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PubMed1992

Sphingosine-1-phosphate, a metabolite of sphingosine, increases phosphatidic acid levels by phospholipase D activation.

Sphingosine and sphingosine-1-phosphate, metabolites of membrane sphingolipids, have recently been shown to stimulate release of calcium from internal sources and to increase proliferation of quiescent Swiss 3T3 fibroblasts (Zhang, H., Desai, N. N., Olivera, A., Seki, T., Brooker, G., and Spiegel, S. (1991) J. Cell Biol. 114, 155-167). The present study demonstrates that mitogenic concentrations of sphingosine induce early increases in sphingosine-1-phosphate levels which precede the increase in the potent mitogen, phosphatidic acid. Sphingosine-1-phosphate itself induces a more rapid increase in phosphatidic acid, thus suggesting that it may mediate the effects of sphingosine on phosphatidic acid accumulation. The concentration dependence for the formation of phosphatidic acid induced by sphingosine-1-phosphate correlates with its effect on DNA synthesis. Similar to sphingosine, sphingosine-1-phosphate also stimulates the activity of phospholipase D, although a significant effect is observed at a much lower concentration. However, in contrast to previous reports with sphingosine, sphingosine-1-phosphate does not inhibit the phosphatidic acid phosphohydrolase activity in cell homogenates. Thus, in addition to its effect on mobilization of calcium, sphingosine-1-phosphate can increase the level of phosphatidic acid, most likely via activation of phospholipase D. We suggest that sphingosine-1-phosphate mediates the effect of sphingosine on phosphatidic acid accumulation in Swiss 3T3 fibroblasts and may regulate cellular proliferation by affecting multiple transmembrane signaling pathways.

3T3 Cells↗
PubMed1996

Phosphatidate phosphatases of mammals, yeast, and higher plants.

Phosphatidate phosphatase (EC 3.1.3.4) catalyzes the hydrolysis of phosphatidate to yield sn-1,2-diacylglycerol and inorganic phosphate. In mammalian systems, forms of phosphatidate phosphatase involved in glycerolipid synthesis and signal transduction have been identified. Forms of the enzyme involved in signal transduction have been purified and partially characterized. In yeast, phosphatidate phosphatases associated with the endoplasmic reticulum and mitochondria have also been purified and partially characterized. Information on phosphatidate phosphatases from mammals and yeast is useful in characterizing the enzyme from plant systems. This review examines progress on the characterization of phosphatidate phosphatases from mammals, yeast, and higher plants. The purification and characterization of the phosphatidate phosphatase involved in glycerolipid synthesis in developing oilseeds may lead to the identification of the encoding gene. Increasing our understanding of the enzymes of lipid synthesis in developing seeds will aid in the development of biotechnological strategies for seed oil modification.

Animals↗
PubMed1979

The influence of charge on phosphatidic acid bilayer membranes.

A complete titration of phosphatidic acid bilayer membranes was possible for the first time by the introduction of a new anaologue, 1,2-dihexadecyl-sn-glycerol-3-phosphoric acid, which has the advantage of a high chemical stability at extreme pH values. The synthesis of the phosphatidic acid is described and the phase transition behaviour in aqueous dispersions is compared with that of three ester phosphatidic acids; 1,2-dimyristoyl-sn-glycerol-3-phosphoric acid, 1,3-dimyristoylglycerol-2-phosphoric acid and 1,2-dipalmitoyl-sn-glycerol-3-phosphoric acid. The phase transition temperatures (Tt) of aqueous phosphatidic acid dispersions at different degrees of dissociation were measured using fluorescence spectroscopy and 90 degrees light scattering. The Tt values are comparable to the melting points of the solid phosphatidic acids in the fully protonated states, but large differences exist for the charged states. The Tt vs. pH diagrams of the four phosphatidic acids are quite similar and of a characteristic shape. Increasing ionisation results in a maximum value for the transition temperatures at pH 3.5 (pK1). The regions between the first and the second pK of the phosphatidic acids are characterised by only small variations in the transition temperatures (extended plateau) in spite of the large changes occurring in the surface charge of the membranes. The slope of the plateau is very shallow with increasing ionisation. A further decrease in the H+ concentration results in an abrupt change of the transition temperature. The slope of the Tt vs. pH diagram beyond pK2 becomes very steep. This is the result of reduced hydrocarbon interaction energy, which was demonstrated by differential scanning calorimetry (Blume, A. and Eibl, H., unpublished data).

