v-Src increases diacylglycerol levels via a type D phospholipase-mediated hydrolysis of phosphatidylcholine.

Activating the protein-tyrosine kinase of v-Src in BALB/c 3T3 cells results in rapid increases in the intracellular second messenger, diacylglycerol (DAG). v-Src-induced increases in radiolabeled DAG were most readily detected when phospholipids were prelabeled with myristic acid, which is incorporated predominantly into phosphatidylcholine. Consistent with this observation, v-Src increased the level of intracellular choline. No increase in DAG was observed when cells were prelabeled with arachidonic acid, which is incorporated predominantly into phosphatidylinositol. Inhibiting phosphatidic acid (PA) phosphatase, which hydrolyzes PA to DAG, blocked v-Src-induced DAG production and enhanced PA production, implicating a type D phospholipase. Consistent with the involvement of a type D phospholipase, v-Src increased transphosphatidylation activity, which is characteristic of type D phospholipases. Thus, v-Src-induced increases in DAG most likely result from the activation of a type D phospholipase/PA phosphatase-mediated signaling pathway.     

Tumor necrosis factor alpha priming of phospholipase D in human neutrophils. Correlation between phosphatidic acid production and superoxide generation.

Tumor necrosis factor alpha (TNF) primes human neutrophils (PMN) for enhanced superoxide (O2-) production if cells are subsequently stimulated with the chemotactic peptide, n-formyl-Met-Leu-Phe (fMLP). fMLP activates phospholipase D to form phosphatidic acid (PA), and a correlation may exist between PA production and O2- generation in PMN. Therefore, we assessed the ability of TNF to prime phospholipase D activation in PMN stimulated with fMLP. TNF (100 units/ml) pretreatment primed enhanced PA production in PMN challenged with 1 microM fMLP, in the absence of cytochalasin B, as demonstrated by increased production of tritiated PA from PMN label with 1-O-[9',10'-3H]hexadecyl-2-lyso-sn-glycero-3-phosphocholine ([3H]LPAF) and by increased PA mass. PA was formed via activation of phospholipase D and occurred with minimal production of diglycerides. Production of O2- was also enhanced in identically treated cells, and we demonstrated a direct correlation between enhanced PA formation and O2- production. Conversely, ethanol inhibition of PA formation led to a comparable reduction in O2- generation. This report of priming of phospholipase D by physiological agonists is the only natural system where enhanced PA formation has been dissociated from diglyceride formation. Our results suggest a link between PA production and NADPH oxidase activation in human PMN.     

Stimulation of Phospholipase D Activity by Phorbol Esters in Cultured Astrocytes

The phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) was found to stimulate phospholipase D activity in cultured primary astrocytes. Both the hydrolysis and the transphosphatidylation reaction catalyzed by phospholipase D were studied in cells labeled with [3H]glycerol. Phosphatidic acid (PA) synthesis was increased after addition of 100 nM TPA. When ethanol was present in the cell culture medium, phosphatidylethanol (Peth), a product of phospholipase D-catalyzed transphosphatidylation, was formed. The half-maximum effective concentrations (EC50) of TPA were 25 nM for PA increase as well as for Peth formation. The formation of Peth in ethanol-treated cells was accompanied by an inhibition of the TPA-induced increase in labeled PA. Increasing ethanol concentrations led to an increase in [3H]Peth and a decrease in [3H]PA. A protein kinase C inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7), inhibited both the synthesis of PA and the formation of Peth observed after TPA addition to the astrocytes. Dioctanoyl-glycerol (100 microM) stimulated the formation of Peth in the presence of ethanol. In addition to the induction of Peth formation in astrocytes, TPA induced Peth formation in ethanol-treated neurons. The present results indicate that phospholipase D activity is stimulated by TPA in cultured primary brain cells. Modulation of phospholipase D activity by protein kinase C is a mechanism that may be important in signal transduction cascades.     

Phospholipase D-catalyzed hydrolysis of phosphatidylcholine occurs with P-O bond cleavage

Mammalian phospholipase D has been implicated in signal-transduction mechanisms, most recently in association with stimuli that enhance phosphatidylcholine (PC) turnover. It was previously unknown whether hydrolysis of PC by phospholipase D proceeds via P-O or C-O bond cleavage. Commercially available phospholipase D isolated from Streptomyces chromofuscus was used to hydrolyse distearoyl phosphatidylcholine (PC) in a detergent-containing buffer consisting of 90% 18O-water. The product of hydrolysis, phosphatidic acid (PA), was purified by thin-layer chromatography and analyzed using californium-252 plasma desorption mass spectrometry. An increase of two mass units was observed, compared to a distearoyl PA control, consistent with a reaction mechanism involving cleavage of the P-O bond.     

