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PubMed1995

Phorbol ester-induced priming of superoxide generation by phosphatidic acid-stimulated neutrophils and granule-free neutrophil cytoplasts.

This study was undertaken to examine the mechanisms involved in polymorphonuclear leukocyte superoxide release stimulated by exogenous phosphatidic acid (PA). Unlike the immediate burst of superoxide release affected by membrane-permeable dioctanoylglycerol (DiC8-DAG), dioctanoyl phosphatidic acid (DiC8-PA) induced superoxide release after a lag period of 5-20 min. This period was considerably reduced or eliminated when cells were primed by substimulatory levels of phorbol myristate acetate (PMA). Granule-depleted neutrophil cytoplasts also responded to DiC8-PA with a burst of superoxide generation. Activation of the cytoplast superoxide generating system in response to DiC8-PA was also significantly faster after cells had been preexposed to substimulatory levels of PMA, indicating that at least a portion of the priming mechanism was independent of PMA-induced degranulation. To further examine the potential mechanism of PMA priming of responses to PA, we evaluated the activity of neutrophil ecto-phosphatidic acid phosphohydrolase (ecto-PA phosphohydrolase), which generates diacylglycerol from exogenous PA. PMA priming had no discernable effect on the activity of this enzyme. In addition, propranolol, an inhibitor of PA phosphohydrolase, did not selectively inhibit PMA priming of neutrophil responses to DiC8-PA, indicating that priming did not result from acceleration of DiC8-PA hydrolysis. We therefore investigated the possibility that activation of protein kinase C was the basis of the primed response. Several semiselective protein kinase C inhibitors (calphostin C, H-7, and acylmethylglycerol) inhibited DiC8-DAG- and DiC8-PA-induced superoxide release as well as PMA-primed responses to approximately the same extent. These results are consistent with the hypothesis that neutrophil responses to phosphatidate are mediated by diglyceride generated by the action of ecto-PA phosphohydrolase. PMA priming does not result from increased catalytic activity of ecto-PA phosphohydrolase but rather seems to result from potentiation of an intermediate involved in the cells' response to multiple stimuli.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1997

Fatty acid binding protein: stimulation of microsomal phosphatidic acid formation.

The effect of fatty acid binding proteins (FABPs) on two key steps of microsomal phosphatidic acid formation was examined. Rat liver microsomes were purified by size-exclusion chromatography to remove endogenous cytosolic fatty acid and fatty acyl-CoA binding proteins while recombinant FABPs were used to avoid cross-contamination with such proteins from native tissue. Neither rat liver (L-FABP) nor rat intestinal fatty acid binding protein (I-FABP) stimulated liver microsomal fatty acyl-CoA synthase. In contrast, L-FABP and I-FABP enhanced microsomal conversion of [14C]oleoyl-CoA and glycerol 3-phosphate to [14C]phosphatidic acid by 18- and 7-fold, respectively. The mechanism for this stimulation, especially by I-FABP, is not known. However, several observations presented here suggest that, like L-FABP, I-FABP may interact with fatty acyl-CoA and thereby stimulate enzyme activity. First, I-FABP decreased microsomal membrane-bound oleoyl-CoA. Second, oleoyl-CoA displaced I-FABP bound fluorescent fatty acid, cis-parinaric acid, with Ki of 5.3 microM and 1.1 sites. Third, oleoyl-CoA decreased I-FABP tryptophan fluorescence with a Kd of 4.2 microM. Fourth, oleoyl-CoA red shifted emission spectra of acrylodated I-FABP, a sensitive marker of I-FABP interactions with ligands. In summary, the results demonstrate for the first time that both L-FABP and I-FABP stimulate liver microsomal phosphatidic acid formation by enhancing synthesis of phosphatidate from fatty acyl-CoA and glycerol 3-phosphate.

Acyl Coenzyme A↗
PubMed1978

The effects of amphiphilic compounds on phosphatidate metabolism.

