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. 1991 Apr;2(4):299–309. doi: 10.1091/mbc.2.4.299

Assessment of receptor-dependent activation of phosphatidylcholine hydrolysis by both phospholipase D and phospholipase C.

T T Dinh 1, D A Kennerly 1
PMCID: PMC361781  PMID: 1829383

Abstract

Enhancement of cellular phospholipase D (PLD)-1 and phospholipase C (PLC)-mediated hydrolysis of endogenous phosphatidylcholine (PC) during receptor-mediated cell activation has received increasing attention inasmuch as both enzymes can result in the formation of 1,2-diacylglycerol (DAG). The activities of PLD and PLC were examined in purified mast cells by quantitating the mass of the water-soluble hydrolysis products choline and phosphorylcholine, respectively. Using an assay based on choline kinase-mediated phosphorylation of choline that is capable of measuring choline and phosphorylcholine in the low picomole range, we quantitated the masses of both cell-associated and extracellular choline and phosphorylcholine. Activating mast cells by crosslinking its immunoglobulin E receptor (Fc epsilon-RI) resulted in an increase in cellular choline from 13.1 +/- 1.2 pmol/10(6) mast cells (mean +/- SE in unstimulated cells) to levels 5- to 10-fold higher, peaking 20 s after stimulation and rapidly returning toward baseline. The increase in cellular choline mass paralleled the increase in labeled phosphatidic acid accumulation detected in stimulated cells prelabeled with [3H]palmitic acid and preceded the increase in labeled DAG. Although intracellular phosphorylcholine levels were approximately 15-fold greater than choline in unstimulated cells (182 +/- 19 pmol/10(6) mast cells), stimulation resulted in a significant fall in phosphorylcholine levels shortly after stimulation. Pulse chase experiments demonstrated that the receptor-dependent increase in intracellular choline and the fall in phosphorylcholine were not due to hydrolysis of intracellular phosphorylcholine and suggested a receptor-dependent increase in PC resynthesis. When the extracellular medium was examined for the presence of water-soluble products of PC hydrolysis, receptor-dependent increases in the mass of both choline and phosphorylcholine were observed. Labeling studies demonstrated that these extracellular increases were not the result of leakage of these compounds from the cytosol. Taken together, these data lend support for a quantitatively greater role for receptor-mediated PC-PLD compared with PC-PLC during activation of mast cells.

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Selected References

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