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PubMed1990

Effects of phosphatidic acid on parathyroid hormone release, intracellular free Ca2+, and inositol phosphates in dispersed bovine parathyroid cells.

The observation that increases in extracellular Ca2+ or the addition of divalent cations, such as Ba2+, Mg2+, Mn2+, or Sr2+, stimulate the accumulation of inositol trisphosphate (InsP3) and its breakdown products in parathyroid cells strongly supports the idea that polyphosphoinositides are hydrolyzed under these conditions. Since phosphatidic acid is produced as a result of polyphosphoinositide hydrolysis, and it has been proposed that phosphatidic acid may be a second messenger for Ca2+ mobilization, we examined the effects of this compound on parathyroid cells. We assessed PTH release, intracellular free Ca2+ ([Ca2+]i), and inositol polyphosphate accumulation in response to phosphatidic acid. Natural phosphatidic acid reduced PTH release at 1.0 mM extracellular Ca2+ by 18 +/- 6%, 48 +/- 5%, 59 +/- 10%, and 79 +/- 6% at concentrations of 1, 10, 50, and 100 micrograms/ml, respectively (n = 5-11). The effect was not dependent on the presence of extracellular Ca2+, since phosphatidic acid (100 micrograms/ml) inhibited PTH secretion by 39 +/- 3% in medium with no added Ca2+ and 1.0 mM EGTA (n = 3). This agent rapidly and transiently increased [Ca2+]i in a dose-dependent manner, as determined by fura-2 fluorescence. At 1.0 mM extracellular Ca2+, [Ca2+]i rose from 309 +/- 8 to a peak of 356 +/- 26, 454 +/- 22, and 587 +/- 57 nM with the addition of 1, 10, and 100 micrograms/ml phosphatidic acid, respectively (n = 2-14). In the absence of extracellular Ca2+ (i.e. medium with 1 or 2 mM EGTA and no added Ca2+), phosphatidic acid produced a quantitatively smaller peak increment of 38 +/- 4% in [Ca2+]i, indicating that this compound could mobilize Ca2+ from intracellular stores (n = 3). At 1.0 mM extracellular Ca2+, phosphatidic acid (200 micrograms/ml) stimulated the accumulation of Inositol trisphosphate (InsP3), Inositol bisphosphate (InsP2), and Inositol monophosphate (InsP1) by 46 +/- 9%, 37 +/- 9%, and 59 +/- 11% after 60 sec, respectively (n = 5-7). Phosphatidic acid had no significant effect on forskolin-stimulated cAMP accumulation. We further determined whether the specific fatty acid composition of phosphatidic acid might influence its effects in parathyroid cells by testing several synthetic compounds. Dipalmitoyl phosphatidic acid (greater than or equal to 50 micrograms/ml) inhibited PTH release in a dose-dependent manner without significantly changing [Ca2+]i. Dioleoyl phosphatidic acid had modest biphasic effects on secretion, with 20 +/- 5% inhibition observed at lower doses (10 micrograms/ml) and a 27 +/- 8% stimulation of secretion at 100 micrograms/ml (n = 6).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1986

Relationship between the displacement of phosphatidate phosphohydrolase from the membrane-associated compartment by chlorpromazine and the inhibition of the synthesis of triacylglycerol and phosphatidylcholine in rat hepatocytes.

Glycerolipid synthesis was studied in isolated hepatocytes by using 177 microM [14C]oleate and 1 mM [3H]glycerol. Chlorpromazine (25-400 microM) inhibited the synthesis of phosphatidylcholine and triacylglycerol. This was accompanied by an average increase of 12-fold in the accumulation of the labelled precursors in phosphatidate at 200 microM chlorpromazine and a decrease in the conversion of phosphatidate to diacylglycerol of 76%. These results indicate that part of the inhibition of the synthesis of phosphatidylcholine and triacylglycerol occurs at the level of phosphatidate phosphohydrolase. The relative rate of triacylglycerol synthesis at different concentrations of chlorpromazine was approximately proportional to the rate of conversion of phosphatidate to diacylglycerol. Phosphatidylcholine synthesis increased at higher rates of conversion of phosphatidate to diacylglycerol, but it was relatively independent of the latter rate when this was inhibited by more than about 30% with chlorpromazine. The addition of oleate to the hepatocytes caused a translocation of phosphatidate phosphohydrolase from the cytosol to the membrane-associated compartment. Chlorpromazine had the opposite effect and displaced the phosphohydrolase from the membranes in the presence or absence of oleate. There was a highly significant correlation between the activity of phosphatidate phosphohydrolase that was associated with the membranes of the hepatocytes and the calculated conversion of [3H]phosphatidate to diacylglycerol. Chlorpromazine also antagonized the association of the phosphohydrolase with microsomal membranes when cell-free preparations were incubated with combinations of oleate and spermine. Furthermore, it inhibited the transfer of the soluble phosphohydrolase to microsomal membranes that were labelled with [14C]phosphatidate and thereby decreased diacylglycerol production. It is concluded that part of the action of chlorpromazine in inhibiting the synthesis of triacylglycerol and phosphatidylcholine occurs because it prevents the interaction of the soluble phosphatidate phosphohydrolase with the membranes on which glycerolipid synthesis occurs. This in turn prevents the conversion of phosphatidate to diacylglycerol.

