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PubMed2002

Purification and properties of a phospholipase A2/lipase preferring phosphatidic acid, bis(monoacylglycerol) phosphate, and monoacylglycerol from rat testis.

Phospholipase A(2) (PLA(2)) was purified to homogeneity from the supernatant fraction of rat testis homogenate. The purified 63-kDa enzyme did not require Ca(2+) ions for activity and exhibited both phosphatidic acid-preferring PLA(2) and monoacylglycerol lipase activities with a modest specificity toward unsaturated acyl chains. Anionic detergents enhanced these activities. Serine-modifying irreversible inhibitors, (p-amidinophenyl) methanesulfonyl fluoride and methylarachidonyl fluorophosphonate, inhibited both activities to a similar extent, indicating a single active site is involved in PLA(2) and lipase activities. The sequence of NH(2)-terminal 12 amino acids of purified enzyme was identical to that of a carboxylesterase from rat liver. The optimal pH for PLA(2) activity (around 5.5) differed from that for lipase activity (around 8.0). At pH 5.5 the enzyme also hydrolyzed bis(monoacylglycerol) phosphate, or lysobisphosphatidic acid (LBPA), that has been hitherto known as a secretory PLA(2)-resistant phospholipid and a late endosome marker. LBPA-enriched fractions were prepared from liver lysosome fractions of chloroquine-treated rats, treated with excess of pancreatic PLA(2), and then used for assaying LBPA-hydrolyzing activity. LBPA and the reaction products were identified by microbore normal phase high performance liquid chromatography/electrospray ionization ion-trap mass spectrometry. These enzymatic properties suggest that the enzyme can metabolize phosphatidic and lysobisphosphatidic acids in cellular acidic compartments.

Acyltransferases↗
PubMed1992

Artifactual Elevation of the Apparent Levels of Phosphatidic Acid and Phosphatidylinositol 4,5-Bisphosphate during Short-Term Labeling of Plant Tissue with Radioactive Precursor.

Some pulvini of Samanea saman Mehr. labeled with radioactive phosphate show apparent remarkable elevations of the levels of phosphatidic acid and phosphatidylinositol 4,5-bisphosphate. The elevated levels, however, appear to be illusory and to result from rapid initial incorporation of label into these phospholipids relative to others. These results demonstrate the need for caution in interpreting apparent changes in the levels of phosphatidic acid or inositol phospholipids in cultures or plants labeled with radioactive precursors.

Journal Article↗
PubMed1977

The effect of pentoxifylline on erythrocyte deformability and on phosphatide fatty acid distribution in the erythrocyte membrane.

Ten maturity-onset diabetics with chronic vascular disease were treated with 400 mg pentoxifylline 3-times daily for 14 days. Erythrocyte deformability (using a filtration technique for whole blood) and phosphatide fatty acid distribution in the erythrocyte membrane were measured before and after the treatment period. Statistical analysis of the data showed that the erythrocyte filtration rate had increased significantly by the end of treatment (2alpha = 0.02), and that there were only slight changes in erythrocyte membrane phosphatide fatty acid levels. The drug was well tolerated, and there were no adverse laboratory findings in the parameters measured. On the basis of results described by other investigators, theimprovement in erythrocyte deformability was attributed to an increase in erythrocyte ATP levels. The authors discuss the importance of red cell fluidity for capillary perfusion.

Aged↗
PubMed1968

Cytidine triphosphate: phosphatidic acid cytidyltransferase in Escherichia coli.

An enzyme has been found in particulate fractions of Escherichia coli that catalyzes the incorporation of cytidine triphosphate (CTP) into lipid in the presence of exogenous phosphatidic acid and Mg(++). The product has been identified enzymatically and by chromatography as cytidine diphosphate diglyceride. The reaction is optimal at a pH of 6.5 and Mg(++) concentration of 5-10 mm. The apparent K(m) for CTP is 7 x 10(-4)M and for phosphatidic acid, 2 x 10(-3)M. The reaction rate falls off rapidly with time and ceases entirely after 1 hr as the result of inactivation of the system by Mg(++).

