Tyrosine kinase inhibitors and Ca2+ signaling: direct interactions with fura-2

Selective inhibitors were used to study the role of tyrosine kinases in alp ha(1A)-adrenoceptor-mediated responses in transfected PC12 cells. Ca2+ resp onses to noradrenaline were measured using fura-2, and the effects of genis tein, tyrphostin A25, and herbimycin A were examined. Neither genistein nor herbimycin A pretreatment altered noradrenaline-induced Ca2+ responses, al though tyrphostin A25 pretreatment caused some reduction. However, acute ad dition of genistein quickly reversed the apparent noradrenaline response, a pparently, through a direct interaction with cytoplasmic fura-2. Both genis tein and tyrphostin A25, at concentrations similar to those used to inhibit tyrosine kinases, markedly reduced fluorescence of fura-2 excited by both 340 and 380 nm, and genistein also reduced the 340/380 ratio. Tyrosine kina se inhibitors did not alter noradrenaline stimulated inositol phosphate for mation in alpha(1A)-PC12 cells. These results suggest that tyrosine kinases are not involved in second messenger responses to alpha(1A)-adrenoceptors, but that tyrosine kinase inhibitors can interact directly with fura-2. (C) 2000 Elsevier Science B.V. All rights reserved.