Animals↗
PubMed1997

Phosphatidic acid elicits calcium mobilization and actin polymerization through a tyrosine kinase-dependent process in human neutrophils: a mechanism for induction of chemotaxis.

Phospholipids mediate important effects as extracellular messengers in diverse biological systems. We investigated the effects of phosphatidic acid, a biologically active phospholipid potentially involved in the inflammatory process, on calcium mobilization and actin polymerization in human neutrophils and correlated these effects with induction of chemotactic migration. Intermediate-chain length phosphatidic acid (DiC10-PA) induced a biphasic increase in intracellular Ca2+ characterized by a rapid rise commencing immediately upon addition of stimulus followed by a secondary increase which, unlike the initial response, was eliminated by chelation of extracellular Ca2+. Neither of these responses were induced by C10-lysophosphatidic acid or diacylglycerol. The tyrosine kinase inhibitor herbimycin-A (5-10 microg/ml) completely blunted the initial but not the delayed response effected by DiC10-PA. Long-chain phosphatidic acid (DiC18:1) induced only an initial rapid increase in intracellular Ca2+ and this response was similarly markedly attenuated by herbimycin-A. Among several physiologically relevant phospholipids, only phosphatidic acid was able to induce Ca2+ mobilization; phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol -- used individually or in mixed liposomes -- were without effect. Phosphatidic acid conferred calcium-mobilizing activity upon inactive liposome preparations and phosphatidic acid-enriched cellular plasma membranes possessed similar calcium-mobilizing activity. Both DiC10-PA and DiC18:1-PA induced actin polymerization in neutrophils at rates which mirrored the influence of each agent on Ca2+ mobilization. Herbimycin-A blunted the initial increase in actin polymerization effected by phosphatidic acid but had no effect on the delayed, EGTA-sensitive phase. DiC10-PA and DiC18:1-PA also induced neutrophil migration along a concentration gradient. Phospholipids that failed to induce a calcium transient, including phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, and phosphatidylinositol, likewise failed to induce either actin polymerization or chemotactic migration. Unlike chemotaxis induced by zymosan-activated human serum, phosphatidate-induced chemotaxis was strongly inhibited by pretreatment of cells with herbimycin-A. Consistent with these observations, phosphatidic acid induced the tyrosine phosphorylation of several proteins as early as 10 s after stimulation. Phosphorylation of two distinct proteins with approximate molecular sizes of 72 and 82 kDa was inhibited by levels of herbimycin A used to effectively inhibit calcium mobilization, actin polymerization and chemotaxis. Thus, in neutrophilic leukocytes, extracellular phosphatidic acid induces a unique tyrosine kinase-based signalling pathway that results in calcium mobilization and actin polymerization. These processes may promote directed cellular migration as a consequence of the interaction of phosphatidic acid with neutrophil plasma membranes.

Actins↗
PubMed2000

A novel pathway for transport and metabolism of a fluorescent phosphatidic acid analog in yeast.

Phosphatidic acid is a central intermediate of biosynthetic lipid metabolism as well as an important signaling molecule in the cell. These studies assess the internalization, or retrograde transport, and metabolism of phosphatidic acid in yeast using a fluorescent analog. An analog of phosphatidic acid fluorescently labeled at the sn-2 position with N-4-nitrobenz-2-oxa-1, 3-diazole-aminocaproic acid (NBD-phosphatidic acid) was introduced to yeast cells by spontaneous transfer from phospholipid vesicles. Transport and metabolism of the NBD-phosphatidic acid were then monitored by fluorescence spectrophotometry, fluorescence microscopy and routine biochemical methods. Primary metabolites of the NBD-phosphatidic acid in yeast were found to be NBD-diacylgycerol and NBD-phosphatidylinositol. Experiments in cells possessing different levels of phosphatidate phosphatase activity suggest that conversion of the NBD-phosphatidic acid to NBD-diacylglycerol is not a pre-requisite for internalization in yeast. Internalization is sensitive to decreased temperature, but neither ATP depletion nor a sec6-4 mutation, which interrupts endocytosis, has an affect. Thus, internalization of NBD-phosphatidic acid apparently occurs via a non-endocytic route. These characteristics of retrograde transport of NBD-phosphatidic acid in yeast differ significantly from transport of other NBD-phospholipids in yeast as well as NBD-phosphatidic acid transport in mammalian fibroblasts.

