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PubMed1983

Phosphatidic acid mimics the muscarinic action of acetylcholine in cultured bovine chromaffin cells.

In cultured bovine chromaffin cells, acetylcholine as well as muscarine stimulated the 32Pi incorporation into phosphatidic acid, induced the efflux of 45Ca2+ from prelabelled cells, and, in parallel, elevated intracellular cyclic GMP content. Phosphatidic acid added to the medium also stimulated the efflux of 45Ca2+ and the synthesis of cyclic GMP in the cells in the same fashion as muscarinic agents, whereas it did not induce the secretion of catecholamines indicating that the effect of phosphatidic acid is specific to muscarinic action. The result supports the hypothesis that phosphatidic acid produced during phosphatidylinositol turnover is linked to the regulation mechanism of Ca2+ mobilization and cyclic GMP synthesis by muscarinic stimulation.

Acetylcholine↗

PubMed1985

Presence of membrane-associated phosphatidate phosphohydrolase activity in cultured islets and its stimulation by glucose.

The cellular location at which exogenous phosphatidic acid is hydrolysed in cultured neonatal rat islets was examined. Phosphatidate phosphohydrolase activity could be demonstrated in both whole cell sonicates and isolated plasma membranes. In the whole cell fraction phosphatidic acid hydrolysis to diacylglycerol was stimulated 43% by the presence of Mg2+. The activity present in isolated membranes was totally dependent on the presence of Mg2+ and was increased in plasma membranes from glucose-stimulated islets. Following exposure of islets to low glucose concentrations, raising the Ca2+ concentration from 150 nM to 40 microM in the presence of Mg2+ did not affect the formation of diacylglycerol in whole cell fractions or plasma membranes. These results indicate the presence within the islet of membrane-bound phosphatidate phosphohydrolase activity and demonstrate its activation by glucose.

Animals↗

PubMed1993

An increase in phosphatidic acid in the absence of changes in diacylglycerol in human platelets stimulated with ADP.

1. ADP caused an increase in radioactivity of phosphatidic acid but not diacylglycerol in human platelets labelled with [3H]arachidonic acid. 2. The radioactivity of phosphatidic acid was significantly increased 10 sec after adding 10 microM ADP and this increase did not depend on production of thromboxane A2. 3. Thrombin (1 U/ml) caused an increase in both diacylglycerol and phosphatidic acid, the latter being much greater than that caused by ADP. 4. The results confirm that ADP stimulates phosphatidic acid production and suggest that a weak stimulus of the phosphatidyl inositol cycle, such as ADP, does not cause accumulation of diacylglycerol.

Adenosine Diphosphate↗

PubMed1983

The role of the phosphatidate-inositide cycle in the action of steroidogenic agents.

Most steroidogenic agents have been found to provoke rapid changes in the metabolism of phospholipids in the phosphatidate-inositide cycle. These changes include: (a) de novo synthesis of phosphatidic acid and its derivatives, phosphatidylglycerol, mono- and polyphosphoinositides and diglyceride; and (b) phosphatidylinositol hydrolysis and consequent generation of diglyceride and phosphatidic acid. The de novo phosphatidate synthesis effect occurs in the action of all tested steroidogenic agents (ACTH, LH, angiotensin, K+, serotonin) and may be induced by either of the "second messengers", cAMP and Ca2+. The phosphatidylinositol hydrolysis effect(s) occurs only in the action of steroidogenic agents that operate via Ca2+ (angiotensin and K+) and may be important in Ca2+ mobilization. The de novo phospholipid effect correlates well with changes in steroidogenesis and, like the latter, requires Ca2+ and protein synthesis; from these and other results, it seems likely that this phospholipid effect plays an important role in the stimulation of steroidogenesis.

Adrenal Glands↗

PubMed1984

Ca(phosphatidate)2 can traverse liposomal bilayers.

Phosphatidic acid can act as Ca2+ cross-membrane ionophore without the necessity of previous autoxidation. The apparent PA-CA2+ dissociation constant is 3 X 10(-3), i.e., in the range of extracellular Ca2+ concentration. There is at least 100-fold preference for Ca2+ over Mg2+. Ca2+ transfer rates are proportional to the square of phosphatidic acid concentration in the bilayer. Removal of the fatty acid ester CO groups reduces the Ca2+ ferrying rate by more than 90 percent. It appears that the cation is held in a cage formed by phosphate and carbonyl oxygens of two PA molecules. In this coordination complex both Ca2+ and the phosphatidic acid headgroups are dehydrated, and the Ca(phosphatide)2 assembly becomes lipid-soluble and can traverse the bilayer.

