Platelet-activating factor stimulates phosphatidic acid formation in cultured rat mesangial cells: Roles of phospholipase D, diglyceride kinase, and de novo phospholipid synthesis

Platelet-activating factor (PAF) stimulates phospholipase C (PLC)-induced hydrolysis of phosphatidylinositol-4,5-bisphosphate (Ptdlns-4,5-P₂). Yet, PAF-stimulated diglycerides (DG) are still elevated at time points where inositol polyphosphates have returned to basal levels. Thus, other signal transduction pathways that hydrolyze phosphatidylcholine (PtdCho) or phosphatidylethanolamine (PtdEth) and form DG and phosphatidic acids (PA) through either PLC or phospholipase D (PLD) may also mediate PAF-stimulated cellular responses. Initially the effects of PAF upon ³²P-PA generation in mesangial cells (MC) were assessed. PA formation may be indicative of several metabolic pathways including PLD and DG kinase activities as well as de novo phospholipid synthesis. PAF (10⁻⁷ M) increased ³²P-PA formation as early as 5 seconds and this elevation persisted up to 15 minutes. When MC were pretreated with the DG kinase inhibitor-R59022, PAF-induced ³²P-PA formation was diminished at early but not late time points, demonstrating that the initial component of PA formation may be due, in part, to PLC activation and subsequent phosphorylation of DG. The reciprocal reaction, PA phosphohydrolase, which dephosphorylates PA to from DG was not stimulated by PAF, suggesting that the sustained elevation of DG induced by PAF is primarily a reflection of PLC. ³H-glycerol pulse-labeling experiments suggest that PAF also stimulates de novo phospholipid synthesis which also contributes to PA formation. Conclusive proof for PLD in the generation of PA was obtained by assessing the formation of ³H-phosphatidylethanol (PEt) from ³H-alkyl-lyso-glycero phosphocholine (GPC) and exogenous ethanol. PAF stimulated alkyl-PEt generation in the presence but not the absence of 0.5% ethanol. Also, PAF induced a concomitant elevation of alkyl-PA at 15 minutes and this elevation of alkyl-PA was reduced when the cells were exposed to exogenous ethanol, reflecting the formation of PEt. Corroborating evidence suggests that PAF stimulates ³H-choline and ³H-ethanolamine release, suggesting that PtdCho and PtdEth are substrates for PLD. Thus, these data demonstrate that MC respond to PAF with elevated PLD and DG kinase activities as well as with an increased rate of de novo lipid synthesis which increases PA, a potential intracellular signal. © 1993 Wiley-Liss, Inc.