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

Selective inhibitors were used to study the role of tyrosine kinases in alpha(1A)-adrenoceptor-mediated responses in transfected PC12 cells. Ca(2+) responses to noradrenaline were measured using fura-2, and the effects of genistein, tyrphostin A25, and herbimycin A were examined. Neither genistein nor herbimycin A pretreatment altered noradrenaline-induced Ca(2+) responses, although tyrphostin A25 pretreatment caused some reduction. However, acute addition of genistein quickly reversed the apparent noradrenaline response, apparently, through a direct interaction with cytoplasmic fura-2. Both genistein 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 kinase inhibitors did not alter noradrenaline stimulated inositol phosphate formation 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.