Fluorescence Polarization↗
PubMed1990

Phosphatidic acid and not diacylglycerol generated by phospholipase D is functionally linked to the activation of the NADPH oxidase by FMLP in human neutrophils.

It is widely accepted that the activation of the NADPH oxidase of phagocytes is linked to the stimulation of protein kinase C by diacylglycerol formed by hydrolysis of phospholipids. The main source would be choline containing phospholipid via phospholipase D and phosphatidate phosphohydrolase. This paper presents a condition where the activation of the respiratory burst by FMLP correlates with the formation of phosphatidic acid, via phospholipase D, and not with that of diacylglycerol. In fact: 1) in neutrophils treated with propranolol, an inhibitor of phosphatidate phosphohydrolase, FMLP plus cytochalasin B induces a respiratory burst associated with a stimulation of phospholipase D, formation of phosphatidic acid and complete inhibition of that of diacylglycerol. 2) The respiratory burst by FMLP plus cytochalasin B lasts a few minutes and may be restimulated by propranolol which induces an accumulation of phosphatidic acid. 3) In neutrophils stimulated by FMLP in the absence of cytochalasin B propranolol causes an accumulation of phosphatidic acid and a marked enhancement of the respiratory burst without formation of diacylglycerol. 4) The inhibition of the formation of phosphatidic acid via phospholipase D by butanol inhibits the respiratory burst by FMLP.

Butanols↗
PubMed1983

Changes in phosphatidylinositol and phosphatidic acid in stimulated human neutrophils. Relationship to calcium mobilization, aggregation and superoxide radical generation.

Human neutrophils aggregate and release mediators of inflammation, such as active oxygen species and lysosomal enzymes, when exposed to the chemoattractant, fMet-Leu-Phe, or the tumor promotor, phorbol myristate acetate. In order to 'stage' events which may lead to such neutrophil responses, we determined the temporal relationship between stimulus-induced changes in the endogenous phospholipids phosphatidylinositol (PI) and phosphatidic acid, the mobilization of calcium, and the onset of aggregation and generation of superoxide anion during the initial 2 min of cell activation. Within 5 s after addition of fMet-Leu-Phe (10(-7) M) neutrophils accumulated phosphatidic acid and the levels of PI decreased, as determined by two-dimensional thin-layer chromatography and phosphorus determinations. By 5 s, phosphatidic acid levels rose approximately 3.5-fold and at 15 s the loss of PI exceeded the quantity of phosphatidic acid generated. In response to phorbol myristate acetate (1 microgram/ml), however, changes in PI or phosphatidic acid were not observed until after 60 s. Accumulation of phosphatidic acid in fMet-Leu-Phe-stimulated cells was not inhibited by chelation of extracellular calcium. Neutrophils exposed to either fMet-Leu-Phe or phorbol myristate acetate also showed rapid decrements in fluorescence of cell-associated chlorotetracycline (used as an indirect probe of mobilization of intracellular membrane-associated calcium) and took up 45Ca2+ from the extracellular medium (under 60 s). The results indicate that changes in calcium mobilization, together with the alterations in phospholipid metabolism (under 5 s) anteceded aggregation and the generation of O2-. (10-15 s) induced by fMet-Leu-Phe. In contrast, when neutrophils were exposed to phorbol myristate acetate, changes in PI and phosphatidic acid (over 60 s) were observed after the mobilization of calcium (under 5 s) and the onset of O2-. generation and aggregation (30-35 s).

