Leukotriene D4-induced signal transduction in human epithelial cells

Sammanfattning: Although the structures of the cysteinyl-leukotrienes have been known for almost twenty years, and their biological effects for almost sixty years, very little is known about the signaling propenies of these compounds. The aim of the present research was to further elucidate the components that are involved in the signaling mechanisms of leukotriene D4 (L TD4) in a human epithelial cell line. Stimulation with LTD4 was found to induce the mobilization of intracellular calcium as well as the influx of calcium through the plasma membrane. Although the LTD4 receptor probably belongs to the seven-transmembrane-spanning G-protein-coupled receptors, phospholipase Cyt is tyrosine phosphorylated and translocated to the plasma membrane upon stimulation with L1D4. The unknown, genistein-insensitive kinase responsible for this effect is directly or indirectly activated by a monomericG-protein that probably belongs to the Rho fantily. We were unable to see any direct association between a Rho protein and phospholipase Cy1, instead we found that the monomeric G-protein Rapl associates with phospholipase Cyt in unstimulated cells. After stimulation the content of Rapl increases in the plasma membrane,simultaneously as it dissociates from phospholipase Cy1 in a manner dependent on protein kinase A. The L1D4-induced calcium influx is mediated through a pertussistoxin- sensitive 0-protein. Engagement of the L TD4 receptor also induces a rapid increase in cAMP and the activation of a genistein-sensitive tyrosine kinase. Both of these events are necessary for the LTD4-induced calcium influx. The increase in cAMP probably precedes the activation of the tyrosine kinase, since preincubation with Rp-cAMPS, which inhibits protein kinase A, interferes with the L 1D4-induced tyrosine phosphorylation. The identities of the kinases involved in the calcium mobilization and influx are still unresolved. Taken together, our findings show that L TD4 stimulation of the intestinal epithelial cell line INT 407 results in the activation of at least three different G-proteins and two kinases of importance for the regulation of cytosolic calcium levels in these cells.