Phorbol myristate acetate stimulates [3H]choline incorporation into phosphatidylcholine independently of the ‘de novo’ pathway in Krebs-II ascitic cells: a unique effect of phorbol ester on choline uptake

Abstract

The effect of phorbol 12-myristate 13-acetate (PMA) on [3H]choline incorporation into phosphatidylcholine (PtdCho) and on the ‘de novo’ pathway of PtdCho synthesis has been investigated, compared with that of oleic acid, in ascitic-strain Krebs-II cells. Both compounds stimulated [3H]choline incorporation into PtdCho, but the PMA-induced incorporation was saturable at concentrations of the agonist around 100 nM, whereas no saturation was noticed with oleic acid up to 1 mM. Chase experiments showed no effect of PMA on the conversion of phosphocholine into CDP-choline. The phorbol ester did not stimulate any of the enzyme activities of the ‘de novo’ pathway, whereas oleic acid increased specifically by 2.5-fold the CTP:phosphocholine cytidylyltransferase (CT, EC 2.7.7.15) activity. In addition, no change in the subcellular distribution of CT was observed upon incubation with PMA, in contrast with oleic acid treatment. Cells challenged with oleic acid showed a 25-fold increase in diradylglycerol (DG) content, which was not modified upon incubation with 200 nM PMA, the most effective concentration of phorbol ester promoting choline incorporation. Subcellular fractionation of Krebs-II cells on Percoll gradients revealed that [3H]PMA and 1-radyl-2-[3H]oleoyl-glycerol, derived from exogenously supplied [3H]oleic acid, both exhibited the same enrichment in the endoplasmic reticulum. We have previously shown that the labelled fatty acid also accumulated in the endoplasmic reticulum [Tercé, Record, Tronchère, Ribbes and Chap (1992) Biochem. J. 282, 333-338]. However, PMA induced a stimulation of choline uptake, which was not provoked by PMA 4-O-methyl ether, which interacts poorly with protein kinase C. Our data provide evidence that the enhancement of [3H]choline incorporation into PtdCho triggered by PMA and oleic acid proceeds via completely distinct mechanism(s).