Calcium↗

PubMed1983

The phosphatidate-phosphoinositide cycle: an intracellular messenger system in the action of hormones and neurotransmitters.

Many hormones and neurotransmitters provoke rapid and striking changes in the metabolism of phospholipids in the phosphatidate-inositide cycle. These changes appear to occur before and after the generation of other accepted "second messengers" (e.g., Ca++ and cAMP), and seem to be important intracellular effector substances for the elicitation of biological effects. The two major mechanisms for perturbing the phosphatidate-inositide cycle are phosphatidylinositol hydrolysis and de novo phosphatidate-inositide synthesis. Phosphatidylinositol hydrolysis occurs in the action of all agents which operate via Ca++ and appears to be provoked both by Ca++-dependent and Ca++-independent mechanisms. Ca++-independent phosphatidylinositol hydrolysis may be triggered directly by receptor activation and may control Ca++ release or entry into the cytosol. Ca++-dependent phosphatidylinositol hydrolysis may be important for further changes in cellular Ca++ distribution and membrane fusion during exocytosis. The de novo phosphatidate-inositide synthesis effect has been observed in the action of most steroidogenic agents (ACTH, luteinizing hormone, angiotensin II, K+, serotonin), parathyroid hormone and insulin. The de novo effect is inhibited by cycloheximide, requires Ca++, and appears to serve as a post-second messenger mechanism to alter membrane structure and the function of membrane associated substances. Considerable evidence suggests that the de novo effect is important in the control of steroidogenesis by the above-mentioned agents; it may also be important in the action of insulin in adipose tissue.

Adrenocorticotropic Hormone↗

PubMed1984

Human platelet activation by bacterial phospholipase C is mediated by phosphatidylinositol hydrolysis but not generation of phosphatidic acid: inhibition by a selective inhibitor of phospholipase C.

We have shown earlier that phospholipase C (PLC) from Clostridium perfringens causes human platelet aggregation and secretion in a concentration dependent manner. The present study was undertaken to further characterize the specificity of the effects of PLC and to better understand the mechanism of the action of this inducer. A methylene-dioxybenzazepine (MDBA) analog of trimetoquinol was synthesized and tested for antiplatelet activity. MDBA (3-30 microM) inhibited PLC-induced aggregation in a concentration dependent manner. Whereas up to 200 microM MDBA did not inhibit aggregation induced by either thrombin, arachidonic acid, or U46619. Effects of PLC (0.05 U/ml) on hydrolysis of phosphatidylinositol, production of phosphatidic acid and thromboxane B2 (TXB2) synthesis were investigated using [32P]-phosphate and [14C]-arachidonic acid labeled platelets. PLC (0.05 U/ml) caused a time dependent decrease in platelet phosphatidylinositol. Up to 50% of labeled phosphatidylinositol was lost from platelets in five minutes. MDBA (3-30 microM) inhibited PLC-induced loss of phosphatidylinositol in a concentration dependent manner. An increase in phosphatidic acid was also observed in PLC-stimulated platelets. Up to 100 microM MDBA did not inhibit production of phosphatidic acid. PLC-treated platelets did not produce any TXB2. In other experiments possible protease contamination of PLC preparations was tested by incubating PLC (0.03-0.5 U/ml) with [14C]-casein. PLC in concentrations up to ten times higher than the concentrations used in aggregation studies did not cause hydrolysis of [14C]-casein, whereas more than 30% of [14C]-casein was hydrolyzed by trypsin. PLC-induced aggregation was not inhibited by up to 300 microM adenosine or ATP. In other experiments, platelet aggregation by ADP was inhibited by adenosine and ATP in a concentration dependent manner. The addition of calcium (0.5- 2.0 mM) increased aggregation by PLC in a concentration dependent manner. These findings suggest that PLC-induced activation of platelets is: (a) dependent on phosphatidylinositol hydrolysis but not on the production of phosphatidic acid, TXB2 or secretion of ADP; (b) not caused by protease contaminants; (c) calcium dependent; and (d) MDBA inhibits PLC-induced aggregation by blocking phosphatidylinositol hydrolysis.