Amphiphilic cations interact with phosphatidate and thereby change its physical properties. This interaction can redirect phospholipid metabolism. In the presence of Mg2+ amphiphilic cations inhibit the activity of phosphatidate phosphohydrolase and stimulate that of phosphatidate cytidylyltransferase. Increasing the concentration of Mg2+ further, or adding Ca2+ have similar effects, except that Ca2+ does not stimulate phosphatidate cytidylyltransferase activity. Amphiphilic anions reverse the effects caused by the amphiphilic cations. The implication of these results are discussed in relation to the pharmacological effects of amphiphilic cationic drugs.

Animals↗
PubMed1978

Cyanamide mediated syntheses under plausible primitive earth conditions. V. The synthesis of phosphatidic acids.

A mixture of ammonium palmitate, 14C-sn-glycero-1(3)-phosphate, cyanimide and imidazole when heated for several hours formed significant quantities of phospholipids. These reaction products were shown by chromatographic, chemical and enzymatic procedures to be monopalmitoylglycerophosphate (MPGP), dipalmitoylglycerophosphate (DPGP) and monopalmitoyl cyclic glycerophosphate (cMPGP). A portion of the MPGP and DPGP possessed the same steric configuration as naturally occurring lysophosphatidic acid and phosphatidic acid. The yield of total phospholipid was maximal at temperatures between 60 degrees and 90 degrees after 8 h. When ratios of reactants were varied, up to 45% of radioactive glycerophosphate was converted into phospholipids. The average proportions of individual phosphatidic acids were: 60% MPGP, 27% DPGP and 13% cMPGP. Evidence was obtained for a synergistic relationship between cyanamide and imidazole in promoting the formation of phosphatidic acids. These results suggest that phosphatidic acids, which are essential precursors for the biochemical synthesis of more complex membrane phospholipids, could have been produced on the primitive Earth.

Cyanamide↗
PubMed1989

An enzymatic assay for picomole levels of phosphatidate.

An assay for phosphatidate that is sensitive, specific, and highly reproducible is described. Phosphatidate is transacylated with methylamine and the glycerol 3-phosphate produced is measured enzymatically. The assay is linear from 50 to 1500 pmol of phosphatidate and can be used to quantitate phosphatidate in small amounts of biological materials.

Animals↗
PubMed1976

Phosphatidic acid phosphatase and phospholipdase A activities in plasma membranes from fusing muscle cells.

Plasma membrane from fusing embryonic muscle cells were assayed for phospholipase A activity to determine if this enzyme plays a role in cell fusion. The membranes were assayed under a variety of conditions with phosphatidylcholine as the substrate and no phospholipase A activity was found. The plasma membranes did contain a phosphatidic acid phosphatase which was optimally active in the presence of Triton X-100 and glycerol. The enzyme activity was constant from pH 5.2 to 7.0, and did not require divalent cations. Over 97% of the phosphatidic acid phosphatase activity was in the particulate fraction. The subcellular distribution of the phosphatidic acid phosphatase was the same as the distributions of the plasma membrane markers, (Na+ + k+)-ATPase and the acetylcholine receptor, which indicates that this phosphatase is located exclusively in the plasma membranes. There was no detectable difference in the phosphatidic acid phosphatase activities of plasma membranes from fusing and non-fusing cells.

Adenosine Triphosphatases↗
PubMed1978

Calcium-induced aggregation and fusion of mixed phosphatidylcholine-phosphatidic acid vesicles as studied by 31P NMR.

