Arachidonic acid liberation induced by phosphatidic acid endogenously generated from membrane phospholipids in rabbit platelets
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Phospholipase D
Phosphatidylethanol
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Phospholipase D has been shown to be a key enzyme in the signal transduction systems involved in neutrophil activation. In the presence of ethanol, the enzyme catalyzes a transphosphatidylation reaction through which phosphatidylethanol is formed instead of the normal product phosphatidic acid. The effects of ethanol on the formation of phosphatidylethanol and phosphatidic acid was studied in neutrophils from human alcoholics in vitro. Neutrophils were isolated and cellular lipids were labeled with [3H]oleate, whereafter the cells were preincubated with cytochalasin B. Subsequently, cells were stimulated with the chemotactic peptide formyl-Met-Leu-Phe in the presence of ethanol concentration ranging from 0 to 200 mM. In the presence of ethanol, both neutrophils from alcoholics and controls produced phosphatidylethanol, with a concomitant reduction of the production of phosphatidic acid. The amounts of phosphatidyl-ethanol and phosphatidic acid formed were dependent on the concentration of ethanol. In neutrophils from alcoholics, a higher apparent Km for the phospholipase D-mediated transphosphatidylation reaction was noted (58 mM ethanol compared with 28 mM in controls). The in vivo mass of phosphatidylethanol in recently drinking alcoholics was also analyzed in neutrophils. Measurable phosphatidyl-ethanol levels (average 5.6 pmol/10(8) neutrophils) were found in alcoholics up to 23 hr after the last intake of ethanol. Thus, in addition to the ethanol-induced changes in the normal production of phosphatidic acid, phosphatidylethanol accumulated in vivo in alcoholics may be expected to influence neutrophil function.
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Phosphatidylethanol is a unique phospholipid which is formed in cell membranes only in the presence of ethanol. The reaction is catalysed by phospholipase D, an enzyme that normally catalyses the hydrolysis of phospholipids leading to the formation of phosphatidic acid. However, phospholipase D also utilizes short-chain alcohols as substrates resulting in the formation of the corresponding phosphatidylalcohol. This is a specific mechanism through which ethanol may interact with cell function. Phospholipase D is activated by several different receptors and has during recent years been suggested to play a role in cellular signalling. Secretory processes as well as long-term changes of cell function have been associated with the activation of phospholipase D. Since ethanol competes with water as a substrate for this enzyme, phosphatidylethanol is formed at the expense of the normal lipid product, phosphatidic acid, in an ethanol concentration-dependent manner. Therefore, the phospholipase D-mediated signal transduction diverges from production of the normal signalling lipid in the presence of ethanol. However, phosphatidic acid may also be formed by other pathways and their relative contribution to the formation of this lipid depends on the cell and receptor type. Thus, it is important to identify the signalling systems where phospholipase D dominates the lipid messenger production since these may be especially vulnerable to ethanol. In addition to a change in phospholipase D-mediated signal transduction, accumulation of phosphatidylethanol in cell membranes may also induce disturbances in cell function. Significant amounts of this abnormal phospholipid have been detected after ethanol exposure in brain and other organs from rat, in cultured cells as well as in human blood cells. The degradation of phosphatidylethanol is relatively slow and it remains in the cells after ethanol has disappeared. It is possible that an abnormal phospholipid that accumulates in cell membranes affects membrane-associated processes. Phosphasidylethanol is a lipid with a small, anionic head group and its biophysical properties are different compared with other phospholipids. Moreover, this lipid has been demonstrated to influence membrane charactenstics, enzyme activities and levels of signalling molecules. Thus, both the inhibition of phospholipase D-mediated signal transduction and the accumulation of phosphatidylethanol represent possible pathways through which ethanol may disturb cell function.