Cytosine Nucleotides↗
PubMed1966

New, partially hydrolyzable synthetic analogues of lecithin, phosphatidyl ethanolamine, and phosphatidic acid.

The synthesis of two new synthetic analogues of lecithin, two of phosphatidyl ethanolamine ("cephalin"), and one new phosphatidic acid analogue is described. They comprise one of each of the following types: the "isosteric" diether lecithin and cephalin analogues ROCH(2)CH(OR)- CH(2)CH(2)P(O) (O(-))OCH(2)CH(2)N(+)R'(3) (R = C(18)H(37); R' = H or CH(3)); and the "hydrocarbon" analogues of phosphatidic acid, lecithin, and cephalin, C(17)H(35)CH(2)CH(C(18)H(37))CH(2)P(O)(R) = (R'); [R = R' = OH; R = O(-), R' = OCH(2)CH(2)N(+)(CH(3))(3); and R = O(-), R' = OCH(2)CH(2)N(+)H(3)]. Infrared spectra and other properties of these compounds are described.

Chemical Phenomena↗
PubMed1977

[Prospects and limitations of the treatment of senile maculopathies with phosphatides (author's transl)].

All kinds of treatment attempted so far in cases of senile maculopathies were unsatisfactory. Intramuscular injections of retinal phosphatides were introduced six years ago. In this follow-up study 216 eyes of patients with senile maculopathy treated by Etaretin have been checked. The former experience was confirmed that an improvement in visual acuity can be obtained only in early cases of the dry type. In such cases i.m. injections of Etaretin are actually the treatment of choice. In fairly advanced cases eventually some stabilization of the process may be obtained. In terminal stages no effect can be expected. Experimental observations revealed that the therapeutic effect of a phosphatide compound depends on the content of the polyunsaturated fatty acid C 22:6, but not on the animal organ which serves as the source of the extract. From an economic point of view these findings are of importance for further experimental work, as well as for broad clinical application.

Age Factors↗
PubMed2005

Phosphatidic acid and diacylglycerol generation is regulated by insulin in cerebral cortex synaptosomes from adult and aged rats.

Insulin receptor associated with the cerebral cortex (CC) has been shown to be involved in brain cognitive functions. Furthermore, deterioration of insulin signaling has been associated with age-related brain degeneration. We have reported previously that aging stimulates phospholipase D/phosphatidate phosphohydrolase 2 (PLD/PAP2) pathway in CC synaptosomes from aged rats, generating a differential availability of their reaction products: diacylglycerol (DAG) and phosphatidic acid (PA). The aim of this work was to determine the effect of aging on DAG kinase (DAGK), as an alternative pathway for PA generation, and to evaluate the effect of insulin on PLD/PAP2 pathway and DAGK. PLD, PAP2, and DAGK activities were measured using specific radiolabeled substrates in CC synaptosomes from adult (4 months old) and aged rats (28 months old). In adult animals, in the presence of the tyrosine phosphatase inhibitor (sodium o-vanadate), insulin stimulated PLD activity at 5 min incubation. DAGK activity was also increased at the same time of incubation and PAP2 was inhibited. In aged animals, PLD activity was not modified by the presence of insulin plus vanadate, PAP2 was inhibited, and DAGK was stimulated by the hormone. Insulin, vanadate, and the combination of both induced protein tyrosine phosphorylation in adult CC synaptosomes. Aged rats showed a lower level of protein phosphorylation with respect to adult rats. Our results show that insulin modulates PA and DAG availability through the regulation of PLD/PAP2 and DAGK pathways in adult rat CC synaptosomes. Additionally, we demonstrated that PA and DAG generation is regulated differentially by insulin during aging.

Aging↗
PubMed2004

Phospholipase D and phosphatidate phosphohydrolase activities in rat cerebellum during aging.