1. The transbilayer distribution of the phospholipids in sonicated egg phosphatidylcholine-phosphatidic acid vesicles and the interaction of Ca2+ with these vesicles was studied by 31P NMR. 2. Over a wide composition range the bilayer of these vesicles has a symmetrical phospholipid composition. 3. With ratios of Ca2+ to phosphatidic acid in the outer monolayer of the vesicles up to 0.3, Ca2+ induces vesicle aggregation. The extent of aggregation is increased by the Ca2+ concentration in the medium and the outer monolayer concentration of phosphatidic acid. The vesicle aggregation can be fully reversed by chelating Ca2+. 4. When the ratio exceeds 0.5 Ca2+ induces vesicle fusion. The fusion is maximal for vesicles containing both phosphatidylcholine and phosphatidic acid. The data suggest that Ca2+-induced lateral phase separations make the bilayer more susceptible to fusion.

Calcium↗
PubMed1985

Sodium-induced aggregation of phosphatidic acid and mixed phospholipid vesicles.

Sodium-induced aggregations of sonicated vesicles prepared from synthetic phosphatidic acid and from its 1:1 mixtures with synthetic phosphatidylethanolamine and phosphatidylcholine were studied by turbidimetric measurements. The aggregation reactions were almost completely reversible on change in the Na+ concentration, pH or temperature. The threshold concentrations of Na+ for aggregations of pure dipalmitoylphosphatidic acid vesicles and mixed dipalmitoylphosphatidylethanolamine- and dimyristoylphosphatidylcholine-dipalmitoylphosphatidic acid vesicles were found to be 200, 310 and 550 mM, respectively, at 25 degrees C and pH 7.2. The hydrocarbon chain lengths of phosphatidic acid and phosphatidylethanolamine had little effect on the threshold concentrations. The threshold concentrations for phospholipid vesicles composed of phosphatidic acid alone or its 1:1 mixture with phosphatidylethanolamine were changed by varying either the pH or temperature, while that for phosphatidylcholine-phosphatidic acid vesicles was almost independent of the pH and temperature, implying that aggregation of the latter vesicles is induced by a somewhat different mechanism.

Dimyristoylphosphatidylcholine↗
PubMed1988

Calcium-induced condensation-reorganization phenomena in multilamellar vesicles of phosphatidic acid. pH potentiometric and 31P-NMR, Raman and ESR spectroscopic studies.

In biological membranes, the anionic characteristics of the polar headgroup of phosphatidic acids are responsible for structural changes induced by Ca2+ in many cellular processes. The very simple headgroup structure of dipalmitoylphosphatidic acid (DPPA) offers particular advantages as a model to study the interactions between Ca2+ and natural phosphatidic acids such as cardiolipin and phosphatidylserine. The effects of calcium ions on DPPA membranes have been studied as a function of temperature by potentiometry and by Raman, ESR and 31P-NMR spectroscopies. The protons in monosodic DPPA liposomes have been considered as a probe to detect pH variations resulting from introduction of Ca2+ inside the membrane. This method has also allowed us to determine the stoichiometry of this reaction: 2 DPPA(H) + Ca2+----Ca(DPPA)2 + 2H+. 31P-NMR spectroscopy has been used to detect reorganization-condensation phenomena in multilamellar vesicles of DPPA under the influence of calcium and temperature. Furthermore, the temperature profiles obtained from Raman spectra for Ca(DPPA)2 membranes provide conclusive evidence that Ca2+ induces major reorganization of the phosphatidic acid component into a highly ordered phase. Quantitative estimates of the degree of motional restriction of spin-labeled soaps embedded inside membranes composed of DPPA with or without Ca2+ have been made using ESR technique. These results are discussed and compared to those found previously for a natural phosphatidic acids such as phosphatidylserine.

Calcium↗
PubMed1983

Phosphatidic acid phosphatase activity in subcellular fractions derived from adult rat type II pneumocytes in primary culture.