Calcium↗
PubMed1994

Arachidonic acid liberation induced by phosphatidic acid endogenously generated from membrane phospholipids in rabbit platelets.

The action of phosphatidic acid generated from membrane phospholipids on phospholipase A2 activation in rabbit platelets was investigated. When [3H]arachidonic acid-labelled platelets were treated with phorbol 12-myristate 13-acetate (PMA) and the membranes isolated from the cells incubated at 37 degrees C with 50 microM CaCl2 and 50 microM guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), both phosphatidic acid production and arachidonic acid liberation increased in PMA- and GTP gamma S-concentration-dependent manners. Ethanol dose-dependently inhibited these responses, accompanied by the formation of phosphatidylethanol. Since propranolol, an inhibitor of phosphatidic acid phosphohydrolase, had no influence on the production of phosphatidic acid, the arachidonic acid liberated does not appear to be derived from diacylglycerol which may be produced from phosphatidic acid through the action of this enzyme. In another approach, treatment of [3H]arachidonic acid-labelled membranes with phospholipase D from Streptomyces chromofuscus induced arachidonic acid liberation as well as phosphatidic acid formation in time- and dose-dependent manners. The former response was suppressed by p-bromophenacyl bromide, a phospholipase A2 inhibitor. These results suggest that phosphatidic acid derived from membrane phospholipids potentiates phospholipase A2 activation and contributes to the amplification of platelet activation.

Animals↗
PubMed2002

Segregation of phosphatidic acid-rich domains in reconstituted acetylcholine receptor membranes.

Purified Acetylcholine Receptor (AcChR) from Torpedo has been reconstituted at low (approximately 1:3500) and high (approximately 1:560) protein to phospholipid molar ratios into vesicles containing egg phosphatidylcholine, cholesterol, and different dimyristoyl phospholipids (dimyristoyl phosphatidylcholine, phosphatidylserine, phosphatidylglycerol and phosphatidic acid) as probes to explore the effects of the protein on phospholipid organization by differential scanning calorimetry, infrared, and fluorescence spectroscopy. All the experimental results indicate that the presence of the AcChR protein, even at the lower protein to phospholipid molar ratio, directs lateral phase separation of the monoanionic phosphoryl form of the phosphatidic acid probe, causing the formation of specific phosphatidic acid-rich lipid domains that become segregated from the bulk lipids and whose extent (phosphatidic acid sequestered into the domain, out of the total population in the vesicle) is protein-dependent. Furthermore, fluorescence energy transfer using the protein tryptophan residues as energy donors and the fluorescence probes trans-parinaric acid or diphenylhexatriene as acceptors, establishes that the AcChR is included in the domain. Other dimyristoyl phospholipid probes (phosphatidylcholine, phosphatidylserine, phosphatidylglycerol) under identical conditions could not mimic the protein-induced domain formation observed with the phosphatidic acid probe and result in ideal mixing of all lipid components in the reconstituted vesicles. Likewise, in the absence of protein, all the phospholipid probes, including phosphatidic acid, exhibit ideal mixing behavior. Since phosphatidic acid and cholesterol have been implicated in functional modulation of the reconstituted AcChR, it is suggested that such a specific modulatory role could be mediated by domain segregation of the relevant lipid classes.

Animals↗
PubMed1976

Production of 1,2-diacylglycerol and phosphatidate in human erythrocytes treated with calcium ions and ionophore A23187.

