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 in the intestinal epithelial cell line Int 407, and this action involves the activation of both different GTP-binding proteins (G-proteins) and phospholipase C of the gamma-subtype (PLC-gamma). With this knowledge as the incentive, we investigated the possible regulatory role of protein tyrosine kinase activities in the calcium signalling system of the LTD(4) receptor. The tyrosine kinase inhibitors genistein and herbimycin A both reduced the LTD(4)-induced calcium signal by 70% when Int 407 cells were stimulated in the presence of extracellular calcium, but had no effect on the signal when the cells were stimulated in a calcium-free medium. in accordance with these findings, pretreatment with a tyrosine kinase inhibitor also blocked thapsigargin-induced cellular influx of calcium. These inhibitors had no effect on the intracellular 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 present. Of possible interest regarding the effect of genistein on LTD(4)-induced calcium influx is that two major tyrosine phosphorylated protein bands were detected in immunoprecipitates obtained with PLC-gamma antibodies from LTD(4)-stimutated cells. These proteins, which associate with PLC-gamma, have estimated molecular weights of 84 and 97 kD. Preincubation with genistein completely abolished the LTD(4)-induced increase in tyrosine phosphorylation of the major 97 kD band, whereas the 84 kD protein band, like the PLC-gamma band, still exhibited an increased phosphorylation of tyrosine residues in response to LTD(4). Neither this effect nor any of the other effects of genistein were induced when cells were preincubated with daidzein, an inactive analogue of genistein. The present results suggest that LTD(4)-induced calcium signalling in epithelial cells involves not only tyrosine phosphorylation of PLC-gamma, but also a tyrosine kinase-dependent step which occurs downstream of PLC-gamma activation and is directly implicated in the regulation of agonist-mediated calcium influx.