Adenosine↗

PubMed1978

Phosphatidic acid releases calcium from a platelet membrane fraction in vitro.

A platelet membrane fraction which actively sequesters calcium in the presence of ATP was prepared and the influence of phosphatidic acid evaluated. At 10--60 micrograms/ml phosphatidic acid caused a concentration dependent release of calcium from the membrane fraction. The calcium was released from inside the vesicles, since release occurred in the presence of EGTA used to bind calcium outside the membrane vesicles. Aspirin failed to inhibit release of calcium by phosphatidic acid. Our results may explain, in part, the prostaglandin and thromboxane independent calcium release which occurs in response to certain aggregating agents. Thus, phosphatidic acid, or a metabolite, may have an important role intracellularly in platelets in promoting calcium movement.

Blood Platelets↗

PubMed1988

Epidermal growth factor stimulates the incorporation of phosphate into phosphatidic acid and phosphoinositides but does not affect phosphoinositide breakdown by phospholipase C in renal cortical slices.

The effects of epidermal growth factor (EGF) on the metabolism of phosphatidic acid and phosphoinositides were examined using renal cortical slices labelled with either sodium [32P]orthophosphate or myo-[3H]inositol. EGF was found to increase the incorporation of phosphate into phosphatidic acid and phosphoinositides. This effect is not dependent on external calcium and is inhibited by 12-O-tetradecanoylphorbol 13-acetate (TPA). When phospholipids were prelabelled, EGF did not decrease the level of 32P in phosphatidic acid and phosphoinositides, and EGF did not affect the formation of inositol phosphates or the concentration of cAMP and cGMP in renal tissue. The results show that EGF stimulates the incorporation of phosphate into phosphatidic acid and phosphoinositides, but does not affect breakdown of phosphoinositides by phospholipase C in renal cortical slices.

Animals↗

PubMed1994

Quantitation of the fatty acid composition of phosphatidic acid by capillary gas chromatography electron-capture detection with picomole sensitivity.

We describe a relatively simple and sensitive method to measure femtomole amounts of phosphatidic acid in cells. Phosphatidic acid was extracted from cells in the presence of 1-heptadecanoyl-2-heptadecanoyl-sn-glycero-3-phosphate as an internal standard, purified by two-dimensional thin-layer chromatography, and hydrolyzed to its constituent free fatty acids which were then derivatized to the corresponding pentafluorobenzyl esters. Pentafluorobenzyl esters of fatty acids were analyzed by gas chromatography with electron-capture detection. Long-chain fatty acids were resolved with excellent signal-to-noise ratios. Using heptadecanoic acid as an internal standard for quantitation, as little as 1 fmol of pentafluorobenzyl ester of stearic acid was detected with a linear response up to 10 pmol. Linear detector responses were obtained for all major classes of fatty acids. For phosphatidic acid measurement, the detection limit was at least 50 fmol thus achieving a 1000-fold increase in sensitivity compared to the most sensitive of the previously described methods. An example is provided of quantitating phosphatidic acid from minute amounts of biological samples such as islets of Langerhans.

Animals↗

PubMed1991

Priming of calcium mobilization in human neutrophils by granulocyte-macrophage colony-stimulating factor: evidence for an involvement of phospholipase D-derived phosphatidic acid.

Human neutrophils pre-incubated with granulocyte-macrophage-colony-stimulating factor (GM-CSF) exhibit an enhanced mobilization of calcium in response to secondary stimuli such as chemotactic factors. The mechanisms underlying this priming effect of GM-CSF were examined. It was first demonstrated that the additional calcium mobilized by chemotactic factors in GM-CSF-treated cells was derived from intracellular stores and was associated neither with an increased permeability to calcium nor with production of inositol 1,4,5-trisphosphate. These results indicated that GM-CSF called upon a novel mechanism in order to enhance the mobilization of calcium in human neutrophils. The growth factor has recently been shown to prime phospholipase D leading to an enhanced activation by chemotactic factors and an augmented production of phosphatidic acid. Furthermore the ability of exogenous phosphatidic acid to mobilize calcium in cell types other than neutrophils has been previously demonstrated. Therefore, we examined the potential involvement of phospholipase D in the priming of the calcium response by GM-CSF in human neutrophils. Inhibition of the production of the fMet-Leu-Phe-stimulated production of phosphatidic acid by ethanol or wortmannin had only marginal effects on the concurrent mobilization of calcium. However, the priming of the mobilization of calcium by GM-CSF was greatly decreased in cells treated with either ethanol or wortmannin. These results provide strong support for the hypothesis that the production of phosphatidic acid, which is enhanced in GM-CSF-treated cells, is linked to an increased mobilization of intracellular calcium. These results may have relevance to the mechanism of action of GM-CSF in mature haematopoeitic cells as well to the mitogenic activity of other growth factors.

