Analysis of Choline and Phosphorylcholine Content in Human Neutrophils Stimulated by f-Met-Leu-Phe and Phorbol Myristate Acetate: Contribution of Phospholipase D and C

We analysed changes in choline (CHO) and phosphorylcholine (PCHO) content of stimulated human polymorphonuclear leukocytes (PMNs) by a chemiluminescence assay to further examine the relative contributions of phospholipase D (PLD) and PLC to phosphatidylcholine (PC) breakdown. PLD activation was also analysed by measuring tritiated phosphatidic acid (PA) and diglycerides (GDs) in PMNs labelled with tritiated alkyl-lyso PC. Stimulation of PMNs with formyl-methionyl-leucyl-phenylalanine fMLP; 0.1 μM induced a weak elevation of mass choline (+25% of basal level) that was strongly potentiated in PMNs primed with cytochalasin B (+350% relative to the control value of 657±53 pmol/10 7 cells). CHO production was rapid and transient, peaking within 1 min, and ran parallel to that of tritiated PA. Thereafter, the amount of tritiated PA declined strongly (40% of maximum by 3 min), whereas the elevated choline content induced by fMLP plateaued for at least 5 min. Phorbol myristate acetate (PMA) sustained the formation of CHO for as long as 20 min, which correlated with that of [ 3H]PA in a time- and concentration-dependent manner. PCHO content of resting PMN leukocytes (1560 ± 56 pmol/10 7 cells) was not modified after stimulation of PMNs with fMLP or PMA for at least 10 min, which argues against breakdown of phosphatidylcholine by PLC. For longer treatment (10–20 min), fMLP stimulated a significant enhancement of PCHO level, which occurred concomitantly with a decrease in CHO level, suggesting that choline kinase rather than PLC may be activated. Unlike fMLP, PMA stimulated a fall in PCHO between 10 and 15 min after PMN stimulation, pointing to different regulatory mechanisms of PCHO level. These data indicate that DG formation from PC in PMNs is mediated by PLD but not by PLC and show that chemiluminescence measurement of choline is a reliable index of PLD activation.