phosphatidylethanol and Leukemia--Erythroblastic--Acute

Phospholipase D (PLD) can be activated by a variety of receptor agonists in different cell types. However, an effect of prostaglandins (PGs) on the activity of this enzyme has not been demonstrated previously. In this study, we found that PGE1 could stimulate PLD in human erythroleukemia cells, as measured by phosphatidylethanol formation, with an ED50 of 3.5 x 10(-7) M. PGE2 was also active, but other PGs including prostacyclin, PGD2 and PGF2, had no effect. PGE1 also elicited cyclic AMP production over the same concentration range that activated PLD. However, it is unlikely that cyclic AMP per se is responsible for PGE-induced PLD activation, because PLD could be substantially activated by PGE2 at concentrations (0.1-1 microM) which did not stimulate cyclic AMP production. Furthermore, no increase of phosphatidylethanol formation could be observed when cells were treated with other adenylyl cyclase-activating agents such as prostacyclin, forskolin and vasoactive intestinal peptide. In Ca+(+)-free medium, PLD activation by PGE1 and PGE2 was greatly reduced, indicating that their effect was through a Ca+(+)-dependent pathway. Pretreatment of cells with pertussis toxin abolished PGE1- and PGE2-stimulated PLD activity, implying the involvement of a G protein in the PGE-mediated signal transduction pathway. Our results not only indicate that E-series PGs may initiate some of their cellular effects through a novel pathway, activation of PLD, but also suggest that PGE-stimulated PLD activity in human erythroleukemia cells is Ca+(+)-dependent and is regulated via a pertussis toxin-sensitive G protein.

Topics: Calcium; Cyclic AMP; Enzyme Activation; Glycerophospholipids; GTP-Binding Proteins; Humans; Leukemia, Erythroblastic, Acute; Phosphatidic Acids; Phospholipase D; Prostaglandins E; Tumor Cells, Cultured

Human erythroleukaemia (HEL) cells were exposed to thrombin and other platelet-activating stimuli, and changes in radiolabelled phospholipid metabolism were measured. Thrombin caused a transient fall in PtdInsP and PtdInsP2 levels, accompanied by a rise in diacylglycerol and phosphatidic acid, indicative of a classical phospholipase C/diacylglycerol kinase pathway. However, the rise in phosphatidic acid preceded that of diacylglycerol, which is inconsistent with phospholipase C/diacylglycerol kinase being the sole source of phosphatidic acid. In the presence of ethanol, thrombin and other agonists (platelet-activating factor, adrenaline and ADP, as well as fetal-calf serum) stimulated the appearance of phosphatidylethanol, an indicator of phospholipase D activity. The Ca2+ ionophore A23187 and the protein kinase C activator phorbol myristate acetate (PMA) also elicited phosphatidylethanol formation, although A23187 was at least 5-fold more effective than PMA. Phosphatidylethanol production stimulated by agonists or A23187 was Ca2(+)-dependent, whereas that with PMA was not. These result suggest that phosphatidic acid is generated in agonist-stimulated HEL cells by two routes: phospholipase C/diacylglycerol kinase and phospholipase D. Activation of the HEL-cell phospholipase D in response to agonists may be mediated by a rise in intracellular Ca2+.

Topics: Adenosine Diphosphate; Calcimycin; Calcium; Cell Line; Enzyme Activation; Epinephrine; Glycerophospholipids; Humans; Kinetics; Leukemia, Erythroblastic, Acute; Myristic Acid; Myristic Acids; Phosphatidic Acids; Phosphatidylinositols; Phospholipase D; Phospholipases; Platelet Activating Factor; Tetradecanoylphorbol Acetate; Thrombin; Tumor Cells, Cultured