LEUKOTRIENE D-4 INDUCES A RAPID INCREASE IN CAMP IN THE HUMAN EPITHELIAL-CELL LINE, INT-407 - A POTENTIAL ROLE FOR THIS SIGNAL IN THE REGULATION OF CALCIUM INFLUX THROUGH THE PLASMA-MEMBRANE

Although the LTD4-induced Ca2+ influx in human epithelial cells has be en shown to be regulated by a pertussis toxin-sensitive heterotrimeric G-protein, most likely a G alpha(i3) protein [Adolfsson J.L.P., Ohd J .F., Sjolander A. Leukotriene D-4-induced activation and translocation of the G-protein alpha(i3)-subunit in human epithelial cells. Biochem Biophys Res Commun 1996; 226. 413-419], the signalling pathway furthe r downstream is still unclear. In the present study, we investigated t he possible involvement of cAMP and protein kinase A activity in the L TD4-induced Ca2+ influx in the epithelial cell line Int 407. Stimulati on with LTD4, but not with the calcium ionophore ionomycin, triggered a rapid increase (peak at 7 s) in the cellular cAMP level, an effect t hat was totally abolished by pertussis toxin. Furthermore, the LTD4-in duced Ca2+ signal was reduced by 60% when cells that had been pre-incu bated with the protein kinase A inhibitor Rp-cAMPS (50 mu M for 30 min ) were stimulated in a calcium containing medium. In contrast, Rp-cAMP S had no apparent effect on the LTD4-induced Ca2+ signal when the cell s were stimulated in a calcium-depleted medium. The immediate LTD4-ind uced protein tyrosine phosphorylation (15 s), previously shown to be n ecessary for the subsequent Ca2+ influx, was abolished not only by pre treatment with pertussis toxin but also by exposure to Rp-cAMPS. Furth ermore, direct activation of the cellular adenylyl cyclase activity by treatment with forskolin alone induced a prompt Ca2+ signal in the pr esence, but not in the absence, of extracellular Ca2+, identical resul ts were obtained with the cell permeable cAMP analogue 8-bromo-cAMP. I n addition, forskolin induced protein tyrosine phosphorylation similar to that seen with LTD4. These results suggest that protein kinase A a ctivity participates in the regulation of the LTD4 induced Ca2+ influx at a site that is downstream of the activation of the pertussis toxin -sensitive G-protein but upstream of a LTD4-stimulated tyrosine kinase (s).