4-Chloro-7-nitrobenzofurazan↗
PubMed1990

Increases in phosphatidic acid levels accompany sphingosine-stimulated proliferation of quiescent Swiss 3T3 cells.

Sphingosine, a breakdown product of cellular sphingolipids, has recently been shown to stimulate DNA synthesis and act synergistically with known growth factors to induce proliferation of quiescent Swiss 3T3 fibroblasts (Hong, Z., Buckley, N. E., Gibson, K., and Spiegel, S. (1990) J. Biol. Chem. 265, 76-81). The present study demonstrates that mitogenic concentrations of sphingosine induce early increases in cytosolic phosphatidic acid, which is a potent mitogen for Swiss 3T3 cells. Structurally related analogs of sphingosine, such as N-stearoylsphingosine and other long chain aliphatic amines, did not mimic the mitogenic effect of sphingosine and did not elevate phosphatidic acid levels. Sphingosine not only stimulated [3H]thymidine incorporation with similar efficiency and kinetics as phosphatidic acid, it also induced similar morphological alterations. Both sphingosine and phosphatidic acid acted synergistically with a variety of growth factors, such as, insulin, epidermal growth factor, fibroblast growth factor, and 12-O-tetradecanoylphorbol 13-acetate. In sharp contrast, sphingosine and phosphatidic acid did not have additive or synergistic effects in either the presence or absence of other growth factors. Both sphingosine and phosphatidic acid stimulated DNA synthesis in cells made protein kinase C-deficient by prolonged treatment with phorbol ester and sphingosine still stimulated similar increases in phosphtidic acid in these cells. Furthermore, similar to the actions of phosphatidic acid on signal transduction in Swiss 3T3 cells, mitogenic concentrations of sphingosine also inhibit cAMP accumulation and trigger the hydrolysis of polyphosphoinositides. Our findings indicate that sphingosine and phosphatidic acid control cellular responses in Swiss 3T3 cells through a common pathway. In view of the prominent role of phosphatidic acid in signal transduction and cellular proliferation, our observations that sphingosine, at mitogenic concentrations, increases the level of phosphatidic acid and also mimics the effects of phosphatidic acid on signal transduction, have important implications for the mechanism of action of sphingosine.

Animals↗
PubMed1987

Phosphatidate accumulation in hormone-treated hepatocytes via a phospholipase D mechanism.

Isolated rat hepatocytes responded to a variety of Ca2+-mobilizing agents (vasopressin, angiotensin II, epinephrine, epidermal growth factor, ATP, and ADP) with a rapid increase in phosphatidate mass, as measured by a sensitive new method. When hepatocytes were incubated with vasopressin (10(-8) M), phosphatidate levels increased 2-3-fold in 2 min, but there was no significant increase in diacylglycerol at this time. Changes in the fatty acid composition of phosphatidate also preceded those in diacylglycerol. De novo synthesis of phosphatidate from [3H]glycerol was unaffected by vasopressin in short-term incubation. Incubation of washed rat liver plasma membranes with GTP gamma S caused a time-dependent increase in phosphatidate. When membranes were incubated with GTP gamma S and [gamma-32P]ATP, no incorporation of 32P into phosphatidate was observed. This excludes the phospholipase C-diacylglycerol kinase pathway and suggests that a phospholipase D activity produced the phosphatidate. At submaximal concentrations of GTP gamma S, ATP and ADP stimulated membrane phosphatidate formation, presumably by acting through P2-purinergic receptors. Only phosphatidylcholine, among the major phospholipids, decreased in the membranes in response to GTP gamma S. The fatty acid composition of the phosphatidate produced in response to vasopressin in hepatocytes also suggests that phosphatidylcholine may be the source of hormonally elicited phosphatidate. We conclude that Ca2+-mobilizing hormones mainly increase phosphatidate levels in hepatocytes by a mechanism that does not involve phosphorylation of diacylglycerol or de novo synthesis but involves a guanine nucleotide-binding protein coupled to phospholipase D.