The development of a method for the subcellular isolation of lamellar body, microsomal, mitochondrial and cytosolic enriched fractions from the adult rat type II pneumocyte is presented. These fractions were used to investigate the distribution and specificity of phosphatidic acid phosphatase (EC 3.1.3.4). The cytosolic enriched fraction possessed the greatest proportion of total phosphatidic acid phosphatase activity and the highest relative specific activity. The small amounts of phosphatase activity in the lamellar body, microsomal and mitochondrial enriched fractions correlated best with a nonspecific phosphatase activity. 1,2-Disaturated phosphatidic acid served as a slightly better substrate than did 1-saturated, 2-unsaturated phosphatidic acid in the cytosolic enriched fraction. This observation indicates that the de novo pathway could contribute to dipalmitoylphosphatidylcholine synthesis in the rat type II pneumocyte.

Animals↗
PubMed1983

Phosphatidylinositol and phosphatidic acid metabolism in rat pancreatic islets in response to neurotransmitter and hormonal stimuli.

Carbamylcholine produced a concentration-dependent stimulation of labelling of phosphatidylinositol and phosphatidic acid in rat islets of Langerhans following preincubation with 32PO43(-). The time course of these effects suggested that the initial action of carbamylcholine was to stimulate phosphatidic acid production, presumably by causing hydrolysis of phosphatidylinositol. This conclusion was substantiated by experiments in which islet phospholipids were pre-labelled with [3H]arachidonic acid. Under these conditions, carbamylcholine caused a loss of radioactivity from phosphatidylinositol, together with an increase in labelling of phosphatidic acid. The effects of carbamylcholine on islet phospholipid labelling were not dependent upon the presence of added Ca2+, but were abolished by EDTA and by atropine. An apparent stimulation of phosphatidylinositol and phosphatidic acid metabolism was also induced by cholecystokinin-pancreozymin, whereas glucagon, arginine, glibenclamide and thyrotropin had no significant effect. The data suggest that enhanced activity of the so-called phosphatidylinositol cycle may be an important event in regulating secretory activity of islets in response to certain neurotransmitter and hormonal stimuli. Furthermore, the results are compatible with the hypothesis that increased phospholipid metabolism may play a role in the modulation of ionic fluxes during stimulation by such agents.

Animals↗
PubMed1984

Escherichia coli membrane vesicles with elevated phosphatidic acid levels. A detergent-free system for in vitro phospholipid synthesis.

A new method for studying phospholipid biosynthesis in Escherichia coli is described. The method makes use of the previously reported observation that E. coli cytidine auxotrophs accumulate phosphatidic acid when starved of cytidine (Ganong, B. and Raetz, C.R.H. (1982) J. Biol. Chem. 257, 389-394). We now show that phosphatidic acid that accumulates in these cells is competent for further biosynthetic use in vivo, if cytidine is re-supplied to the cells. Furthermore, phosphatidic acid-rich membranes prepared from such cells can be used for in situ assays of the later steps of phospholipid biosynthesis. Since this system does not require detergent, our in situ assays more accurately reflect the conditions of an intact membrane. We have used this system to probe the regulation of the branch-point of the biosynthetic pathway for phospholipid polar headgroups. Phosphatidic acid-rich membranes prepared from cells that overproduce either phosphatidylserine synthase or phosphatidylglycerolphosphate synthase do not have increased rates of lipid synthesis in our in situ assays. This correlates with synthetic rates measured in vivo and, thus, our in situ assays accurately reflect conditions in a growing cell's membrane.

Cell Membrane↗
PubMed1984

De novo biosynthesis of docosahexaenoyl-phosphatidic acid in bovine retinal microsomes.

Lysophosphatidic acid stimulated several-fold the formation of docosahexaenoyl-phosphatidic acid from 14C-labeled docosahexaenoic acid (22:6 (n-3] in the bovine retina. 1-Palmitoyl- and 1-oleoyl-sn-glycerol 3-phosphate were the preferred acceptors. Most of the activity was localized in the 105 000 X g microsomal fraction. Despite the very high content of 22:6 in the phospholipids of photoreceptor membranes, only about 1% of the microsomal activity was found in discs isolated from rod outer segments. The newly synthesized docosahexaenoyl-phosphatidic acid was further metabolized to diacylglycerols, triacylglycerols, phosphatidylcholine and phosphatidylserine. The de novo synthesis of docosahexaenoyl-phosphatidylcholine was stimulated by 1 mM CDPcholine. Lysophosphatidic acid and lysophosphatidylcholine up to 50 microM do not compete with each other for 22:6 in the formation of their respective diacylated lipids. This suggests that this fatty acid is introduced into phosphatidic acid and phosphatidylcholine via different acylation systems. We conclude that, in addition to the deacylation-acylation cycle, there is also an active pathway for the acylation of 22:6 into glycerolipids during the de novo biosynthesis of phosphatidic acid.