Adult↗

PubMed1991

Glucose and carbachol synergistically stimulate phosphatidic acid accumulation in pancreatic islets.

Phosphatidic acid has been previously implicated as an intracellular mediator of insulin secretion. Very little is known, however, about endogenous phosphatidic acid levels in islets. We now show, for the first time, that glucose and carbachol, at concentrations which stimulate insulin secretion, significantly increase endogenous phosphatidic acid levels in pancreatic islets by 2-fold at 1 min, nearly 3-fold at 2 min, and over 3-fold at 30 min compared to control. Possible mechanisms include de novo synthesis from glucose and/or activation of phospholipase D. Our data, taken together with previous studies, suggest that phosphatidic acid may have a central role in insulin secretion as an intracellular mediator.

Animals↗

PubMed1998

Interleukin-2 stimulates a late increase in phosphatidic acid production in the absence of phospholipase D activation.

The signal transduction pathways involving phospholipid metabolism during T-cell proliferation remain partly undefined. Herein we show that interleukin-2 caused a late (> 12 h) rise in the intracellular phosphatidic acid content of CTLL-2 cells which was a consequence of the activation of the enzyme diacylglycerol kinase. No activation of phospholipase D was observed at similar times. Incubation of the cells with a recognized diacylglycerol kinase a isoform inhibitor, R59499, prior to interleukin-2 stimulation was able to block cell cycle entry, diacyglycerol kinase activation and phosphatidic acid accumulation. In contrast, when R59499 was added 3 h after interleukin-2, few or no observable effects on the above three parameters were noticed. These results suggest that the early signaling employed by IL-2 involving the alpha isoform of diacylglycerol kinase is sufficient to control the late increase in phosphatidic acid and that phosphatidic acid is a mitogenic agent in T-cells.

3T3 Cells↗

PubMed1978

Phosphatidic and lysophosphatidic acid production in phospholipase C-and thrombin-treated platelets. Possible involvement of a platelet lipase.

Incubation of 32P-labelled platelets with Clostridium welchii phospholipase C greatly stimulates 32P-incorporation into phosphatidic and lysophosphatidic acids. A net synthesis is demonstrated for both phospholipids, which exhibit identical specific radioactivities. Phosphatidic acid production roughly parallels the phospholipase C-induced aggregation, whereas lysophosphatidic acid appears secondarily during cell lysis. The same qualitative variations are observed during thrombin-induced aggregation. At the physiological pH used throughout the incubations, platelets display no phospholipase A activity towards phosphatidic acid, whereas diglycerides are deacylated by platelet lysates. On the basis of these findings, a mechanism for phosphatidic and lysophosphatidic acid production is proposed, involving a phosphorylation of the di- and monoglycerides formed upon phospholipase C and lipase action. The possible role of such a pathway in regulating arachidonic acid release from phospholipids during platelet activation is discussed.

Animals↗

PubMed2001

Phosphatidic acid: an emerging plant lipid second messenger.

Evidence is accumulating that phosphatidic acid is a second messenger. Its level increases within minutes of a wide variety of stress treatments including ethylene, wounding, pathogen elicitors, osmotic and oxidative stress, and abscisic acid. Enhanced signal levels are rapidly attenuated by phosphorylating phosphatidic acid to diacylglycerol pyrophosphate. Phosphatidic acid is the product of two signalling pathways, those of phospholipases C and D, the former in combination with diacylglycerol kinase. Families of these genes are now being cloned from plants. Several downstream targets of phosphatidic acid have been identified, including protein kinases and ion channels.

Diphosphates↗

PubMed1999

Effect of calcium and phosphatidic acid binding on the C2 domain of PKC alpha as studied by Fourier transform infrared spectroscopy.