Adenine Nucleotides↗
PubMed1993

Regulation of phosphatidate phosphatase activity from the yeast Saccharomyces cerevisiae by sphingoid bases.

The regulation of Saccharomyces cerevisiae membrane-associated phosphatidate phosphatase (3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4) activity by sphingoid bases was examined using Triton X-100/lipid-mixed micelles. Sphingosine, phytosphingosine, and sphinganine inhibited purified preparations of the 104- and 45-kDa forms of phosphatidate phosphatase in a dose-dependent manner. The structural requirements for the sphingoid base inhibition of phosphatidate phosphatase activity were a free amino group and a long chain hydrocarbon. A detailed kinetic analysis was performed to determine the mechanism of phosphatidate phosphatase inhibition by sphingoid bases. The phosphatidate phosphatase dependence on phosphatidate was cooperative (Hill numbers of approximately 2) in the absence and presence of sphingoid bases. Sphingosine, phytosphingosine, and sphinganine were parabolic competitive inhibitors of phosphatidate phosphatase activity. This indicated that more than one inhibitor molecule contributed to the exclusion of phosphatidate from the enzyme. The aKi values (inhibitor constants) for sphingosine, phytosphingosine, and sphinganine were 1.5, 0.4, and 0.2 mol %, respectively, and the Km value for phosphatidate was 2.2 mol %. The cellular concentrations of free phytosphingosine and sphinganine were 0.16 and 0.53 mol %, respectively, relative to the total phospholipids in S. cerevisiae. The cellular concentrations of phytosphingosine and sphinganine were in the range of the aKi values for these sphingoid bases. These results raised the suggestion that phosphatidate phosphatase activity may be regulated in vivo by sphingoid bases.

Amines↗
PubMed1985

The molecular species of phosphatidic acid, diacylglycerol and phosphatidylcholine synthesized from sn-glycerol 3-phosphate in rat lung microsomes.

The species pattern of phosphatidic acid, diacylglycerol and phosphatidylcholine synthesized from [14C]glycerol 3-phosphate was measured using a newly developed HPLC technique yielding 13 molecular species. A direct comparison of these species patterns presupposes determination of the lipolytic activity of lung microsomes. The lipolytic activity was quantitatively determined by measuring the changes of the endogenous concentration of diacylglycerol, triacylglycerol and free fatty acids. The species pattern of endogenous diacylglycerol measured in the time-course of lipolysis did not show any changes up to an incubation period of 20 min, suggesting that the lipolytic activity showed only a very low selectivity for individual substrate species. Diisopropylfluorophosphate (5 mumol/mg microsomal protein) strongly decreased the lipolytic activities as well as the microsomal phosphatidate phosphohydrolase activity, as measured by means of exogenous phosphatidic acid, and also the generation of phosphatidic acid from [14C]glycerol 3-phosphate. In lung microsomes, labeled phosphatidic acid and diacylglycerols were synthesized from the endogenous free fatty acids and sn-[14C]glycerol 3-phosphate, which had previously been added. By addition of CDPcholine to the prelabeled microsomes the synthesis of phosphatidylcholine was measured. After hydrolysis of phosphatidic acid and phosphatidylcholine with cytoplasmatic phosphatidate phosphohydrolase or phospholipase C, respectively, the de novo synthesized species patterns of these two lipids and of the diacylglycerol were determined. Comparison of the species pattern of de novo synthesized phosphatidic acid with that of diacylglycerol largely showed the same distribution of radioactivity among the individual species, except that the relative proportion of label was higher in the 16:0/16:0 and 16:0/18:0 species of phosphatidic acid and lower in the 16:0/20:4 and 18:0/20:4 species than in the corresponding species of diacylglycerol. The species pattern of de novo-synthesized diacylglycerol showed no differences from that of the phosphatidylcholine synthesized from it. From this result we concluded that the cholinephosphotransferase of lung microsomes is nonselective for individual species of the diacylglycerol substrate. The 16:0/18:1 and 16:0/18:2 species of phosphatidic acid, diacylglycerol and phosphatidylcholine showed a higher synthesis rate than their 18:0 counterparts, whereas the 16:0 or 18:0 analogues of species containing 20:4 and 22:6 fatty acids showed nearly the same synthesis rates.(ABSTRACT TRUNCATED AT 400 WORDS)

1-Acylglycerol-3-Phosphate O-Acyltransferase↗
PubMed2001

Depressed responsiveness of phospholipase C isoenzymes to phosphatidic acid in congestive heart failure.

