Requirement of phosphatidylinositol 3-kinase activation and calcium influx for leukotriene B4-induced enzyme release

Abstract

Leukotriene B4 (LTB4) is a potent lipid mediator involved in host defense and inflammatory responses. It causes chemotaxis, generation of reactive oxygen species, and degranulation. However, only little is known of the molecular mechanisms by which LTB4 induces these biological activities. To analyze the intracellular signaling pathways to mediate lysosomal enzyme release through the cloned LTB4, receptor (BLT1), we transfected BLT1 to rat basophilic leukemia cells (RBL-2H3). LTB4 dose-dependently released β-hexosaminidase, and the release was mostly inhibited when the cells were pretreated with pertussis toxin, indicating that the degranulation is mediated by Gi proteins. LTB4 activated phosphatidylinositol 3-kinase (PI3-K) through Gi, and inhibition of PI3-K by wortmannin or LY290042 inhibited degranulation. Granulocytes from PI3-Kγ-deficient mice showed reduced LTB4-induced degranulation, suggesting that this isozyme of PI3-K is involved in the degranulation. LTB4 also caused calcium release from intracellular stores and calcium influx from the outside milieu through Gi, but only the calcium influx is critical for the lysosomal enzyme release. Calcium influx and PI3-K activation are both downstream events of Gi, since they were inhibited by pertussis toxin. These two events are in essence independent each other, because calcium depletion did not affect PI3-K, and inhibition of PI3-K did not attenuate calcium influx significantly. Thus, our results have clearly shown that LTB4 binds BLT1 and activates Gi-like protein, and both PI3-Kγ activation and a sustained calcium elevation by calcium influx are necessary for enzyme release in these cells.