Leukotriene D4 and E4 Induce Transmembrane Signaling in Human Epithelial Cells SINGLE CELL ANALYSIS REVEALS DIVERSE PATHWAYS AT THE G-PROTEIN LEVEL FOR THE INFLUX AND THE INTRACELLULAR MOBILIZATION

Leukotrienes are recognized as important mediators of the inflammatory process. Recently, increasing attention has been paid to the role of noninflammatory cells in the regulation of the inflammatory process. To further increase our knowledge of this matter we have, in the present study, investigated leukotriene-induced Ca2* signaling, using a single cell technique in a human epithelial cell line, Intestine 407. It was evident that both LTD4 and LTE,, at physiological concentrations (10 nM), triggered rapid and pronounced cytosolic free Ca2+ transients, due to both influx across the plasma membrane and intracellular mobilization. Preincubation with pertussis toxin (1200 rig/ml) decreased the level of agonist-induced Ca2+ transients to an extent similar to that caused by depletion of extracellular Ca’+, suggesting that the toxin affected the influx but not the intracellular mobilization of Ca2+. Indeed, by using the Mn2+ quenching technique, it could be shown that pertussis toxin totally inhibited the influx of Ca’+. The fact that, even after pertussis toxin treatment, direct G-protein activation by AlF; was still able to trigger a cytosolic free Ca2+ transient, indicates that, in these cells, G-proteins (GTP-binding proteins) that are insensitive to pertussis toxin are capable of mediating a Ca2+ signal. In order to test the idea that such G-proteins regulate mobilization of intracellular Ca’+ induced by LTD, and LTEI, we electropermeabilized and preincubated the Intestine 407 cells with guanosine-5’-0-(2-thiodiphosphate) (GDPBS), let them reseal, and, after loading with fura2, investigated the effects on agonist-stimulated Ca’+ signaling. Electropermeabilization and resealing alone did not significantly affect the Ca2+ responses triggered by LTD4 or LTE,. Addition of GDP@, in the presence of extracellular Ca2+, reduced the Ca2+ responses by approximately 60-70%. In Ca2+-depleted medium, GDP@ also impaired the LTD,-induced response by 65%, however, it had no effect on the Ca2+ response induced by LTE.,. In conclusion, LTD., and LTE, trigger cytosolic free Ca2+ signaling in a human epithelial cell line by causing both an influx of Ca2+ and mobilization of intracellular Ca’+. The Ca2+-signaling mechanism appears to consist of dual pathways, since the influx is regulated by a pertussis toxin-sensitive G-protein, but,