The cardiac sarcolemmal membrane cis -unsaturated fatty acid-sensitive phospholipase D hydrolyzes phosphatidylcholine to form phosphatidic acid. The functional significance of phosphatidic acid is indicated by its ability to increase [Ca(2+)](i)and augment cardiac contractile performance via the activation of phospholipase C. Accordingly, we tested the hypothesis that a defect occurs in the membrane level of phosphatidic acid and/or the responsiveness of cardiomyocytes to phosphatidic acid in congestive heart failure due to myocardial infarction. Myocardial infarction was produced in rats by ligation of the left coronary artery while sham-operated animals served as control. At 8 weeks after surgery, the experimental animals were at a stage of moderate congestive heart failure. Compared to sham controls, phosphatidic acid-mediated increase in [Ca(2+)](i), as determined by the fura 2-AM technique, was significantly reduced in failing cardiomyocytes. Immunoprecipitation of sarcolemmal phospholipase C isoenzymes using specific monoclonal antibodies revealed that the stimulation of phospholipase C gamma(1)and delta(1)phosphatidylinositol 4,5-bisphosphate hydrolyzing activities by phosphatidic acid was decreased in the failing heart. Although the activity of phospholipase C beta(1)in the failing heart was higher than the control, phosphatidic acid did not stimulate this isoform in control sarcolemma, and produced an inhibitory action in the failing heart preparation. Furthermore, the specific binding of phosphatidic acid to phospholipase C gamma(1)and delta(1)isoenzymes was decreased, whereas binding to phospholipase beta(1)was absent in the failing heart. A reduction in the intramembranal level of phosphatidic acid derived via cis -unsaturated fatty acid-sensitive phospholipase D was also seen in the failing heart. These findings suggest that a defect in phosphatidic acid-mediated signal pathway in sarcolemma may represent a novel mechanism of heart dysfunction in congestive heart failure.

Animals↗
PubMed1994

Angiotensin II induces phosphatidic acid formation in neonatal rat cardiac fibroblasts: evaluation of the roles of phospholipases C and D.

Phosphatidic acid has been proposed to contribute to the mitogenic actions of various growth factors. In 32P-labeled neonatal rat cardiac fibroblasts, 100 nM [Sar1]angiotensin II was shown to rapidly induce formation of 32P-phosphatidic acid. Levels peaked at 5 min (1.5-fold above control), but were partially sustained over 2 h. Phospholipase D contributed in part to phosphatidic acid formation, as 32P- or 3H-phosphatidylethanol was produced when cells labeled with [32P]H3PO4 or 1-O-[1,2- 3H]hexadecyl-2-lyso-sn-glycero-3-phosphocholine were stimulated in the presence of 1% ethanol. [Sar1]angiotensin II-induced phospholipase D activity was transient and mainly mediated through protein kinase C (PKC), since PKC downregulation reduced phosphatidylethanol formation by 68%. Residual activity may have been due to increased intracellular Ca2+, as ionomycin also activated phospholipase D in PKC-depleted cells. Phospholipase D did not fully account for [Sar1]angiotensin II-induced phosphatidic acid: 1) compared to PMA, a potent activator of phospholipase D, [Sar1]angiotensin II produced more phosphatidic acid relative to phosphatidylethanol, and 2) PKC downregulation did not affect [Sar1]angiotensin II-induced phosphatidic acid formation. The diacylglycerol kinase inhibitor R59949 depressed [Sar1]angiotensin II-induced phosphatidic acid formation by only 21%, indicating that activation of a phospholipase C and diacylglycerol kinase also can not account for the bulk of phosphatidic acid. Thus, additional pathways not involving phospholipases C and D, such as de novo synthesis, may contribute to [Sar1]angiotensin II-induced phosphatidic acid in these cells. Finally, as previously shown for [Sar1]angiotensin II, phosphatidic acid stimulated mitogen activated protein (MAP) kinase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin II↗
PubMed1995

Leukotriene B4 stimulation of an early elevation of phosphatidic acid mass in human neutrophils.

