Phenylarsine Oxide (PAO)-Mediated Activation of Phospholipase D in Rat Basophilic Leukemia (RBL-2H3) Cells: Possible Involvement of Calcium and Protein Kinase C

Addition of phenylarsine oxide (PAO) to [ 3H]oleic acid-labeled rat basophilic leukemia (RBL-2H3) cells gave rise to the remarkable formation of [ 3H]phosphatidylbutanol (PBut), a specific product of phospholipase D (PLD) activation. Preincubation of cells with 2,3-dimercaptopropanol (DMP) or dithiothreitol (DTT), compounds containing sulfhydryls, prevented PAO-stimulated [ 3H]PBut formation, indicating that PAOstimulated PLD through interacting with vicinal thiol groups. Treatment of cells with PAO resulted in increase in intracellular Ca 2+, concentration without significant production of inositol phosphates. Removal of extracellular free Ca 2+ by chelating with EGTA was found to inhibit [ 3H]PBut formation by PAO. Incubation of cells with 20 nM phorbol 12-myristate 13-acetate (PMA) for 6 h caused down-regulation of protein kinase C (PKC) α and β isozymes, whereas it had no effect on PKC δ, ε, and ζ isozymes. Under this condition, decrease in PAO-stimulated [ 3H]PBut formation was observed to occur with a concomitant decrease in the level of PKC α and β isozymes. These results suggest that a covalent bridge between vicinal thiol groups of cell surface proteins induced by PAO potentiates PLD activation and that PAO-induced PLD activation is regulated by Ca 2+ and PKC α and/or β isozymes.