Aging is a process that affects different organs, of which the brain is particularly susceptible. PA and DAG are central intermediates in the phosphoglyceride as well as in the neutral lipid biosynthetic pathway, and they have also been implicated in signal transduction. Phospholipase D (PLD) and phosphatidate phosphohydrolase (PAP) are the enzymes that generate PA and DAG. The latter can be transformed into MAG by diacylglycerol lipase (DGL). In the present study, we examine how aging modulates the PLD, PAP, and DGL isoforms in cerebellar subcellular fractions from 4- (adult), 28-, and 33-mon-old (aged) rats. PI-4,5-bisphosphonate (PIP2)-dependent PLD, PAP1, and DGL1 were distributed in different percentages in all cerebellum subcellular fractions. On the other hand, PAP2 and DGL2 activities were observed in all subcellular fractions except in the cytosolic fraction. Aging modified the enzyme distribution pattern. In addition, aging decreased nuclear (45%), mitochondrial-synaptosomal (55%), and cytosolic (71%) PAP1 activity and increased (28%) microsomal PAP1 activity. DGL1 activity was decreased in nuclear (85%) and mitochondrial-synaptosomal (63%) fractions by aging. On the other hand, PIP2-dependent PLD activities were increased in the mitochondrial-synaptosomal fraction. PAP2 and DGL2 were increased in the microsomal fraction by 87 and 114%, respectively, and they were decreased in the nuclear fraction. The changes observed in cerebellum PAP1 and DGL1 activities from aged rats with respect to adult rats could be related to modifications in lipid metabolism. Differential PA metabolization during aging through PIP2-dependent PLD/PAP2/DGL2 activities could be related to alterations in the neural signal transduction mechanisms.

Age Factors↗
PubMed1980

A sensitive assay for phosphatidate phosphohydrolase in mouse liver microsomes.

A technique is described for the assay of phosphatidate phosphohydrolase using 1,2-[9,10-3H]dioleoyl-sn-glycero-3-phosphate as a substrate. This substrate was prepared enzymatically using mouse liver microsomes washed with 0.5 M NaCl, which synthesize minimal amounts of neutral lipids at high enzyme concentrations. Measurement of the product, 1,2-[9,10-3H]dioleoylglycerol, was 10-fold more sensitive than the usual colorimetric assay for inorganic phosphate release. In addition, the assay provides information about the relative contribution of other activities which limit the availability of diacylglycerols for further esterification to triacylglycerols and/or phospholipids.

Animals↗
PubMed1988

Formation of diacylglycerol by a phospholipase D-phosphatidate phosphatase pathway specific for phosphatidylcholine in endothelial cells.

The conversion of phosphatidylcholine (PC) to diacylglycerol (DAG) was studied in sonicated endothelial cells and in subcellular fractions in the presence of 0.05% Triton X-100 and 2 mM EDTA. DAG formation occurred predominantly in an organelle fraction that sedimented at 15,000 x g. In parallel reactions with exogenous 1-oleoyl-2-[3H]oleoyl-PC (sn-2-[3H]DOPC) and phosphatidyl[3H]choline ([choline-3H]PC), [3H]DAG was formed by a reaction pathway in which [3H]choline was the only product derived from [choline-3H]PC. [3H]Choline was not formed secondarily from [3H]glycerophosphocholine or [3H]phosphocholine. Small amounts of [3H]phosphatidate ([3H]PA) were isolated from reactions with sn-2-[3H]DOPC at short incubation times, and substantial PA phosphatase activity was demonstrated. These data, taken together, supported a phospholipase D-PA phosphatase pathway of DAG formation. Kinetic data established that the low ratio of [3H]PA/[3H]DAG formed in reactions with sn-2-[3H]DOPC was due to a 15-fold higher Vmax and 7-fold lower apparent Km of the PA phosphatase. The [3H]PA/[3H]DAG product ratio was increased by addition of unlabeled PA or by selective extraction of phospholipase D with Triton X-100. The characteristics of the phospholipase D indicated a unique enzyme. Activity was optimal in the presence of EDTA and was almost totally dependent upon Triton X-100. The pH profile displayed a peak at 7.0. Of particular significance was the stringent substrate specificity. Phosphatidylinositol was not hydrolyzed, and activities towards phosphatidylethanolamine and sphingomyelin were at most 30- to 50-fold lower than those towards PC. Phospholipase D and PA phosphatase were identified in a number of rat tissues and other cells. The highest activities of phospholipase D were present in lung and endothelial cells. Phospholipase D was partially purified from rat lung by Triton X-100 extraction and anion exchange chromatography. When linked with PA phosphatase, the phospholipase D could initiate a pathway of DAG formation that is highly specific for PC.