1. When the ionophore A23187 and Ca2+ were added to normal human erythrocytes, the incorporation of 32P into phosphatidate was enhanced within 1 min, but there was only slight labelling of other phospholipids. 2. Labelling of phosphatidate in these cells did not continue to increase after about 20min at 37 degrees C; by this time, radioactivity in phosphatidate was about ten times higher inionophore A23187-treated cells than in controls. A net synthesis of phosphatidate was measured in response to the increase in intracellular Ca2+ concentration; the content of this phospholipid in the cell was increased by approximately 50%. 3. In the presence of 2.5 mM-Ca2+ a maximum effect was seen with about 0.5 mug of ionophore/ml. 4. The concentration of Ca2+ giving half-maximal labelling of phosphatidate in the presence of 10 mug of ionophore A23187/ml was about 10 muM. 5. A rapid decrease of ATP content in the cell occurred in ionophore-treated cells. 6. Labelling of phosphatidate appeared to be secondary to the production of 1,2-diacylglycerol in the cells; accumulation of 1,2-diacylglycerol was only seen after about 15 min. After 60 min, the 1,2-diacylglycerol content of the cells was five to seven times that of untreated control cells. 7. The change in the shape of erythrocytes treated with Ca2+ and ionophore appeared to be related to accumulation of 1,2-diacylglycerol. 8. The source of 1,2-diacylglycerol has not been definitely identified, but its fatty acid compositon was similar to that of phosphatidylcholine. However, it has an unusually high content of hexadecenoic acid, a fatty acid not common in the major erythrocyte phospholipids. 9. Accumulation of 1,2-diacyglycerol also occurred in energy-starved cells, even in the absence of calcium; in this case it appeared to be produced by phosphatidate breakdown.

Adenosine Triphosphate↗
PubMed1977

The effects of amphiphilic cationic drugs and inorganic cations on the activity of phosphatidate phosphohydrolase.

1. Phosphatidate phosphohydrolase from the particle-free supernatant of rat liver was assayed by using emulsions of phosphatidate as substrate. 2. The inhibition of the phosphohydrolase by chlorpromazine was of a competitive type with respect to phosphatidate. The potency of various amphiphilic cationic drugs as inhibitors of this reaction was related to their partition coefficients into a phosphatidate emulsion. 3. The effect of chlorpromazine on the phosphohydrolase activity was complementary rather than antagonistic towards Mg2+. Chlorpromazine stimulated the phosphohydrolase activity in the absence of added Mg2+ and was able to replace the requirement for Mg2+. However, at optimum concentrations of Mg2+, chlorpromazine inhibited the reaction, as did Ca2+. The phosphohydrolase activity was also stimulated by Co2+ and to a lesser extent by Mn2+, Fe2+, Fe3+, Ca2+, spermine and spermidine when Mg2+ was not added to the assays. 4. It is concluded that the inhibition of phosphatidate phosphohydrolase by amphiphilic cations can largely be explained by the interaction of these compounds with phosphatidate, which changes the physical properties of the lipid, making it less available for conversion into diacylglycerol. 5. The implications of these results to the effects of amphiphilic cations in redirecting glycerolipid synthesis at the level of phosphatidate are discussed.

Animals↗
PubMed1978

Mechanisms for the effects of ethanol on hepatic phosphatidate phosphohydrolase.

1. The effects of the intramuscular administration of glycerol and dihydroxyacetone (40mmol per kg body wt.), sorbitol and glucose (20mmol per kg body wt.) or NaCl (1.5mmol per kg body wt. in 10ml of water per kg body wt.) were investigated on soluble phosphatidate phosphohydrolase and certain metabolites in rat liver. 2. The effects of ethanol and glycerol on phosphatidate phosphohydrolase were also studied in isolated perfused livers. 3. The administration of glycerol, sorbitol and dihydroxyacetone in vivo increased hepatic phosphatidate phosphohydrolase activity by 137, 63 and 32% respectively in 4h. 4. A significant positive correlation was found between the hepatic sn-glycerol 3-phosphate concentration and phosphatidate phosphohydrolase after the administration of various substrates in vivo. 5. The soluble phosphatidate phosphohydrolase activity tended to increase during perfusions of isolated rat livers without added substrates, and neither ethanol nor glycerol produced additional effects. 6. The activity of soluble phosphatidate phosphohydrolase was 2.5 times higher in the livers of hyperthyroid rats than in normal rats. This activity was not influenced by intragastric ethanol or glycerol administration, nor was the concentration of sn-glycerol 3-phosphate changed by these compounds. 7. It is concluded that the ethanol-induced increase in hepatic phosphatidate phosphohydrolase may at least in part be mediated by the hepatic concentration of metabolites, probably by the concentration of sn-glycerol 3-phosphate.

