed BK-stimulated calcium influx, while an inactive analogue of geniste in (daidzein) did not, strongly suggests the involvement of tyrosine k inases in the regulation of a BK-induced calcium entry pathway. To our knowledge, this is the first report of tyrosine kinase involvement in the regulation of calcium entry following activation of a receptor th at lacks endogenous tyrosine kinase activity and is known to be couple d to phosphatidylinositol turnover. We found that calcium entry in HSW P (human foreskin fibroblast) cells can also be stimulated by emptying the intracellular calcium stores with thapsigargin. Genistein also in hibits the plateau phase of the thapsigargin-induced calcium response while leaving the transient phase intact. This suggests that the Ca2influx pathway induced by depletion of intracellular calcium stores wi th thapsigargin also may be regulated via a tyrosine kinase pathway.