Fourier transform infrared (FTIR) spectroscopy was used to investigate the structural and thermal denaturation of the C2 domain of PKC alpha (PKC-C2) and its complexes with Ca(2+) and phosphatidic acid vesicles. The amide I regions in the original spectra of PKC-C2 in the Ca(2+)-free and Ca(2+)-bound states are both consistent with a predominantly beta-sheet secondary structure below the denaturation temperatures. Spectroscopic studies of the thermal denaturation revealed that for the PKC-C2 domain alone the secondary structure abruptly changed at 50 degrees C. While in the presence of 2 and 12.5 mM Ca(2+), the thermal stability of the protein increased to 60 and 70 degrees C, respectively. Further studies using a mutant lacking two important amino acids involved in Ca(2+) binding (PKC-C2D246/248N) demonstrated that these mutations were inherently more stable to thermal denaturation than the wild-type protein. Phosphatidic acid binding to the PKC-C2 domain was characterized, and the lipid-protein binding became Ca(2+)-independent when 100 mol% phosphatidic acid vesicles were used. The mutant lacking two Ca(2+) binding sites was also able to bind to phosphatidic acid vesicles. The effect of lipid binding on secondary structure and thermal stability was also studied. Beta-sheet was the predominant structure observed in the lipid-bound state, although the percentage represented by this structure in the total area of the amide I band significantly decreased from 60% in the lipid-free state to 47% in the lipid-bound state. This decrease in the beta-sheet component of the lipid-bound complex correlates well with the significant increase observed in the 1644 cm(-1) band which can be assigned to loops and disordered structure. Thermal stability after lipid binding was very high, and no sign of thermal denaturation was observed in the presence of lipids under the conditions that were studied.

Animals↗

PubMed1978

Problems encountered in measuring the activity of phosphatidate phosphohydrolase.

The measurement of phosphate release from phosphatidate overestimates the microsomal activity of phosphatidate phosphohydrolase from rat liver, since phosphate is also produced via the glycerol phosphate that results from the deacylation of phosphatidate. The determination of phosphate production can be a reliable assay for the soluble phosphatidate phosphohydrolase in rat liver, because the glycerol phosphate formed is not hydrolysed under the conditions used.

Animals↗

PubMed1984

Thromboxane-induced phosphatidate formation in human platelets. Relationship to receptor occupancy and to changes in cytosolic free calcium.

The inter-relationships between receptor occupancy, inositol phospholipid metabolism and elevation of cytosolic free Ca2+ in thromboxane A2-induced human platelet activation were investigated by using the stable thromboxane A2 mimetic, 9,11-epoxymethanoprostaglandin H2, and the thromboxane A2 receptor antagonist, EPO45. 9,11-Epoxymethanoprostaglandin H2 stimulated platelet phosphatidylinositol metabolism as indicated by the rapid accumulation of [32P]phosphatidate and later accumulation of [32P]phosphatidylinositol in platelets pre-labelled with [32P]Pi. These effects of 9,11-epoxymethanoprostaglandin H2 were concentration-dependent and half-maximal [32P]phosphatidate formation occurred at an agonist concentration of 54 +/- 8 nM. With platelets labelled with the fluorescent Ca2+ indicator quin 2, resting cytosolic free Ca2+ was 86 +/- 12 nM. 9,11-Epoxymethanoprostaglandin H2 induced a rapid, concentration-dependent elevation of cytosolic free Ca2+ to a maximum of 300-700 nM. Half-maximal stimulation was observed at an agonist concentration of 80 +/- 23 nM. The thromboxane A2 receptor antagonist EPO45 selectively inhibited 9,11-epoxymethanoprostaglandin H2-induced [32P]phosphatidate formation and elevation of cytosolic free Ca2+, indicating that both events are sequelae of receptor occupancy. Human platelets contain a single class of stereospecific, saturable, high affinity (KD = 70 +/- 13 nM) binding sites for 9,11-epoxymethano[3H]prostaglandin H2. The concentration-response curve for receptor occupancy (9,11-epoxymethano-[3H]prostaglandin H2 binding) is similar to that for 9,11-epoxymethanoprostaglandin H2-induced [32P]phosphatidate formation and for elevation of cytosolic free Ca2+. These observations indicate that human platelet thromboxane A2 receptor occupation is closely linked to inositol phospholipid metabolism and to elevation of cytosolic free Ca2+. Both such events may be necessary for thromboxane A2-induced human platelet activation.

Blood Platelets↗