Animals↗
PubMed1985

The acylation of sn-glycerol 3-phosphate and the metabolism of phosphatidate in microsomal preparations from the developing cotyledons of safflower (Carthamus tinctorius L.) seed.

Microsomal preparations from the developing cotyledons of safflower (Carthamus tinctorius) catalysed the acylation of sn-glycerol 3-phosphate in the presence of acyl-CoA. The resulting phosphatidate was further utilized in the synthesis of diacyl- and tri-acylglycerol by the reactions of the so-called 'Kennedy pathway' [Kennedy (1961) Fed. Proc. Fed. Am. Soc. Exp. Biol. 20, 934-940]. Diacylglycerol equilibrated with the phosphatidylcholine pool when glycerol backbone, with the associated acyl groups, flowed from phosphatidate to triacylglycerol. The formation of diacylglycerol from phosphatidate through the action of a phosphatidate phosphohydrolase (phosphatidase) was substantially inhibited by EDTA and, under these conditions, phosphatidate accumulated in the microsomal membranes. The inhibition of the phosphatidase by EDTA was alleviated by Mg2+. The presence of Mg2+ in all incubation mixtures stimulated quite considerably the synthesis of triacylglycerol in vitro. Microsomal preparations incubated with acyl-CoA, sn-glycerol 3-phosphate and EDTA synthesized sufficient phosphatidate for the reliable analysis of its intramolecular fatty acid distribution. In the presence of mixed acyl-CoA substrates the sn-glycerol 3-phosphate was acylated exclusively in position 1 with the saturated fatty acids, palmitate and stearate. The polyunsaturated fatty acid linoleate was, however, utilized largely in the acylation of position 2 of sn-glycerol 3-phosphate. The affinity of the enzymes involved in the acylation of positions 1 and 2 of sn-glycerol 3-phosphate for specific species of acyl-CoA therefore governs the non-random distribution of the different acyl groups in the seed triacylglycerols. The acylation of sn-glycerol 3-phosphate in position 1 with saturated acyl components also accounts for the presence of these groups in position 1 of sn-phosphatidylcholine through the equilibration of diacylglycerol with the phosphatidylcholine pool, which occurs when phosphatidate is utilized in the synthesis of triacylglycerol. These results add further credence to our previous proposals for the regulation of the acyl quality of the triacylglycerols that accumulate in developing oil seeds [Stymne & Stobart (1984) Biochem. J. 220, 481-488; Stobart & Stymne (1985) Planta 163, 119-125].

Acyl Coenzyme A↗
PubMed1983

Acetal phosphatidic acids: novel platelet aggregating agents.