Animals↗
PubMed1998

Priming of phosphatidic acid production by staurosporine in f-Met-Leu-Phe-stimulated human neutrophils--correlation with respiratory burst.

Staurosporine, a microbial alkaloid known as a potent though non specific PKC inhibitor, enhances the production of superoxide anion (respiratory burst) of human polymorphonuclear leukocytes (PMN) stimulated by chemoattractants such as f-Met-Leu-Phe (fMLP). To gain insights into the mechanisms of this priming, we analysed staurosporine effects on formation of second messengers issued from phospholipase D (PLD), i.e., phosphatidic acid (PA) and its dephosphorylated form, diglycerides (DG). PA and DG were measured by two methods, in mass and after the labelling of PMN with a phosphatidylcholine precursor, [3H]-1-O-alkyl-2-lyso-3-phosphatidylcholine. Treatment of labelled PMN with low concentrations of staurosporine (12.5 and 50 nM) which prime respiratory burst had no significant effect on basal amounts of tritiated PA and DG, but potentiated fMLP-mediated formation of [3H]PA and phosphatidylethanol (PEt) pointing to a priming of PLD activity. PA mass in resting PMN increased (approximately 80 +/- 7%) in the presence of high drug concentrations only (250-500 nM), with no change in basal DAG mass. Low staurosporine concentrations (6.25-25 nM) markedly potentiated PA mass formation induced by fMLP and positive correlation (R = 0.95) was found between enhanced superoxide formation and generation of PA but not DG. Furthermore, cytochalasin B, which is known to prime PA production induced by fMLP, synergised the priming of respiratory burst by staurosporine, which further suggests a functional role of PA. In contrast to staurosporine, the more selective PKC inhibitor GF109203X neither stimulated PLD nor primed fMLP-induced PLD or respiratory burst. These data indicate that priming of fMLP-mediated PMN respiratory burst by staurosporine correlates with PA formation. This priming may be linked to alteration of early signalling events upstream of PLD rather than to feedback inhibition of PKC.

Diglycerides↗
PubMed2000

Role of phosphatidic acid phosphatase 2a in uptake of extracellular lipid phosphate mediators.

Phosphatidic acid (PA) phosphatase 2a (PAP2a) is an integral membrane glycoprotein that hydrolyzes a number of structurally related lipid phosphate substrates when presented in mixed phospholipid and detergent micelles. The physiological substrate specificity and functions of this enzyme are unclear. Using reconstitution studies we demonstrate that PAP2a hydrolyses both PA and LysoPA substrates in a lipid bilayer. To investigate the activity of PAP2a against cellular substrates we generated HEK293 cell variants stably overexpressing the enzyme. Although one of these lines exhibited a 27-fold increase in PAP2 activity measured in vitro, levels of PA were not significantly reduced in comparison with control cells. Cell surface labeling and activity measurements demonstrate that a portion of the enzyme was localized to the cell surface. Pagano and Longmuir (J. Biol. Chem. 260 (1985) 1909) described the rapid uptake of PA by cultured cells, but the mechanisms and proteins involved were not identified. We found that overexpression of PAP2a was accompanied by a 2.1-fold increase in uptake of a fluorescent PA analog but that uptake of other phospholipids and diacylglycerols was unaltered. The increase in lipid uptake was completely dependent on PAP activity and unaffected by endocytosis inhibitors. Our results indicate that PAP2a is a cell surface enzyme that plays an active role in the hydrolysis and uptake of lipids from the extracellular space.

Cell Line↗
PubMed2005

Regulation of cell survival by lipid phosphate phosphatases involves the modulation of intracellular phosphatidic acid and sphingosine 1-phosphate pools.

