Electrospray ionization/mass spectrometric analyses of human promonocytic U937 cell glycerolipids and evidence that differentiation is associated with membrane lipid composition changes that facilitate phospholipase A(2) activation

Upon differentiation, U937 promonocytic cells gain the ability to release a large fraction of arachidonate esterified in phospholipids when stimulated , but the mechanism is unclear. U937 cells express group IV phospholipase A (2) (cPLA(2)), but neither its level nor its phosphorylation state increase s upon differentiation. A group VI PLA(2) (iPLA(2)) that is sensitive to a bromoenol lactone inhibitor catalyzes arachidonate hydrolysis from phosphol ipids in some cells and facilitates arachidonate incorporation into glycero phosphocholine (GPC) lipids in others, but it is not known whether U937 cel ls express iPLA(2). We confirm that ionophore A23187 induces substantial [H -3]arachidonate release from differentiated but not control U937 cells, and electrospray ionization mass spectrometric (ESI/MS) analyses indicate that differentiated cells contain a higher proportion of arachidonate-containin g GPC species than control cells. U937 cells express iPLA(2) mRNA and activ ity, but iPLA(2) inhibition impairs neither [H-3]arachidonate incorporation into nor release from U937 cells. Experiments with phosphatidate phosphohy drolase (PAPH) and phospholipase D (PLD) inhibitors coupled with ESI/MS ana lyses of PLD-PAPH products indicate that differentiated cells gain the abil ity to produce diacylglycerol (DAG) via PLD-PAPH. DAG promotes arachidonate release by a mechanism that does not require DAG hydrolysis, is largely in dependent of protein kinase C, and requires cPLA(2) activity. This may refl ect DAG effects on cPLA(2) substrate state.