Animals↗
PubMed1987

Triton X-100 promotes the accumulation of phosphatidic acid and inhibits the synthesis of phosphatidylcholine in human decidua and chorion frondosum tissues in vitro.

Triton X-100 is known to affect phospholipid metabolism and the generation of various signal molecules from cellular phospholipids. In the present work the effect of Triton X-100 on phospholipid metabolism of human decidua and of the primordial placenta (chorion frondosum) was studied. Triton X-100 (0.05%, v/v) added to tissue mince 30 min before the end of a 60 min incubation stimulated 2-4-fold (decidua) and 4-6-fold (placenta) the incorporation of [32P]phosphate ([32P]Pi) into phosphatidic acid, while markedly decreasing the labeling of phosphatidylcholine. Triton X-100 had no effect on the labeling of phosphatidylinositol in the decidua, and only a slight increase was observed in the placenta. When labeled glucose was used to assess phospholipid synthesis, the addition of Triton had no effect on phosphatidic acid, while decreasing the synthesis of phosphatidylcholine. Incorporation of [32P]Pi into phosphatidic acid was not accelerated by a submicellar concentration (0.01%) of Triton, whereas the synthesis of phosphatidylcholine was decreased irrespective of detergent concentration. Anionic or cationic detergents could not mimic the action of Triton on phosphatidic acid synthesis. Although Triton inhibited the synthesis of ATP in a dose-dependent manner, this could not account for the above results. Instead, it is suggested that diacylglycerol kinase and phosphocholine:CTP cytidylyltransferase are possible targets of the action of Triton X-100.

Chorion↗
PubMed1988

Age-related changes in the translocation of phosphatidate phosphohydrolase from the cytosol to microsomal membranes in rat liver.

The effects of oleate, spermine and chlorpromazine were assayed in the presence or absence of 0.15 M KCl on the translocation of phosphatidate phosphohydrolase activity from cytosol to endoplasmic reticulum membranes in liver homogenates obtained from rats aged 1, 30, 60, 180 and 360 days. Marked age-associated decreases in phosphatidate phosphohydrolase distribution onto the membranes were demonstrated under nearly all conditions. In liver homogenates taken from 1-day-old rats and incubated with 0.15 M KCl, most of the enzyme was active (associated with the membranes). Physiological salt concentration (0.15 M KCl) produced a 2-fold increase of oleate-induced translocation of phosphatidate phosphohydrolase activity in liver homogenates from 1-day-old rats; it had no effect on those from 60-day-old rats, and produced a notable decline in liver homogenates obtained from 180- and 360-day-old rats. The promoting effect of spermine on oleate-induced translocation of this enzyme activity was higher in younger rats when incubated in the absence of 0.15 M KCl. Chlorpromazine did not show its usual antagonizing effect on oleate-induced translocation of phosphatidate phosphohydrolase when added to homogenates taken from 1-day-old rats. The antagonizing effect was slightly apparent in liver homogenates from 30-day-old rats and was more pronounced in those from 60-day-old rats in which the values diminished to one-half and to one-third either in the presence or absence of 0.15 M KCl.

Aging↗
PubMed1989

Activation of rat liver cytosolic phosphatidic acid phosphatase by nucleoside diphosphates.

