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.