Leukotriene B4 stimulation of an early elevation of phosphatidic acid mass in human neutrophils.

The signal transduction pathway of leukotriene B4 involves phospholipase D activation in cytochalasin B-primed neutrophils, but leukotriene B4 stimulation of increased phosphatidic acid mass in neutrophils has not been demonstrated. Employing the NIH Image program, we have examined the effect of leukotriene B4 on phosphatidic acid mass in human neutrophils incubated with or without cytochalasin B. Our results show that 0.15 microM leukotriene B4 without cytochalasin B was capable of increasing phosphatidic acid mass in neutrophils by 2-fold after 5 s, 2.5-fold after 1 min, and 2-fold after 5 min incubation. Leukotriene B3, leukotriene B4, and leukotriene B5 were equipotent stimuli for phosphatidic acid mass elevation. Leukotriene B4 induced phosphatidylethanol formation at the expense of phosphatidic acid in cells preincubated with 0.25-1% ethanol, indicating phospholipase D activation. Cytochalasin B enhanced leukotriene B4 stimulation of phosphatidic acid mass elevation and phosphatidylethanol formation. There were no measurable changes in 1,2-diglyceride mass after 5 s, but a 1.7-fold increase occurred after 1 min and declined thereafter. Leukotriene B4 stimulation of [3H]glycerol incorporation into phosphatidic acid, diglyceride and phosphatidylinositol was detectable after a 1-min incubation, suggesting increased de novo synthesis of these lipids. These results suggest that leukotriene B4 stimulation of phospholipase D activity contributes to part of the early increased phosphatidic acid mass and that combined actions of stimulated phospholipases C and D, and de novo phosphatidic acid synthesis contribute to the total increased phosphatidic acid mass.