1 Palmitaldehyde, olealdehyde and linolealdehyde acetal phosphatidic acids induced rapid shape change and dose-dependent biphasic aggregation of human platelets in platelet-rich plasma; aggregation was reversible at low doses and irreversible at high doses of the acetal phosphatidic acids. The palmitaldehyde congener elicited monophasic dose-dependent aggregation of sheep platelets in platelet-rich plasma.2 The threshold concentration for palmitaldehyde acetal phosphatidic acid (PGAP)-induced platelet aggregation was 2.5-5 muM for human platelets and 0.25-0.5 muM for sheep platelets. PGAP was 4-5 times as potent versus human platelets as the olealdehyde and linolealdehyde acetal phosphatidic acids, which were equipotent.3 PGAP-induced irreversible aggregation of [(14)C]-5-hydroxytryptamine ([(14)C]-5-HT)-labelled human platelets in platelet-rich plasma was accompanied by release of 44.0+/-2.4% (s.e.) of the platelet [(14)C]-5-HT; reversible aggregation was not associated with release. In contrast, PGAP-induced release of [(14)C]-5-HT-labelled sheep platelets was dose-dependent.4 The adenosine diphosphate (ADP) antagonist, 2-methylthio-AMP, and the cyclo-oxygenase inhibitor, aspirin, abolished PGAP-induced second phase aggregation and release in human platelets but did not affect the first, reversible, phase of aggregation. Both the first and second phases of PGAP-induced aggregation were abolished by chlorpromazine, by the phospholipase A(2) inhibitor, mepacrine, and by nmolar concentrations of prostaglandin E(1) (PGE(1)); these agents abolished the second, but not the first phase of ADP-induced aggregation.5 The related phospholipids, lecithin, lysolecithin and phosphatidic acid, at <100 muM, neither induced aggregation of human platelets in platelet-rich plasma, nor modified PGAP-induced aggregation; 1-palmityl lysophosphatidic acid elicited aggregation of human platelets at a threshold concentration of 100 muM.6 It is concluded that the acetal phosphatidic acids induce platelet aggregation per se by direct action at the platelet membrane, and that the acetal function is of primary importance in their potent platelet-stimulating activity. Moreover, as the acetal phosphatidic acids are the major components of the smooth muscle-contracting acidic phospholipid tissue extract ;Darmstoff' (Vogt, 1949), their potent platelet-aggregating properties may be of physiological or pathological significance.

Adenosine Diphosphate↗
PubMed1988

Regulation of phosphatidate phosphatase activity by inositol in Saccharomyces cerevisiae.

Regulation of phosphatidate phosphatase (EC 3.1.34) activity was examined in Saccharomyces cerevisiae cells supplemented with phospholipid precursors. Addition of inositol to the growth medium of wild-type cells resulted in a twofold increase in phosphatidate phosphatase activity. The increase in phosphatidate phosphatase activity was not due to soluble effector molecules, and inositol did not have a direct effect on enzyme activity. The phosphatidate phosphatase activity associated with the mitochondrial, microsomal, and cytosolic fractions of the cell was regulated by inositol in the same manner. Cells supplemented with inositol had elevated phospholipid levels and reduced triacylglycerol levels compared with unsupplemented cells. Serine, ethanolamine, and choline did not significantly affect the phosphatidate phosphatase activity of cells grown in the absence or presence of inositol. Enzyme activity was not regulated in inositol biosynthesis regulatory mutants, suggesting that regulation by inositol is coupled to regulation of inositol biosynthesis. Phosphatidate phosphatase activity was pleiotropically expressed in structural gene mutants defective in phospholipid biosynthesis. These results suggested that phosphatidate phosphatase was regulated by inositol at a genetic level.

Culture Media↗
PubMed1986

Translocation of Mg2+-dependent phosphatidate phosphohydrolase between cytosol and endoplasmic reticulum in a permanent cell line from human lung.

Incubation of A549 cells with digitonin for 4 min resulted in the release of over 90% of the lactate dehydrogenase activity into the medium. Approximately 80% of the Mg2+-dependent but only 7% of the Mg2+-independent phosphatidate phosphohydrolase activity was released in the presence of digitonin. Pretreatment of the cells with oleate reduced the efflux of the Mg2+-dependent phosphatidate phosphohydrolase activity to approximately 5% of total. Oleate did not affect the release of lactate dehydrogenase or the release of the Mg2+-independent phosphohydrolase activity. Incubation of A549 cells with [3H]oleate for 60 min led to incorporation of the label into phosphatidic acid, phosphatidylethanolamine, phosphatidylcholine, diacylglycerol, monoacylglycerol, and triacylglycerol, in ascending order. When the level of exogenous oleate was increased to over 2.0 mM, there was a marked increase in the incorporation into monoacylglycerol and diacylglycerol. Only small amounts of radioactivity were associated with phosphatidic acid. Time course studies revealed that the amount of radioactive phosphatidate remained low throughout the incubation period. These investigations were interpreted to indicate that free fatty acids can promote the translocation of the Mg2+-dependent phosphatidate phosphohydrolase activity from cytosol to membrane fractions. This translocation could, at least theoretically, function to facilitate the metabolism of increased amounts of phosphatidate.

