Differentiation of U937 cells enables a phospholipase D-dependent pathway of cytosolic phospholipase A(2) activation

Treatment with dibutyryl cyclic AMP (dBcAMP) of the human, premonocytic U93 7 cell line results in differentiation toward a monocyte/granulocyte-like c ell. This differentiation enables the cell to activate cytosolic phospholip ase A(2) (cPLA(2)) to release arachidonate upon stimulation. In contrast, u ndifferentiated cells are unable to release arachidonate even when stimulat ed with calcium ionophores. In the present research, a role for phospholipa se D (PLD) in the regulation of cPLA(2) was shown based on a number of obse rvations. First, the ionomycin- and fMLP-stimulated production of arachidon ate in differentiated cells was sensitive to ethanol (2% (v/v)). Ethanol ac ts as an alternate substrate in place of water for PLD producing phosphatid ylethanol (PEt) instead of phosphatidic acid. Indeed, ionomycin stimulation of differentiated cells produced a 14-fold increase in PEt levels. Further evidence for the involvement of PLD in the regulation of cPLA(2) came from the observation that the stimulated production of diacylglycerol (for whic h phosphatidic acid is a major source) was greatly diminished in undifferen tiated cells as compared to differentiated cells. Moreover, the normally de ficient activation of cPLA(2) in undifferentiated cells could be stimulated to release arachidonate if the cells were electroporated in the presence o f GTP[gamma]S and MgATP. This treatment stimulates phosphatidylinositol-4,5 -bisphosphate (PIP2) production which appears to activate PLD and cPLA(2) i n subsequent steps. The phosphatidic acid (and diacylglycerol derived from phosphatidic acid) appears to greatly regulate the action of cPLA(2) by an unknown mechanism, and undifferentiated cells lack the ability to stimulate PLD activity due to a dysfunction of PLP2 production. (C) 1999 Academic Pr ess.