Phosphatidic acid regulation of phosphatidylinositol 4-phosphate 5-kinases

Phosphatidic acid (PA) production by receptor-stimulated phospholipase D is believed to play an important role in the regulation of cell function. The second messenger function of PA remains to be elucidated. PA can bind and affect the activities of different enzymes and here we summarise the current status of activation of Type I phosphatidylinositol 4-phosphate 5-kinase by PA. Type 1 phosphatidylinositol 4-phosphate 5-kinase is also regulated by ARF proteins as is phospholipase D and we discuss the contributions of ARF and PA towards phosphatidylinositol(4,5)bisphosphate synthesis at the plasma membrane.     

Cardiolipin-specific phospholipase D of Haemophilus parainfluenzae. II. Characteristics and possible significance

A phospholipase specific for cardiolipin (CL) was found in the membrane of Haemophilus parainfluenzae. The enzyme hydrolyzed CL to phosphatidic acid (PA) and phosphatidylglycerol (PG), indicating that it was a phospholipase D (an enzyme activity believed to be confined to higher plants). In addition to its substrate specificity, this enzyme was unusual in its requirement for Mg(2+) (K(m) of 1.3 mm) for maximal activity and its inhibition by chelating agents, heavy metals, some detergents, and organic solvents. When inhibitors of phospholipase activity were added to the growth medium, CL accumulated and PG disappeared in the membrane, suggesting that the phospholipase D was active in vivo. The activity of phospholipase D in cell-free homogenates was greater than expected from earlier studies of CL metabolism and greater than the other phospholipase activities detected in the homogenate. The high activity of the CL-specific phospholipase D suggests there might be a very active degradation of CL to PG and PA and an active resynthesis of CL from the hydrolysis products.     

Elicitation of suspension-cultured tomato cells triggers the formation of phosphatidic acid and diacylglycerol pyrophosphate

Phosphatidic acid (PA) and its phosphorylated derivative diacylglycerol pyrophosphate (DGPP) are lipid molecules that have been implicated in plant cell signaling. In this study we report the rapid but transient accumulation of PA and DGPP in suspension-cultured tomato (Lycopersicon esculentum) cells treated with the general elicitors, N,N',N",N"'-tetraacetylchitotetraose, xylanase, and the flagellin-derived peptide flg22. To determine whether PA originated from the activation of phospholipase D or from the phosphorylation of diacylglycerol (DAG) by DAG kinase, a strategy involving differential radiolabeling with [(32)P]orthophosphate was used. DAG kinase was found to be the dominant producer of PA that was subsequently metabolized to DGPP. A minor but significant role for phospholipase D could only be detected when xylanase was used as elicitor. Since PA formation was correlated with the high turnover of polyphosphoinositides, we hypothesize that elicitor treatment activates phospholipase C to produce DAG, which in turn acts as substrate for DAG kinase. The potential roles of PA and DGPP in plant defense signaling are discussed.     

Characteristics and subcellular localization of phospholipase d and phosphatidic Acid phosphatase in mung bean cotyledons

Mixed micelles of ³²P-labeled phosphatidylcholine or phosphatidic acid (PA) and the nonionic detergent octylphenol polyethylene oxide (NP-40 Nonidet) were used to assay the activities of phospholipase D and PA phosphatase in crude extracts of mung bean (Vigna radiata) cotyledons. Together these enzymes degrade phosphatidylcholine to free choline, inorganic phosphate, and sn-1,2-diacylglycerol. Both enzymes have pH optima around 5.0. The enzymes are present in fully imbibed cotyledons and increase in activity during seedling growth. Fractionation of cotyledon extracts on sucrose gradients showed that the cells contain two PA phosphatases. One enzyme with a pH optimum of 7.5 has the same distribution on sucrose gradient as the endoplasmic reticulum marker enzyme NADH-cytochrome c reductase. The other, PA phosphatase, with a pH optimum of 5.0, was present in a protein body-rich fraction and in the load portion of the gradient. Fractionation of broken protoplasts on Ficoll gradients (a method which allows for the isolation of a high proportion of intact protein bodies) indicates that most of the cellular phospholipase D and PA phosphatase (pH 5.0) are associated with the protein bodies. Using column chromatography (DEAE-cellulose and Sephadex G-200), PA phosphatase (pH 5.0) was found to be a different enzyme from the major acid phosphatase in the cotyledons. Apparent molecular weights of phospholipase D and PA phosphatase were 150,000 and 37,000, respectively. The activity of phospholipase D was not affected by free choline, but was markedly inhibited by the choline analog and plant growth retardant isopropyl 4′-(trimethylammonium chloride-5′-methylphenyl piperidine-1-carboxylate (AMO 1618). The finding that these acid hydrolases are located in the protein bodies supports the conclusion that protein bodies form the general lytic compartment in the storage parenchyma cells.     

