THE REGULATION OF LEUKOTRIENE D-4-INDUCED CALCIUM INFLUX IN HUMAN EPITHELIAL-CELLS INVOLVES PROTEIN-TYROSINE PHOSPHORYLATION

Leukotriene D-4 (LTD(4)) has been found to induce calcium signalling i n the intestinal epithelial cell line Int 407, and this action involve s the activation of both different GTP-binding proteins (G-proteins) a nd phospholipase C of the gamma-subtype (PLC-gamma). With this knowled ge as the incentive, we investigated the possible regulatory role of p rotein tyrosine kinase activities in the calcium signalling system of the LTD(4) receptor. The tyrosine kinase inhibitors genistein and herb imycin A both reduced the LTD(4)-induced calcium signal by 70% when In t 407 cells were stimulated in the presence of extracellular calcium, but had no effect on the signal when the cells were stimulated in a ca lcium-free medium. in accordance with these findings, pretreatment wit h a tyrosine kinase inhibitor also blocked thapsigargin-induced cellul ar influx of calcium. These inhibitors had no effect on the intracellu lar mobilisation of calcium, which was supported by the findings that LTD(4) was able to induce an increase in the tyrosine phosphorylation of PLC-gamma even when one of the tyrosine kinase inhibitors was prese nt. Of possible interest regarding the effect of genistein on LTD(4)-i nduced calcium influx is that two major tyrosine phosphorylated protei n bands were detected in immunoprecipitates obtained with PLC-gamma an tibodies from LTD(4)-stimutated cells. These proteins, which associate with PLC-gamma, have estimated molecular weights of 84 and 97 kD. Pre incubation with genistein completely abolished the LTD(4)-induced incr ease in tyrosine phosphorylation of the major 97 kD band, whereas the 84 kD protein band, like the PLC-gamma band, still exhibited an increa sed phosphorylation of tyrosine residues in response to LTD(4). Neithe r this effect nor any of the other effects of genistein were induced w hen cells were preincubated with daidzein, an inactive analogue of gen istein. The present results suggest that LTD(4)-induced calcium signal ling in epithelial cells involves not only tyrosine phosphorylation of PLC-gamma, but also a tyrosine kinase-dependent step which occurs dow nstream of PLC-gamma activation and is directly implicated in the regu lation of agonist-mediated calcium influx.