We have shown previously that LPPs (lipid phosphate phosphatases) reduce the stimulation of the p42/p44 MAPK (p42/p44 mitogen-activated protein kinase) pathway by the GPCR (G-protein-coupled receptor) agonists S1P (sphingosine 1-phosphate) and LPA (lysophosphatidic acid) in serum-deprived HEK-293 cells [Alderton, Darroch, Sambi, McKie, Ahmed, N. J. Pyne and S. Pyne (2001) J. Biol. Chem. 276, 13452-13460]. In the present study, we now show that this can be blocked by pretreating HEK-293 cells with the caspase 3/7 inhibitor, Ac-DEVD-CHO [N-acetyl-Asp-Glu-Val-Asp-CHO (aldehyde)]. Therefore LPP2 and LPP3 appear to regulate the apoptotic status of serum-deprived HEK-293 cells. This was supported further by: (i) caspase 3/7-catalysed cleavage of PARP [poly(ADP-ribose) polymerase] was increased in serum-deprived LPP2-overexpressing compared with vector-transfected HEK-293 cells; and (ii) serum-deprived LPP2- and LPP3-overexpressing cells exhibited limited intranucleosomal DNA laddering, which was absent in vector-transfected cells. Moreover, LPP2 reduced basal intracellular phosphatidic acid levels, whereas LPP3 decreased intracellular S1P in serum-deprived HEK-293 cells. LPP2 and LPP3 are constitutively co-localized with SK1 (sphingosine kinase 1) in cytoplasmic vesicles in HEK-293 cells. Moreover, LPP2 but not LPP3 prevents SK1 from being recruited to a perinuclear compartment upon induction of PLD1 (phospholipase D1) in CHO (Chinese-hamster ovary) cells. Taken together, these data are consistent with an important role for LPP2 and LPP3 in regulating an intracellular pool of PA and S1P respectively, that may govern the apoptotic status of the cell upon serum deprivation.

Animals↗
PubMed1993

Phosphatidylcholine is a major source of phosphatidic acid and diacylglycerol in angiotensin II-stimulated vascular smooth-muscle cells.

In cultured vascular smooth-muscle cells, angiotensin II produces a sustained formation of diacylglycerol (DG) and phosphatidic acid (PtdOH). Since the fatty acid composition of these molecules is likely to determine their efficacy as second messengers, it is important to ascertain the phospholipid precursors and the biochemical pathways from which they are produced. Our experiments suggest that phospholipase D (PLD)-mediated phosphatidylcholine (PtdCho) hydrolysis is the major source of both DG and PtdOH during the late signalling phase. First, in cells labelled with [3H]myristate, which preferentially labels PtdCho, formation of [3H]PtdOH precedes formation of [3H]DG. Second, in contrast with phospholipase C (PLC) activation, DG mass accumulation is dependent on extracellular Ca2+. Similarly, DG mass accumulation is not attenuated by protein kinase C activation, which we have previously shown to inhibit the phosphoinositide-specific PLC. Third, the fatty acid composition of late-phase DG and PtdOH more closely resembles that of PtdCho than that of phosphatidylinositol. Finally, in cells labelled for a short time with [3H]glycerol, the radioactivity incorporated into [3H]DG and PtdOH was greater than that incorporated into PtdIns, but not into PtdCho. We found no evidence that synthesis de novo or phosphatidylethanolamine breakdown contributes to sustained DG and PtdOH formation. Thus, in angiotensin II-stimulated cultured vascular smooth-muscle cells, PLD-mediated PtdCho hydrolysis is the major source of sustained DG and PtdOH, whereas phosphoinositide breakdown is a minor contributor. Furthermore, PtdOH phosphohydrolase, which determines the relative levels of DG and PtdOH, appears to be regulated by protein kinase C. These results have important implications for the role of these second messengers in growth and contraction.

Angiotensin II↗
PubMed2005

An important role of phosphatidic acid in ABA signaling during germination in Arabidopsis thaliana.