Cell Line↗
PubMed1991

Phosphatidate phosphatase from Saccharomyces cerevisiae. Isolation of 45- and 104-kDa forms of the enzyme that are differentially regulated by inositol.

Immunoblot analysis of cell extracts using antibodies specific for the 91-kDa form of membrane-associated phosphatidate phosphatase from Saccharomyces cerevisiae (Lin, Y.-P., and Carman, G.M. (1989) J. Biol. Chem. 264, 8641-8645) revealed the existence of a 45-kDa form of the enzyme. Immunoblot analysis also showed that the 91-kDa form of the enzyme was a proteolytic product of a 104-kDa enzyme. The mitochondrial fraction contained the 45-kDa enzyme, whereas the microsomal fraction contained the 45- and 104-kDa enzymes. In vivo labeling experiments showed that the 104-kDa form of phosphatidate phosphatase was not a precursor of the 45-kDa form of the enzyme. The 45- and 104-kDa forms of phosphatidate phosphatase were purified and characterized. The enzymological properties of both enzymes were similar. However, the phosphatidate phosphatase 45- and 104-kDa proteins differed with respect to their isoelectric points and peptide fragments resulting from V8 proteolysis and cyanogen bromide cleavage. The expression of the phosphatidate phosphatase 45- and 104-kDa enzymes were regulated differentially in cells supplemented with inositol. The addition of inositol to the growth medium resulted in the induction of the phosphatidate phosphatase 45-kDa enzyme. The expression of the 104-kDa enzyme was not affected by inositol. Both forms of phosphatidate phosphatase were induced when cells entered the stationary phase of growth.

Blotting, Western↗
PubMed1985

Molecular species of phosphatidylinositol, phosphatidic acid and diacylglycerol in a phytohemagglutinin-stimulated T-cell leukemia line.

Addition of phytohemagglutinin to JURKAT cells, a human T-cell leukemia line, induced a rapid breakdown of phosphatidylinositol 4,5-bisphosphate (and may also be phosphatidylinositol 4-phosphate) and an accumulation of phosphatidic acid. The accumulation and disappearance of the various molecular species of phosphatidic acid, diacylglycerol and phosphatidylinositol (PtdIns) in response to phytohemagglutinin was studied in JURKAT cells. The cells were prelabeled with [2-3H]glycerol for 2 days and 3H-labeled lipids were isolated from the cells after incubation for 2 min at 37 degrees C in the absence or in the presence of phytohemagglutinin. The isolated 3H-labeled lipids were separated into individual molecular species by reverse-phase HPLC after conversion to their 1,2-[3H]diacylglycerol acetate derivatives either by acetolysis or by acetylation. Stimulation with phytohemagglutinin induced a 2-fold increase in [3H]phosphatidic acid. The molecular species of the accumulated [3H]phosphatidic acid consisted of polyenoic species, which were almost absent in the [3H]phosphatidic acid of the unstimulated cells. Stearoylarachidonoyl species of [3H]phosphatidic acid accumulated most prominently. Although an accumulation of [3H]diacylglycerol was hardly measurable in the phytohemagglutinin-stimulated cells, the HPLC analysis of the molecular species of [3H]diacylglycerol showed a 2-fold increase in the stearoylarachidonoyl species in the stimulated cells. Stimulation with phytohemagglutinin had almost no effect on the composition of molecular species of [3H]PtdIns. The stearoylarachidonyl species is the most abundant molecular species of PtdIns in JURKAT cells. These results suggest that the [3H]diacylglycerol moiety of [3']phosphatidic acid originates from inositol lipid(s). The results also suggest a rapid and preferential phosphorylation of the diacylglycerol formed by receptor-stimulated hydrolysis of inositol lipid(s).