Induction of neutrophil calcium mobilization by phosphatidic acid-enriched plasma membranes

The purpose of this investigation was to determine if phosphatidic acid (PA) confined to biological membranes could induce physiological responses similar to those induced by exogenous PA. Plasma membranes were treated with phospholipase D (PLD) to increase concentrations of PA within the membranes. Membranes were also treated with other phospholipases including phospholipase A2 (PLA2), and phospholipase C (PLC), which degrade phospholipids without generating PA. A phosphatidylinositol (PI) 3'-kinase inhibitor, LY294002, strongly and selectively inhibited intracellular calcium mobilization induced by PLD-treated membranes. This study suggests that PA-enriched plasma membranes, which exert their effects by activating a unique signaling pathway mediated by PI 3'-kinase, are potent, physiologically relevant initiators of neutrophil activation.     

Calcium uptake by isolated nerve endings: evidence for a rapid component mediated by the breakdown of phosphatidylinositol

Several physiological stimuli, including neuronal depolarization, increase the production of phosphatidate (PA) from phosphatidylinositol (PI) and increase calcium fluxes across cell membranes. To determine if breakdown of PI is required for neuronal calcium uptake, we tested inhibitors of PI-specific phospholipase C on depolarization-dependent uptake of calcium by isolated brain synaptosomes. At a concentration of 0.1 mM these inhibitors reduced calcium uptake produced by depolarization for 1 to 3 sec, but did not affect uptake due to more prolonged depolarization. Exogenous PA also stimulated calcium accumulation by synaptosomes and this uptake was not reduced by the enzyme inhibitors. These results suggest that the rapid calcium influx produced by neuronal depolarization may be mediated by the breakdown of PI.     

Phosphatidic acid and phospholipase D both stimulate phosphoinositide turnover in cultured human keratinocytes

Phosphatidic acid (PA) induced a rapid dose-dependent increase in production of inositol phosphates in cultured adult human keratinocytes, peaking at 30 s. Natural and dioleoyl PA were equally effective, while other phospholipid classes had no effect. Lipid A was also active. Lyso-PA also induced inositol phosphate production, but contamination of the PA preparation by lyso-PA could not account for the effect of PA. The effect of PA could not be reproduced by treatment of cells with calcium ionophore. PA-induced inositol phosphate production could be inhibited (> 50%) by pre-treatment of cells with either pertussis toxin or 12-O-tetradecanoylphorbol 13-acetate, suggesting the involvement of a GTP-binding protein and a protein kinase C-mediated negative feedback mechanism. PA also stimulated release of arachidonic acid from keratinocytes. Treatment of cells with exogenous phospholipase D similarly induced inositol phosphate production in the keratinocytes. Since PA may be formed by receptor-mediated activation of phospholipase D, or by phosphorylation of diacylglycerol, the results suggest that PA may play a significant role in signalling mechanisms of human keratinocytes.     

Enhanced apoptosis through farnesol inhibition of phospholipase D signal transduction

Farnesol is a catabolite of the cholesterol biosynthetic pathway that preferentially causes apoptosis in tumorigenic cells. Phosphatidylcholine (PC), phosphatidic acid (PA), and diacylglycerol (DAG) were able to prevent induction of apoptosis by farnesol. Primary alcohol inhibition of PC catabolism by phospholipase D augmented farnesol-induced apoptosis. Exogenous PC was unable to prevent the increase in farnesol-induced apoptosis by primary alcohols, whereas DAG was protective. Farnesol-mediated apoptosis was prevented by transformation with a plasmid coding for the PA phosphatase LPP3, but not by an inactive LPP3 point mutant. Farnesol did not directly inhibit LPP3 PA phosphatase enzyme activity in an in vitro mixed micelle assay. We propose that farnesol inhibits the action of a DAG pool generated by phospholipase D signal transduction that normally activates an antiapoptotic/pro-proliferative target.     

Phosphatidic acid that accumulates in platelet-derived growth factor-stimulated Balb/c 3T3 cells is a potential mitogenic signal.