The signal transduction pathway of leukotriene B4 involves phospholipase D activation in cytochalasin B-primed neutrophils, but leukotriene B4 stimulation of increased phosphatidic acid mass in neutrophils has not been demonstrated. Employing the NIH Image program, we have examined the effect of leukotriene B4 on phosphatidic acid mass in human neutrophils incubated with or without cytochalasin B. Our results show that 0.15 microM leukotriene B4 without cytochalasin B was capable of increasing phosphatidic acid mass in neutrophils by 2-fold after 5 s, 2.5-fold after 1 min, and 2-fold after 5 min incubation. Leukotriene B3, leukotriene B4, and leukotriene B5 were equipotent stimuli for phosphatidic acid mass elevation. Leukotriene B4 induced phosphatidylethanol formation at the expense of phosphatidic acid in cells preincubated with 0.25-1% ethanol, indicating phospholipase D activation. Cytochalasin B enhanced leukotriene B4 stimulation of phosphatidic acid mass elevation and phosphatidylethanol formation. There were no measurable changes in 1,2-diglyceride mass after 5 s, but a 1.7-fold increase occurred after 1 min and declined thereafter. Leukotriene B4 stimulation of [3H]glycerol incorporation into phosphatidic acid, diglyceride and phosphatidylinositol was detectable after a 1-min incubation, suggesting increased de novo synthesis of these lipids. These results suggest that leukotriene B4 stimulation of phospholipase D activity contributes to part of the early increased phosphatidic acid mass and that combined actions of stimulated phospholipases C and D, and de novo phosphatidic acid synthesis contribute to the total increased phosphatidic acid mass.

Cytochalasin B↗
PubMed1996

Regulation of phosphatidate phosphatase activity from the yeast Saccharomyces cerevisiae by phospholipids.

Regulation of Saccharomyces cerevisiae membrane-associated phosphatidate phosphatase (3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4) activity by phospholipids was examined using purified enzyme and Triton X-100/phospholipid-mixed micelles. Anionic phospholipids activated phosphatidate phosphatase activity whereas zwitterionic phospholipids had a slight inhibitory effect on activity. Cardiolipin (A0.5 = 1.9 mol %), CDP-diacylglycerol (A0.5 = 2.6 mol %), and phosphatidylinositol (A0.5 = 5.5 mol %) were the most potent anionic phospholipid activators. Enzyme activation by cardiolipin (n=2.8), CDP-diacylglycerol (n=2.1), and phosphatidylinositol (n=3.3) followed positive cooperative kinetics. A kinetic analysis was performed to determine the mechanism of phosphatidate phosphatase activation by anionic phospholipids. The dependence of phosphatidate phosphatase on phosphatidate was cooperative (n approximately 2.2) in the absence and presence of phospholipid activators. Cardiolipin, CDP-diacylglycerol, and phosphatidylinositol were mixed competitive activators of phosphatidate phosphatase activity. The major effect of the activators was to cause a decrease in the Km for phosphatidate. Sphinganine, a positively charged sphingoid base, inhibited phosphatidate phosphatase activity and antagonized the activation of the enzyme by cardiolipin and phosphatidylinositol. Sphinganine caused an increase in the cooperativity of cardiolipin activation, but had little effect on the A0.5 value for cardiolipin. On the other hand, sphinganine had little effect on the cooperativity of phosphatidylinositol activation, but caused an increase in the A0.5 value for phosphatidylinositol. The activation constants for cardiolipin, CDP-diacylglycerol, and phosphatidylinositol were within the range of their cellular concentrations. These results suggested that the activation of phosphatidate phosphatase activity by anionic phospholipids may be physiologically relevant.

Cardiolipins↗
PubMed1994

Phosphatidic acid increases in response to noradrenaline and endothelin-1 in adult rabbit ventricular myocytes.

OBJECTIVE: The aim was to assess whether noradrenaline and endothelin-1 can stimulate endogenous production of phosphatidic acid in adult ventricular myocytes. METHODS: After stimulation of rabbit ventricular myocytes with noradrenaline and endothelin-1, total lipids were extracted using the Bligh and Dyer procedure and separated by thin layer chromatography, and phosphatidic acid was quantified using photodensitometric analysis of visualised lipids with CuSO4/H3PO4. RESULTS: Noradrenaline (10(-5) M) elicited a rapid increase in phosphatidic acid at 2 min, followed by a decrease at 5 min. A second delayed and sustained increase in phosphatidic acid occurred at 10 min. The response to noradrenaline (10(-9) to 10(-5) M) was concentration dependent with a half maximum response (EC50) of 3.1 x 10(-8) M and the maximum effect at 10(-6) M. The increase in phosphatidic acid production in response to noradrenaline was abolished by an alpha 1 adrenergic receptor blocking agent (2-[beta-(4-hydroxyphenyl)-ethylaminomethyl]tetralone) but unaffected by the beta adrenergic blocking agent L-propranolol. An increase in phosphatidic acid was also elicited in rabbit ventricular myocytes in response to endothelin-1. The response was time and concentration dependent with the maximal increase at 12 min, EC50 5.3 x 10(-9) M, and maximum effect at 10(-6) M. Both noradrenalin and endothelin-1 stimulated phosphatidylbutanol production in the presence of butanol (100 mM), indicating that both agonists activate phospholipase D. CONCLUSIONS: Noradrenaline at physiological concentrations elicits both a rapid and a delayed increase in phosphatidic acid in adult rabbit ventricular myocytes. Endothelial-1, at physiological concentrations, also stimulates an increase in the mass of phosphatidic acid in myocytes, but the increase induced by endothelin-1 is monophasic, in contrast to the biphasic response seen during stimulation with noradrenaline. Activation of phospholipase D contributes to the increase in phosphatidic acid seen during stimulation of myocytes with either noradrenaline or endothelin-1. These are the first data to characterise endogenous production of phosphatidic acid in isolated adult ventricular myocytes.