Phosphatidic acid (PA) functions as a lipid signaling molecule in plants. Physiological analysis showed that PA triggers early signal transduction events that lead to responses to abscisic acid (ABA) during seed germination. We measured PA production during seed germination and found increased PA levels during early germination. To investigate the role of PA during seed germination, we focused on the PA catabolic enzyme lipid phosphate phosphatase (LPP). LPP catalyzes the conversion of PA to diacylglycerol (DAG). There are 4 LPP genes in the Arabidopsis genome. Among them, AtLPP2 and AtLPP3 are expressed during seed germination. Two AtLPP2 T-DNA insertional mutants (lpp2-1 and lpp2-2) showed hypersensitivity to ABA and significant PA accumulation during germination. Furthermore, double-mutant analysis showed that ABA-insensitive 4 (ABI4) is epistatic to AtLPP2 but ABA-insensitive 3 (ABI3) is not. These results suggest that PA is involved in ABA signaling and that AtLPP2 functions as a negative regulator upstream of ABI4, which encodes an AP2-type transcription factor, in ABA signaling during germination.

Abscisic Acid↗
PubMed1992

Role of phospholipase D-derived diradylglycerol in the activation of the human neutrophil respiratory burst oxidase. Inhibition by phosphatidic acid phosphohydrolase inhibitors.

An agonist-activated phospholipase D/phosphatidic acid phosphohydrolase (PAH) pathway was recently demonstrated in human neutrophils, and evidence suggests that phosphatidic acid (PA) and/or diradylglycerol (DG) generated from this pathway participates in activation of the O2(-)-generating respiratory burst. We have used a series of cationic amphiphilic compounds (sphingosine, propranolol, chlorpromazine, and desipramine) and antibiotics (clindamycin, trimethoprim, and roxithromycin) all of which inhibit the respiratory burst, to investigate the role of the phospholipase D/PAH pathway in neutrophil activation. The phosphatidylcholine (PC) pool in intact cells was first labeled using [3H]-1-O-alkyl-lysoPC; released [3H]-PA and [3H]-DG were then quantified after the addition of either chemo-attractant or PMA. Using either agonist, all compounds showed a dose-dependent inhibition of [3H]-DG generation which correlated with inhibition of O2- generation, but compounds failed to inhibit directly the NADPH oxidase in a cell-free system. For either activator, a plot of the ID50 values for O2- generation vs those for DG generation was linear over four orders of magnitude. In many cases, inhibition of [3H]-DG generation corresponded to an increase in [3H]-PA, implicating PAH as the locus of inhibition. Superoxide generation was inhibited under conditions where PA was either elevated or minimally affected. Neither O2- release nor DG generation showed any selectivity for stereoisomers of propranolol, suggesting that this inhibition does not act via a specific binding site on PAH. No evidence was obtained for an effect of the inhibitors on PA mobility as monitored by electron spin resonance studies of spin-labeled PA in a model membrane system. Data are consistent with an effect of the inhibitors at the level of the interaction of PAH with the membrane and/or its substrate. These data imply that DG produced via the phospholipase D/PAH pathway functions in the activation or maintenance of the respiratory burst.

Diglycerides↗
PubMed1990

Trifluoperazine enhances accumulation and inhibits phosphohydrolysis of phosphatidate in thrombin-stimulated platelets.

Trifluoperazine (TFP) in concentrations up to 10-15 microM increased the formation of phosphatidic acid (PA) in platelets treated with 0.5 U/ml of thrombin, while higher concentrations of TFP inhibited formation of PA. Liberation of arachidonate (AA) from platelet phospholipids was progressively inhibited as the concentration of TFP increased. At thrombin doses lower than 0.1 U/ml TFP, (less than or equal to 25 microM) enhanced PA formation with either no effect of AA liberation (6 donors) or with much greater enhancement of PA formation than the decrease in liberation of AA (3 donors). The enhancement of PA formation by TFP did therefore not seem to be due to inhibition of phospholipase A2 (PLA2) by the phenothiazine, which has been suggested. We show further that TFP inhibits PA phosphohydrolase in platelet lysates, although with complex kinetics. It is therefore concluded that the enhancement of thrombin-induced PA production by TFP is not caused by inhibition of PLA2 but could be due to TFP-induced inhibition of PA phosphohydrolase.

Arachidonic Acid↗