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Rapid activation of phospholipase D (PLD) in response to cell stimulation was recently demonstrated in many systems, raising the hypothesis that PLD participates in transduction of extracellular signals across the plasma membrane. In the present study, we describe the identification of a neutral PLD activity in purified rat brain synaptic plasma membranes, and the in vitro conditions required to assay its catalytic activity with exogenous [3H]phosphatidylcholine as substrate. Production of [3H]phosphatidic acid, the natural lipid product of PLD and of [3H]phosphatidylethanol, catalyzed by PLD in the presence of ethanol via transphosphatidylation, were measured. The synaptic membrane PLD exhibited its highest activity at pH 7.2 and was thus defined as a neutral PLD. Enzyme activity was absolutely dependent on the presence of sodium oleate and was strongly activated by Mg2+ ions (at 1 mM). Ca2+ at concentrations up to 0.25 mM was as stimulatory as Mg2+, but at 2 mM it completely inhibited enzyme activity. Mg2+ extended the linear phase of PLD activity from 2 to 15 min, suggesting that it may stabilize the enzyme under our assay conditions. The production of [3H]phosphatidylethanol was a saturable function of ethanol concentration. Production of [3H] phosphatidic acid was inversely related to the concentration of ethanol and to the accumulation of phosphatidylethanol, indicating that the two phospholipids are indeed produced by the competing hydrolase and transferase activities of the same enzyme. beta,beta-Dimethylglutaric acid, utilized previously as a buffer in studies of rat brain PLD, inhibited enzyme activity at neutral pH but not at acidic pH. The properties of the neutral synaptic membrane PLD and its relationships with other in vitro, acid, and neutral PLD activities, as well as with the signal-dependent PLD detected in intact cells, are discussed.
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Abstract Objective. To investigate the involvement of phospholipase D in the signaling pathways activated by 2 pathologically relevant inflammatory microcrystals, monosodium urate (MSU) and calcium pyrophosphate dihydrate (CPPD). Methods. Human peripheral blood neutrophils were used throughout. Phospholipase D activity was monitored by measuring 3 separate indices: 1) the mass of phosphatidic acid, 2) the levels of alkyl‐phosphatidic acid, and 3) the levels of formation, in the presence of ethanol, of phosphatidylethanol. The latter 2 parameters were measured in cells labeled with 1‐0‐ 3 H–alkyl‐2‐acetyl‐sn‐glycero‐3‐phosphocholine. The cells were stimulated with microcrystals of triclinic morphology. Results. Both MSU and CPPD crystals induced a time‐ and concentration‐dependent accumulation of phosphatidic acid mass and elevation in levels of alkyl‐phosphatidic acid and phosphatidylethanol in prelabeled cells. The activation of phospholipase D by the microcrystals was partially sensitive to colchicine and largely resistant to pertussis toxin. Inhibition of phosphatidic acid formation by wortmannin or ethanol reduced the microcrystal‐stimulated production of superoxide anions. Conclusion. These results indicate that microcrystals stimulate phospholipase D in human neutrophils and that at least some of the functional consequences of neutrophil‐microcrystal interactions may be dependent on this biochemical pathway.
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A substrate specificity of cabbage phospholipase D (PLD) was studied using the synthetic phospholipids having different head groups. The phospholipids were synthesized from phosphatidylcholine and appropriate bases by transphosphatidylation of PLD. The bases used were ethanolamine, serine, ethanol and γ-hydroxybutyric acid. The phosphatidic acid, the product of PLD, was separated in TLC and measured densitometrically. The kinetic parameters were estimated for each substrate and the effects of pH, SDS, Ca2+ and other metal ions were examined. Vmax values found were 3.75, 2.36, 5.59, 1.63, 2.30 nmol/min/μg protein for phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylethanol, and phosphatidylburytic acid, respectively. These results indicate a broad specificity of cabbage PLD toward phospholipids with different head groups. Particularly phosphatidylserine was most easily hydrolyzed by PLD and its activity did not depend on Ca2+.
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To evaluate the role of the C2 domain in protein kinase Cepsilon (PKCepsilon) localization and activation after stimulation of the IgE receptor in RBL-2H3 cells, we used a series of mutants located in the phospholipid binding region of the enzyme. The results obtained suggest that the interaction of the C2 domain with the phospholipids in the plasma membrane is essential for anchoring the enzyme in this cellular compartment. Furthermore, the use of specific inhibitors of the different pathways that generate both diacylglycerol and phosphatidic acid has shown that the phosphatidic acid generated via phospholipase D (PLD)-dependent pathway, in addition to the diacylglycerol generated via phosphoinosite-phospholipase C (PLC), are involved in the localization of PKCepsilon in the plasma membrane. Direct stimulation of RBL-2H3 cells with very low concentrations of permeable phosphatidic acid and diacylglycerol exerted a synergistic effect on the plasma membrane localization of PKCepsilon. Moreover, the in vitro kinase assays showed that both phosphatidic acid and diacylglycerol are essential for enzyme activation. Together, these results demonstrate that phosphatidic acid is an important and essential activator of PKCepsilon through the C2 domain and locate this isoenzyme in a new scenario where it acts as a downstream target of PLD.
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Phospholipase D
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