Adrenergic alpha-Antagonists↗
PubMed1992

The 45- and 104-kDa forms of phosphatidate phosphatase from Saccharomyces cerevisiae are regulated differentially by phosphorylation via cAMP-dependent protein kinase.

Evidence is presented that demonstrated that the 45- and 104-kDa forms of phosphatidate phosphatase from Saccharomyces cerevisiae (Morlock, K. R., McLaughlin, J. J., Lin, Y.-P., and Carman, G. M. (1991) J. Biol. Chem. 266, 3586-3593) were regulated differentially by phosphorylation. Purified 45-kDa phosphatidate phosphatase was phosphorylated by cAMP-dependent protein kinase whereas purified 104-kDa phosphatidate phosphatase was not phosphorylated. cAMP-dependent protein kinase catalyzed the phosphorylation of pure 45-kDa phosphatidate phosphatase at a serine residue which resulted in a stimulation (2.4-fold) of phosphatidate phosphatase activity. Alkaline phosphatase catalyzed the dephosphorylation of pure 45-kDa phosphatidate phosphatase which resulted in an inhibition (1.3-fold) of phosphatidate phosphatase activity. Results of studies using mutants (bcy1 and cyr1) defective in cAMP-dependent protein kinase activity corroborated the results of the phosphorylation studies using pure preparations of phosphatidate phosphatase. The 45-kDa phosphatidate phosphatase phosphorylated in vitro and in vivo had phosphopeptides in common. The activation of the GAL10-RAS2val19 allele in mutant cells resulted in an increase in the synthesis of diacylglycerols and triacylglycerols. These results were consistent with the phosphorylation and activation of 45-kDa phosphatidate phosphatase by cAMP-dependent protein kinase in vivo.

Alleles↗
PubMed1994

Regulation of phosphatidate phosphatase activity from the yeast Saccharomyces cerevisiae by nucleotides.

Regulation of Saccharomyces cerevisiae membrane-associated phosphatidate phosphatase (3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4) activity by nucleotides was examined using pure enzyme and Triton X-100/phosphatidate-mixed micelles. Adenosine, guanosine, cytidine, and uridine nucleotides inhibited phosphatidate phosphatase activity in a dose-dependent manner. ATP and CTP were the most potent inhibitors of the enzyme. A kinetic analysis was performed to determine the mechanism of enzyme inhibition by nucleotides. The mechanism of inhibition by ATP and CTP with respect to phosphatidate (the substrate) was complex. The dependence of phosphatidate phosphatase activity on phosphatidate was cooperative, and nucleotides affected both Vmax and Km. ATP did not inhibit phosphatidate phosphatase activity by binding to the enzyme or to phosphatidate. Phosphatidate phosphatase dependence on Mg2+ ions (the cofactor) followed saturation kinetics, and the mechanism of nucleotide inhibition with respect to Mg2+ ions was competitive. Thus, the mechanism of enzyme inhibition by nucleotides was the chelation of Mg2+ ions. The inhibitor constant for ATP was lower than its cellular concentration in glucose-grown cells. However, the inhibitor constant for ATP was higher than its cellular concentration in glucose-starved cells. Changes in the cellular concentration of ATP affected the proportional synthesis of triacylglycerols and phospholipids. These results were consistent with the regulation of phosphatidate phosphatase activity by ATP through a Mg2+ ion chelation mechanism.

Binding Sites↗
PubMed1967

The formation and continuous turnover of a fraction of phosphatidic acid on stimulation of NaC1 secretion by acetylcholine in the salt gland.