Phosphatidic acid phosphatase (EC 3.1.3.4) was purified 30-fold by ammonium sulfate fractionation and hydroxyapatite chromatography from the soluble fraction of rat liver. ADP was found to stimulate the enzyme activity with half-maximal stimulation at 0.2 mM. Similar effects were seen when ADP was replaced by GDP or CDP. In contrast, ATP inhibited the enzyme; half-maximal inhibition observed at 0.2 mM. Again, the degree of inhibition did not differ when GTP or CTP replaced ATP. Thus, the structure of the base part of the nucleotide was not critical for mediating these effects. The positions of the phosphate groups in the nucleotide structure were however found to be of importance for the enzyme activity. Variations in the structure of the phosphate ester bound at the 5'-position had a pronounced effect on phosphatidic acid phosphatase activity. The effect of nucleotides depended on pH, and the inhibition by ATP was more pronounced at pH levels lower than 7.0, whereas the stimulatory effect of ADP was virtually the same from pH 6.0 to pH 8.0. The enzyme showed substrate saturation kinetics with respect to phosphatidic acid, with an apparent Km of 0.7 mM. Km increased in the presence of ATP, whereas both apparent Vmax and Km increased in the presence of ADP, suggesting different mechanisms for the action of the two types of nucleotides. The results indicated that physiological levels of nucleotides with a diphosphate or a triphosphate ester bound at the 5'-position of the ribose moiety influenced the activity of phosphatidic acid phosphatase. The possibility is discussed that these effects might be of importance for the regulation of triacylglycerol biosynthesis.

Adenosine Diphosphate↗
PubMed1984

Prostacyclin inhibition of phosphatidic acid synthesis in human platelets is not mediated by protein kinase C.

The activation of protein kinase C in human platelets by phorbol-12, 13- dibutyrate (PDBu) results in the phosphorylation of a 40,000 dalton protein. This phosphorylation is time- and concentration-dependent. Maximal phosphorylation is rapid and is not affected by indomethacin or prostacyclin. PDBu does not promote activation of the phosphodiesteratic cleavage (phospholipase C) of the inositol phospholipids and the subsequent formation of 1,2-diacylglycerol or its phosphorylated product, phosphatidic acid. If platelets exposed to PDBu are subsequently stimulated with thrombin, this stimulus does not initiate further 40,000 dalton protein phosphorylation but will promote the formation of phosphatidic acid and also the phosphorylation of a 20,000 dalton protein (myosin light chain). However, prostacyclin will prevent the subsequent stimulation of phosphatidic acid synthesis by thrombin in a concentration-dependent manner. The fact that prostacyclin can affect the response to thrombin, even in the presence of phorbol ester, supports the idea that the enzymes related to the formation of phosphatidic acid or inhibition of its synthesis are not related to the phosphorylated 40K protein.

Blood Platelets↗
PubMed1991

Divergent effects of propranolol on neutrophil superoxide release: involvement of phosphatidic acid and diacylglycerol as second messengers.

Relatively high levels of propranolol (170 microM) markedly attenuated the generation of 1,2 diacylglycerol in neutrophils stimulated with either FMLP plus cytochalasin B or with 20.0 mM NaF. This effect resulted from inhibition of phosphatidic acid phosphohydrolase as it was accompanied by a corresponding increase in the recovery of phosphatidic acid in organic extracts of stimulated cells. Although propranolol enhanced phosphatidic acid levels in neutrophils treated with FMLP alone, the drug had only a slight inhibitory influence on diglyceride generation in these cells. The effect of propranolol on enhancement of PA levels in neutrophils treated with FMLP alone strongly correlated with enhancement of FMLP-induced O2- generation. However, propranolol induced a similar dose-dependent inhibition of O2- generation in neutrophils stimulated with either FMLP + cytochalasin B or with 20.0 mM NaF. These results are consistent with the hypothesis that both phosphatidic acid and diacylglycerol are required for optimal initiation of neutrophil O2- release.

Cytochalasin B↗