Stimulation of cells with certain agonists often activates both phospholipases C and D. These generate diacylglycerol and phosphatidate, respectively, although the two lipids are also apparently interconvertable through the actions of phosphatidate phosphohydrolase and diacylglycerol kinase. Diacylglycerol activates protein kinase C while one role for phosphatidate is the activation of actin stress fiber formation. Therefore, if the two lipids are interconvertable, it is theoretically possible that an uncontrolled signaling loop could arise. To address this issue structural analysis of diacylglycerol, phosphatidate, and phosphatidylbutanol (formed in the presence of butan-1-ol) from both Swiss 3T3 and porcine aortic endothelial cells was performed. This demonstrated that phospholipase C activation generates primarily polyunsaturated species while phospholipase D activation generates saturated/monounsaturated species. In the endothelial cells, where phospholipase D was activated by lysophosphatidic acid independently of phospholipase C, there was no activation of protein kinase C. Thus we propose that only polyunsaturated diacylglycerols and saturated/monounsaturated phosphatidates function as intracellular messengers and that their interconversion products are inactive.Stimulation of cells by particular agonists which occupy either heterotrimeric G-protein-coupled receptors or those with an intrinsic tyrosine kinase activity induce an increase in the mass of diradylglycerols (collectively diacylglycerol, alkyl, acylglycerol and alkenyl, acylglycerol; DRG), 1 in particular sn-1,2-diacylglycerol (DAG), the physiological activator of protein kinase C (PKC) (1). DAG is produced, together with inositol 1,4,5-trisphosphate which stimulates the elevation of intracellular free calcium concentration, by phospholipase C (PLC)-catalyzed phosphatidylinositol 4,5-bisphosphate hydrolysis. Agonist stimulation of this pathway is rapidly desensitized, DAG generation has been demonstrated to be rapid, but transient, declining toward basal levels within 1-2 min (2, 3). However, there is frequently a second sustained phase of DAG generation. This phase has been associated with an increase in the activation of phospholipase D (PLD)-catalyzed phosphatidylcholine (PC) hydrolysis, producing phosphatidate (PA) which can be converted to DAG by the action of phosphatidate phosphohydrolase. It has also been proposed that DAG can be derived from other pathways, e.g. through a PC-PLC pathway, although the evidence for stimulation of this pathway in mammalian cells remains mostly circumstantial (4, 5).Cells contain multiple species of DAG, however, a limited subset of these change following stimulation. Comparison of the acyl chain DAG structures with those of the cellular phospholipids indicated that the initial phase of DAG increase was predominantly from inositol phospholipids, while the sustained phase, which was accompanied by an increase in choline release, was probably produced from PC (6 -9). The initial phase of DAG generation was ma...

The mechanism of inhibition of phospholipase D (PLD) by ceramides was determined using granulocytes differentiated from human promyelocytic leukemic (HL-60) cells. In a cell-free system, hydrolysis of phosphatidylcholine by membrane-bound PLD depended upon phosphatidylinositol 4,5-bisphosphate, guanosine 5-3-O-(thio)triphosphate) (GTP␥S), and cytosolic factors including ADP-ribosylating factor (ARF) and RhoA. C 2 -(N-acetyl-), C 8 -(N-octanoyl-), and long-chain ceramides, but not dihydro-C 2 -ceramide, inhibited PLD activity. Apyrase or okadaic acid did not modify the inhibition of PLD by ceramides, indicating that the effect in the cellfree system was unlikely to be dependent upon a ceramide-stimulated kinase or phosphoprotein phosphatases. C 2 -and C 8 -ceramides prevented the GTP␥S-induced translocation of ARF1 and RhoA from the cytosol to the membrane fraction. In whole cells, C 2 -ceramide, but not dihydro-C 2 -ceramide, inhibited the stimulation of PLD by N-formylmethionylleucylphenylalanine and decreased the amounts of ARF1, RhoA, CDC42, Rab4, and protein kinase C-␣ and -␤ 1 that were associated with the membrane fraction, but did not alter the distribution of protein kinase C-⑀ and -. It is concluded that one mechanism by which ceramides prevent the activation of PLD is inhibition of the translocation to membranes of G-proteins and protein kinase C isoforms that are required for PLD activity. PLD1 in mammalian cells plays a key role in signal transduction and its activation occurs in a wide range of cell types in response to hormones and growth factors. Several components such as G-proteins, PKC, and Ca 2ϩ are involved in regulating PLD (for review, see Refs. 1 and 2). PLD catalyzes the hydrolysis of cell phospholipids, mainly PC, resulting in the formation of PA, which may also be the precursor of lysoPA. These two lipids are bioactive, and they have many effects that are similar. PA or lysoPA been reported to stimulate the respiratory burst in neutrophils (3), monoacylglycerol acyltransferase (4), phospholipase C␥ (5), phosphatidylinositol-4-phosphate kinase (6), PKC- (7), and the Ras-Raf-mitogen-activated protein kinase pathway (8) and to inhibit adenylate cyclase (9). PA may also be dephosphorylated by phosphatidate phosphohydrolase to diacylglycerol (10), a well characterized activator of PKC (2).In cell-free assays, the activation of membrane-associated PLD by GTP␥S is dependent on the presence of both membranes and cytosol components. The latter consist of small G-proteins of the Ras superfamily, such as ARF (11, 12), RhoA (13, 14), and CDC42 (15, 16). ARF was first identified as a cofactor necessary for the ADP-ribosylation of the ␣-subunit of heterotrimeric G-proteins, i.e. G s , by cholera toxin (17). ARF has also been implicated in vesicular transport in the Golgi (18) and in endocytosis (19). ARF-stimulated PLD has been partially purified from HL-60 cell membranes, and this stimulation was dependent on the presence of PIP 2 (12). Subsequently, ARF-stimulated PLD was separated from oleate-stim...

Concentrations of the bioactive lipids, phosphatidate and diacylglycerol, increased with time in culture in ras-and tyrosine kinase (fps)-transformed ®broblasts but not in control ®broblasts. On Day 3, diacylglycerol and phosphatidate concentrations were about 3.3-and 5.5-fold higher respectively in the ras-transformed compared to control ®broblasts. These concentrations in fpstransformed ®broblasts were increased about twofold. The changes in phosphatidate and diacylglycerol resulted from enhanced phospholipid turnover rather than from synthesis de novo. The increased ratio of phosphatidate to diacylglycerol is explained by decreased activities of two distinct phosphatidate phosphohydrolases and increased diacylglycerol kinase in ras-transformed ®bro-blasts. Ceramide concentrations were about 2.5-and threefold higher in the fps-and ras-transformed cells respectively on Day 3 compared to the controls. Incubating control ®broblasts from Days 1 to 3 with phosphatidylcholine-speci®c phospholipase C increased diacylglycerol, phosphatidate and ceramide concentrations, and decreased Mg 2+ -independent-phosphatidate phosphohydrolase activity. 8-(4-chlorophenylthio)-cAMP had a cytostatic e ect in ras-transformed cells, it decreased the concentrations of phosphatidate and diacylglycerol, but increased that of ceramide. The consequences of increased ceramide and phosphatidate concentrations in ras-transformed cells are discussed in relation to signal transduction, cell division and the transformed phenotype.

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