We developed a monoclonal antibody specific to phosphatidic acid (PA). Using this antibody, a novel method to quantify trace amounts of PA was achieved. With the method, PA can be measured in the range of 20-500 pmol. We applied this method to quantify changes in PA levels in Balb/c 3T3 cells stimulated by platelet-derived growth factor. PA contents were very low in quiescent cells and dramatically increased with time up to 15 min. On the other hand, a biphasic diacylglycerol (DG) increase was found. The early phase showed a transient small peak of DG at 30 s followed by a decrease to 1 min. In the second phase, DG accumulated gradually but very markedly up to 15 min. Treatment with propranolol, a PA phosphohydrolase inhibitor, enhanced the accumulation of PA and inhibited the formation of DG in the second phase. However, R59022, a DG kinase inhibitor, did not influence the accumulation of DG or PA, suggesting that platelet-derived growth factor stimulates mainly phospholipase D-catalyzed hydrolysis of phospholipids rather than phospholipase C-catalyzed hydrolysis in the second phase. PA, even after contaminating lyso-PA was removed, could stimulate DNA synthesis, although lyso-PA was 25 times more potent. Moreover, phospholipase D was found to be a much stronger mitogen than phospholipase C. Phospholipase D treatment caused a biphasic accumulation of PA. PA levels reached a maximum at 1 h, and then decreased between 1 and 2 h; finally, there was a gradual elevation up to 10 h. In this case, there was no significant DG accumulation. On the other hand, phospholipase C treatment induced only DG accumulation without any significant change in PA. These results indicate that PA accumulation, rather than an increase in DG, correlates well with mitogenesis.     

Modulation of cyclooxygenase-2 expression by phosphatidylcholine specific phospholipase C and D in macrophages stimulated with lipopolysaccharide

Lipopolysaccharide (LPS) enhances the expression of cyclooxygenase 2 (COX-2) in macrophages, and stimulates production of prostaglandins that cause endothelial dysfunction in septic shock. In an effort to identify strategies for reducing LPS-inducible expression of COX-2, inhibitors of the phospholipases involved in LPS dependent over-expression of COX-2 were studied. LPS enhances expression of COX-2 mRNA and protein by activating sequentially phosphatidylcholine-specific phospholipase C (PC-PLC), protein kinase C (PKC) and phosphatidylcholine-specific phospholipase D (PC-PLD). This stimulates production of phosphatidic acid (PA), which increases expression of COX-2 mRNA and protein. Inhibition of PC-PLC by D609 (tricyclodecanoyl xanthogenate), and of PC-PLD activity by 1-butanol, reduced LPS-dependent over-production of PA and suppressed the increase of COX-2 mRNA and protein. Activation of PKC, normally seen in LPS-treated cells, was mimicked with phorbol myristic acid (PMA), and this also increased PA production and enhanced COX-2 expression. Propranolol inhibition of phosphatidic acid phosphohydrolase (PPH) increased PA accumulation and enhanced LPS-dependent COX-2 protein synthesis. These results suggest that inhibitors of PC-PLC, PKC and PC-PLD, or activators of PPH could be useful in the management of LPS-induced overproduction of prostaglandins and of vascular dysfunction in septic shock.     

Age-related changes in the metabolization of phosphatidic acid in rat cerebral cortex synaptosomes

In this study, phosphatidic acid (PA) metabolization is found to generate diacylglycerol (DAG), monoacylglycerol (MAG) and glycerol by the sequential action of lipid phosphate phosphatase (LPP), diacylglycerol lipase (DAGL), and monoacylglycerol lipase (MAGL) in cerebral cortex (CC) synaptosomes. It is also demonstrated that PA is metabolized by phospholipases A (PLA)/lysophosphatidic acid phosphohydrolase (LPAPase) in synaptic endings. Age-related changes in the metabolization of PA have been observed in rat cerebral cortex synaptosomes in the presence of the alternative substrates for LPP, namely LPA, sphingosine 1-phosphate (S1P) and ceramide 1-phosphate (C1P). In addition, LPA and C1P up to concentrations of about 50 μM favor the metabolism in the direction of MAG and glycerol in aged and adult synaptosomes, respectively. At equimolecular concentrations with PA, LPA decreases DAG formation in adult and aged synaptosomes, whereas S1P decreases it and C1P increases it only in aged synaptosomes. Sphingosine (50 μM) or ceramide (100 μM) increase PA metabolism by the pathway that involves LPP/DAGL/MAGL action in aged membranes. Using RHC-80267, a DAGL inhibitor, we could observe that 50% and 33% of MAG are produced as a result of DAGL action in adult and aged synaptosomes, respectively. Taken together, our findings indicate that the ageing modifies the different enzymatic pathways involved in PA metabolization.     

