Sequential degradation of choline phosphoglycerides by phospholipase D and phosphatidate phosphohydrolase in dibutyryl cAMP-differentiated U937 cells.

Dibutyryl-cAMP-differentiated U937 cells incorporate alkyllyso-sn-glycero-3-[32P]phosphocholine (alkyllyso-[32P]GPC) into cellular alkylacyl-sn-glycero-3-phosphocholine (alkylacyl-GPC). Upon stimulation with fMet-Leu-Phe (fMLP), recombinant C5a, or phorbol 12-myristate 13-acetate (PMA), these cells produce alkylacyl-sn-glycero-3-[32P]phosphate (alkylacyl-[32P]GP). In the presence of ethanol (0.5%), alkylacyl-sn-glycero-3-[32P]phosphoethanol (alkylacyl-[32P]GPet) is also formed with a concomitant reduction in alkylacyl-[32P]GP accumulation. Because cellular ATP is not labeled with 32P, alkylacyl-[32P]GP and alkylacyl-[32P]GPet must be formed by phospholipase D (PLD)-catalyzed hydrolysis and transphosphatidylation, respectively. Activation by receptor agonists, but not by PMA, requires extracellular Ca2+ and is augmented by cytochalasin B pretreatment. Upon stimulation, dibutryl cAMP-differentiated U937 cells labeled with alkylacyl-[32P]GPC produce [32P]PO4 but not [32P]phosphocholine. Furthermore, when these cells were labeled in alkylacyl-GPC by incubation with [3H]alkyllyso-GPC and then stimulated, [3H]alkylacyl-glycerol ([3H]alkylacyl-Gro) is produced with a time-course similar to that of [32P]PO4 formation and coincident with the decline in alkylacyl-GP accumulation. These results demonstrate that alkylacyl-GP formed by PLD is dephosphorylated by phosphatidate phosphohydrolase to produce PO4 and alkylacyl-Gro. Upon stimulation with fMLP or C5a, U937 cells labeled in diacyl-sn-glycero-3-phosphocholine (diacyl-GPC) by incubation with [3H]acyllyso-GPC generate [3H]diacyl-GP, [3H]diacyl-GPEt, and [3H]diacyl-Gro with kinetics similar to those for the generation of the [3H]alkyl products. Thus, in differentiated U937 cells stimulated with receptor agonists, both alkylacyl-GPC and diacyl-GPC are sequentially metabolized by PLD and phosphatidate phosphohydrolase.