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Multiple sources of sn-1,2-diacylglycerol in platelet-derived-growth-factor-stimulated Swiss 3T3 fibroblasts. Evidence for activation of phosphoinositidase C and phosphatidylcholine-specific phospholipase D.

ABSTRACT: Platelet-derived growth factor (PDGF) stimulated sn-1,2-diacylglycerol (DAG) mass formation in Swiss 3T3 fibroblasts with a lag time of some 30 s. The response was biphasic, with the second phase being sustained over time. PDGF also stimulated the formation of Ins(1,4,5)P3 with a similar lag time to the DAG response, suggesting that DAG is derived from PtdIns(4,5)P2 hydrolysis at this time point. PDGF-stimulated phosphatidylcholine (PtdCho) hydrolysis in Swiss 3T3 fibroblasts, as measured by the formation of water-soluble choline metabolites and phosphatidylbutanol (PtdBut) accumulation, was by a phospholipase D (PLD)-catalysed pathway which was kinetically downstream of initial PtdIns(4,5)P2 hydrolysis. Accumulation of PtdBut increased up to 15 min, suggesting that PLD activity is not rapidly densitized in response to PDGF. The kinetics of PtdCho hydrolysis closely paralleled the second phase of DAG formation, strongly suggesting that during prolonged stimulation periods PtdCho is a major source of DAG in these cells. However, since PtdIns(4,5)P2 breakdown was also prolonged, PDGF-stimulated DAG may be derived from both phospholipids. Down-regulation of protein kinase C (PKC), by pre-treatment with phorbol 12-myristate 13-acetate, abolished both [3H]choline and [3H]PtdBut formation, suggesting that PLD-catalysed PtdCho hydrolysis may be dependent on PKC activation, supporting its dependence on prior PtdIns(4,5)P2 hydrolysis.

SUBMITTER: Plevin R

PROVIDER: S-EPMC1151640 | biostudies-other | 1991 Oct

REPOSITORIES: biostudies-other

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Project description:Treatment of Swiss-mouse 3T3 fibroblasts with a tumour-promoting phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), increases the absolute amount of 1,2-diacylglycerol (1,2-DG) in a time- and dose-dependent manner. Within 1 min of TPA (0.1 microM) addition to confluent monolayer cells, cellular 1,2-DG content increases significantly, and reaches a value nearly 4-fold above control at 10 min. The content of 1,2-DG then falls, but still remains at twice the basal value for up to 60 min. The effect of TPA on 1,2-DG content is dose-dependent, with a half-maximal effect obtained with 6 nM. Studies with a series of tumour promoters have revealed that stimulating effect on 1,2-DG formation is closely related to their relative potency as a tumour promoter, as well as their mitogenic effect on 3T3 cells. TPA does not have any stimulatory effect on phosphoinositide turnover, which is a major mechanism of 1,2-DG formation by natural agonists, including a peptide mitogen, bombesin, but significantly inhibits bombesin-induced phosphoinositide turnover. Nevertheless, the content of 1,2-DG is even higher in cells treated with both bombesin and TPA than in cells treated with bombesin alone. In addition, a diacylglycerol kinase inhibitor, R59022, increases 1,2-DG content synergistically with bombesin, but not with TPA. Measurements of the absolute amounts of various phospholipids show that TPA does not increase phosphatidic acid, despite a remarkable increase in 1,2-DG. Further studies on phospholipid metabolism reveal that TPA stimulates metabolic turnover of phosphatidylcholine (PC), without changing the mass of PC. TPA stimulates the incorporation of both [3H]choline and [32P]Pi into PC, and the release of [3H]choline metabolites from prelabelled cells. These findings suggest that TPA increases cellular 1,2-DG content mainly via increased PC turnover and diacylglycerol kinase inhibition. The present study provides evidence for a novel effect of TPA on the metabolism of 1,2-DG in Swiss-mouse 3T3 cells.

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Multiple sources of sn-1,2-diacylglycerol in platelet-derived-growth-factor-stimulated Swiss 3T3 fibroblasts. Evidence for activation of phosphoinositidase C and phosphatidylcholine-specific phospholipase D.

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