Acetylcholine, which stimulates NaCl secretion in the avian salt gland, causes the rapid formation of a fraction of phosphatidic acid, as measured by (32)P incorporation, which amounts maximally to about 0.18 micromoles per g of fresh tissue. This does not appear to involve synthesis of the diglyceride moiety of phosphatidic acid, as measured by glycerol-1-(14)C incorporation. It presumably involves formation of phosphatidic acid by the diglyceride kinase pathway from preformed diglyceride and ATP. The specific activity of the AT(32)P of the tissue is not increased in the presence of acetylcholine. At time intervals after addition of acetylcholine during which a full response, measured as increased O(2) uptake, may be observed, phosphatidic acid appears to be the only phosphatide which shows any increase either in total (32)P radioactivity or in net specific acitvity. This responsive fraction of phosphatidic acid undergoes continuous turnover of its phosphate moiety. There is no evidence that this turnover is due to the phosphatidic acid acting as a pool of intermediate for the synthesis of other phospholipids or glycerides. The responsive fraction amounts to not more than 20% of the total phosphatidic acid of the tissue; it does not mix with the other (non-responsive) phosphatidic acid of the tissue. The observations suggest that this phosphatidic acid plays some role in the over-all secretory process.

Acetylcholine↗
PubMed1980

Factors controlling the metabolism of phosphatidate by phosphohydrolase and phospholipase A-type activities. Effects of magnesium, calcium and amphiphilic cationic drugs.

1. The simultaneous deacylation and dephosphorylation of 1,2-diacyl-sn-[3H]glycerol 3-phosphate by the microsomal and soluble fraction of rat liver was studied. The substrate was either in the form of an emulsion or bound to microsomal membranes. 2. Mg2+ stimulated the deacylation and dephosphorylation of phosphatidate emulsions by both fractions, although the stimulation of both microsomal activities was less than that in the soluble fraction. The preparations of membrane-bound phosphatidate contained Mg2+. Further addition of Mg2+ inhibited dephosphorylation, whereas low concentrations of EDTA stimulated. Additional Mg2+ had little effect on the deacylation of membrane-bound phosphatidate and EDTA inhibited it. 3. Ca2+ inhibited the phosphohydrolase reactions in both fractions, but had little effect on the deacylation of phosphatidate emulsions or membrane-bound phosphatidate. 4. In the absence of Mg2+, lower concentrations of amphiphilic cations (chlorpromazine and benfluorex) stimulated the deacylation and dephosphorylation of phosphatidate emulsions by the soluble fraction. They also stimulated deacylation by the microsomal fraction, but inhibited dephosphorylation. In the present of 5 mM MgCl2, these drugs inhibited the dephosphorylation and deacylation of phosphatidate emulsions, the deacylation reaction being slightly less sensitive. Chlorpromazine (0.4 and 0.8 mM) also inhibited the dephosphorylation of membrane-bound Mg2+-phosphatidate by microsomal and microsomal plus soluble fractions. The deacylation was stimulated by 0.4 mM chlorpromazine and by 1 and 2 mM norfenfluramine. Chlorpromazine (0.8 mM) inhibited the deacylation by microsomal plus soluble fractions, but not by microsomal fractions alone. 5. The possible importance of the deacylation of phosphatidate in the physiological and pharmacological control of glycerolipid synthesis is discussed.

Animals↗
PubMed1997

Increased concentrations of phosphatidate, diacylglycerol and ceramide in ras- and tyrosine kinase (fps)-transformed fibroblasts.

Concentrations of the bioactive lipids, phosphatidate and diacylglycerol, increased with time in culture in ras- and tyrosine kinase (fps)-transformed fibroblasts but not in control fibroblasts. On Day 3, diacylglycerol and phosphatidate concentrations were about 3.3- and 5.5-fold higher respectively in the ras-transformed compared to control fibroblasts. These concentrations in fps-transformed fibroblasts were increased about twofold. The changes in phosphatidate and diacylglycerol resulted from enhanced phospholipid turnover rather than from synthesis de novo. The increased ratio of phosphatidate to diacylglycerol is explained by decreased activities of two distinct phosphatidate phosphohydrolases and increased diacylglycerol kinase in ras-transformed fibroblasts. Ceramide concentrations were about 2.5- and threefold higher in the fps- and ras-transformed cells respectively on Day 3 compared to the controls. Incubating control fibroblasts from Days 1 to 3 with phosphatidylcholine-specific phospholipase C increased diacylglycerol, phosphatidate and ceramide concentrations, and decreased Mg2+-independent-phosphatidate phosphohydrolase activity. 8-(4-chlorophenylthio)-cAMP had a cytostatic effect in ras-transformed cells, it decreased the concentrations of phosphatidate and diacylglycerol, but increased that of ceramide. The consequences of increased ceramide and phosphatidate concentrations in ras-transformed cells are discussed in relation to signal transduction, cell division and the transformed phenotype.

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