ROLE FOR TYROSINE KINASES IN CARBACHOL-REGULATED CA ENTRY INTO COLONIC EPITHELIAL-CELLS

We studied a possible role of tyrosine kinases in the regulation of Ca entry into colonic epithelial cells HT-29/B6 using digital image proc essing of fura 2 fluorescence. Both carbachol and thapsigargin increas ed Ca entry to a similar extent and Ca influx was reduced by the tyros ine kinase inhibitor genistein (50 mu M). Further experiments were per formed in solutions containing 95 mM K to depolarize the membrane pote ntial, and the effects of different inhibitors on influx of Ca, Mn, an d Ba were compared. Genistein, but not the inactive analogue daidzein nor the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2-methy lpiperazine, decreased entry of all three divalent cations by 47-59%. In high-K solutions, carbachol or thapsigargin both caused intracellul ar Ca to increase to a plateau of 223 +/- 19 nM. This plateau was redu ced by the tyrosine kinase inhibitors genistein (to 95 +/- 8 nM), lave ndustin A (to 155 +/- 17 nM), and methyl-2,5-dihydroxycinnamate (to 39 +/- 3 nM). Orthovanadate, a protein tyrosine phosphatase inhibitor, p revented the inhibitory effect of genistein. Ca pumping was unaffected by genistein. Carbachol increased tyrosine phosphorylation (immunoblo ts with anti-phosphotyrosine antibodies) of 110-, 75-, and 70-kDa prot eins, and this phosphorylation was inhibited by genistein. We conclude that carbachol and thapsigargin increase Ca entry, and tyrosine phosp horylation of some key proteins may be important for regulating this p athway.