A novel mechanism for acetylcholine to generate diacylglycerol in brain

The classical scheme involving inositol phospholipid breakdown by phospholipase C as the sole source of diacylglycerol (DAG) has recently been challenged by evidence that phosphatidylcholine (PC) is an alternative source. In synaptic membranes of canine cerebral cortex, cholinergic agonists caused rapid accumulation of [3H]phosphatidic acid (PA) from [3H]PC within 15 s, whereas [3H]DAG formation showed a transient lag period before becoming elevated and then exceeding the amount of [3H]PA. Additional evidence shows that DAG is produced from PC by the action of phospholipase D to yield PA, which is further dephosphorylated to DAG by PA phosphatase. Our results indicate that this muscarinic acetylcholine receptor-regulated PC phospholipase D-PA phosphatase pathway may be a novel mechanism in cell signal transduction processes for activation of protein kinase C in brain.     

磷脂酸的诱导和降解在植物逆境响应中的作用

磷脂酸(phosphatidic acid,PA)是植物中重要的脂质信号分子,被称为"脂质第二信使",参与多种逆境胁迫相关的信号传导途径.植物体内的PA可通过直接的磷脂酶D途径和间接的磷脂酶C途径产生.当植物受到胁迫刺激后,细胞内的PA含量会在几分钟内升高,在胁迫消失后经磷酸化作用形成甘油二酯焦磷酸降解,恢复到正常水平...     

Insulin effect on isolated rat hepatocytes: diacylglycerol-phosphatidic acid interrelationship.

It is widely accepted that insulin action does not involve inositol phospholipid hydrolysis through the stimulation of a phosphatidylinositol-specific phospholipase C (PI-PLC). This consideration prompted us to investigate the insulin effect on the mechanism leading to the accumulation of diacylglycerol (DAG) and phosphatidic acid (PA) in rat hepatocytes. Basically, insulin induces: (i) a significant increase of both [3H]glycerol and fatty acid labelling of DAG; (ii) a significant increase of PA labelling preceding DAG labelling and paralleled by a decrease of phosphatidylcholine (PC) labelling. These observations, which suggest an insulin-dependent involvement of a phospholipase D, are strengthened by the increase of PC-derived phosphatidylethanol in presence of ethanol. Finally, the observation that the PA levels do not return to basal suggests that other mechanisms different from PC hydrolysis, such as the stimulation of direct synthesis of PA, may be activated.     

Nod factor and elicitors activate different phospholipid signaling pathways in suspension-cultured alfalfa cells

Lipo-chitooligosaccharides (Nod factors) are produced by symbiotic Rhizobium sp. bacteria to elicit Nod responses on their legume hosts. One of the earliest responses is the formation of phosphatidic acid (PA), a novel second messenger in plant cells. Remarkably, pathogens have also been reported to trigger the formation of PA in nonlegume plants. To investigate how host plants can distinguish between symbionts and pathogens, the effects of Nod factor and elicitors (chitotetraose and xylanase) on the formation of PA were investigated in suspension-cultured alfalfa (Medicago sativa) cells. Theoretically, PA can be synthesized via two signaling pathways, i.e. via phospholipase D (PLD) and via phospholipase C in combination with diacylglycerol (DAG) kinase. Therefore, a strategy involving differential radiolabeling with [(32)P]orthophosphate was used to determine the contribution of each pathway to PA formation. In support, PLD activity was specifically measured by using the ability of the enzyme to transfer the phosphatidyl group of its substrate to a primary alcohol. In practice, Nod factor, chitotetraose, and xylanase induced the formation of PA and its phosphorylated product DAG pyrophosphate within 2 min of treatment. However, whereas phospholipase C and DAG kinase were activated during treatment with all three different compounds, PLD was only activated by Nod factor. No evidence was obtained for the activation of phospholipase A(2).     

Synaptosomal Phospholipase D Potential Role in Providing Choline for Acetylcholine Synthesis

The phospholipase D of the rat brain synaptic membrane possesses the highest activity of this enzyme of any mammalian tissue examined. The synaptic phospholipase D activity is latent and barely detectable in the absence of 4 mM sodium oleate. Several other fatty acids were either less effective or ineffective as stimulators of activity compared to this monounsaturated fatty acid. The activity was decreased by hemicholinium-3, an inhibitor of choline uptake and slightly activated by neostigmine, an acetylcholinesterase inhibitor. Incubation of synaptosomes in the presence of sodium oleate and acetyl-coenzyme A resulted in the formation of a product chromatographing with acetylcholine. Acetylcholine formation was nearly undetectable in the absence of sodium oleate or acetyl-coenzyme A. These results implicate synaptosomal phospholipase D in releasing choline from phosphatidylcholine for acetylcholine formation.