Stimulation of phosphatidylcholine turnover by β-phorbol ester and diacylglycerol in the primordial human placenta: the suggested role of phospholipase D activation

Abstract

The effects of 4beta-phorbol-12-myristate-13-acetate (PMA) and 1,2-(sn)-dioctanoylglycerol (DOCG) on the phosphatidylcholine (PC) turnover (defined as degradation to diacylglycerol followed by PC resynthesis) and on the activity of PC-specific phospholipase D were investigated in placental mince incubated with various radiolabelled precursors in vitro. Experiments with [32P]phosphate indicated that 1 microM PMA and 125-250 microM DOCG were the lowest concentrations that led to maximal and selective stimulation of PC labelling. Moreover, PMA and DOCG acted along different time courses: PMA enhanced labelling after 60 min incubation, with a lag period of at least 30 min, whereas DOCG stimulated PC labelling after only 30 min with no further increase in the next 30 min. The following findings suggest that increased labelling of PC with [32P]phosphate in PMA-treated tissue reflects an increased rate of PC turnover: (1) the effects of PMA and DOCG were additive and PMA did not have any effect on the labelling of PC(DOCG) indicating that it stimulated PC labelling even if it did not activate CTP:choline cytidylyl transferase, the regulatory enzyme of PC synthesis de novo; (2) PMA did not increase the labelling of PC from [3H]glycerol or [3H]glucose ruling out a PMA-promoted availability of glycolytic and/or lipolytic intermediates for PC formation; and (3) the PMA effect was attended by an increased labelling of phosphatidic acid whereas there was no change in the labelling of lyso-PC, indicating the activation of phospholipase D. Experiments in which the transphosphatidylation reaction between [3H]myristic acid-labelled PC and ethanol was used to estimate phospholipase D activity showed 2.4-fold and 1.4-1.8-fold activations by PMA and DOCG, respectively, with no additivity noted. These results suggest that PMA stimulates PC turnover in the early human placenta via the activation of phospholipase D. Rapid metabolic conversion decreases the capacity of DOCG to accelerate PC-turnover and to activate phospholipase D. The early DOCG-induced stimulation of PC labelling with [32P]phosphate is attributed mainly to its known activating effect on CTP: choline cytidylyl transferase.