Cell Line↗
PubMed1982

Alpha1-adrenergic stimulation of phosphatidylinositol-phosphatidic acid turnover in rat parotid cells.

The regulation of phosphatidylinositol turnover by alpha-adrenergic agonists in rat parotid acinar cell aggregates was examined with respect to kinetics and agonist-antagonist interactions. Phosphatidylinositol turnover was followed by the changes in the specific activities of [32P]phosphatidic acid and [32P]phosphatidylinositol. The specific activity of phosphatidic acid increased rapidly (within 1 min) after addition of epinephrine (10(-5) M), reached a maximal level within 12-16 min, and then decreased. Incorporation of 32P into phosphatidylinositol exhibited a lag phase of about 5 min and then increased continuously for an additional 40 min. The absolute amounts of phosphatidic acid and phosphatidylinositol did not change. The concentrations of epinephrine needed to stimulate 32P incorporation into phosphatidic acid and phosphatidylinositol, measured at 15 and 30 min, respectively, were similar; Ka values of 2.05 +/- 0.46 X 10(-6) M for phosphatidic acid and 2.98 +/- 0.30 X 10(-6) M for phosphatidylinositol were found. The effects of agonists on 32P labeling of phosphatidylinositol, in order of potency, were epinephrine greater than or equal to norepinephrine greater than phenylephrine much greater than normetanephrine. When various adrenergic antagonists were evaluated for their ability to inhibit 10(-5) M epinephrine-stimulated 32P incorporation into both phosphatidic acid and phosphatidylinositol, the order of antagonist potency was prazosin greater than or equal to phenoxybenzamine greater than phentolamine greater than or equal to yohimbine greater than much greater than propranolol. These findings indicate that phosphatidylinositol-phosphatidic acid turnover in the rat parotid gland is mediated by the alpha 1-adrenergic receptor system.

Adrenergic alpha-Agonists↗
PubMed1997

Dependence on intracellular Ca2+ on mass and turnover of phosphoinositides and phosphatidate in human erythrocytes.

Effects of a calcium-load on mass and turnover of phosphoinositides and phosphatidate were investigated in human erythrocytes by short-term labeling with [32P]Pi. The labeling of phosphatidate was accelerated at normal mass by short-term elevation of free intracellular [Ca2+] up to 1 microM and inhibited by the reduction of normal free [Ca2+]. Thus, the labeling of phosphatidate is a Ca2+-regulated process and not only the consequence of a net synthesis of diacylglycerol by other Ca2+-dependent reactions. Persisting elevation of free intracellular [Ca2+] between 1-40 microM induced an increase of the mass of phosphatidylinositol 4-phosphate with a concomitant decrease of the mass of phosphatidylinositol 4,5-bisphosphate. Under these conditions, the normal steady-state turnover of phosphoinositides was not altered by Ca2+, but mass and turnover of phosphatidate continuously rose. The increase in phosphatidate mass by far exceeded the decrease of the mass of phosphoinositides, indicating that phosphatidate was generated to a great extent by hydrolysis of other phospholipids in addition to the action of phosphoinositidase C with subsequent phosphorylation of diacylglycerol to phosphatidate. The results demonstrate that different phospholipid phosphodiesterases of human erythrocytes are activated by Ca2+-concentrations in the microM range as is known from various other cell types. In contrast to current explanations, Ca2+-dependent phospholipid phosphodiesterases of human erythrocytes did not exhibit an unusually low affinity against rising cytosolic Ca2+-concentrations.

Binding Sites↗