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PubMed2006

Protease-activated receptors differentially regulate human platelet activation through a phosphatidic acid-dependent pathway.

Pathological conditions such as coronary artery disease are clinically controlled via therapeutic regulation of platelet activity. Thrombin, through protease-activated receptor (PAR) 1 and PAR4, plays a central role in regulation of human platelet function in that it is known to be the most potent activator of human platelets. Currently, direct thrombin inhibitors used to block platelet activation result in unwanted side effects of excessive bleeding. An alternative therapeutic strategy would be to inhibit PAR-mediated intracellular platelet signaling pathways. To elucidate the best target, we are studying differences between the two platelet thrombin receptors, PAR1 and PAR4, in mediating thrombin's action. In this study, we show that platelet activation by PAR1-activating peptide (PAR1-AP) requires a phospholipase D (PLD)-mediated phosphatidic acid (PA) signaling pathway. We show that this PAR1-specific PA-mediated effect is not regulated through differential granule secretion after PAR-induced platelet activation. Perturbation of this signaling pathway via inhibition of lipid phosphate phosphatase-1 (LPP-1) by propranolol or inhibition of the phosphatidylcholine-derived phosphatidic acid (PA) formation by PLD with a primary alcohol significantly attenuated platelet activation by PAR1-AP. Platelet activation by thrombin or PAR4-AP was insensitive to these inhibitors. Furthermore, these inhibitors significantly attenuated activation of Rap1 after stimulation by PAR1-AP but not thrombin or PAR4-AP. Because PA metabolites such as diacylglycerol play an important role in intracellular signaling, identifying crucial differences in PA regulation of PAR-induced platelet activation may lead to a greater understanding of the role of PAR1 versus PAR4 in progression of thrombosis.

Cytoplasmic Granules↗
PubMed1990

Complement C5a activation of phospholipase D in human neutrophils. A major route to the production of phosphatidates and diglycerides.

The contribution of phospholipase D (PLD) to the production of phosphatidic acid (PA) and diglyceride (DG) by C5a-stimulated human neutrophils has been studied. Membrane-associated 1-O-alkyl-phosphatidylcholine (alkyl-PC) was double labeled with 3H and 32P by incubating neutrophils with [3H]alkyl-lysoPC and alkyl-[32P]lysoPC. Upon stimulation with recombinant C5a, these labeled neutrophils produce 1-O-alkyl-phosphatidic acid (alkyl-PA) and, in the presence of ethanol, 1-O-alkyl-phosphatidyl-ethanol (alkyl-PEt), containing both 3H and 32P. Formation of radiolabeled alkyl-PEt parallels that of radiolabeled alkyl-PA and requires both extracellular Ca2+ and cytochalasin B. Furthermore, the 3H/32P ratios of alkyl-PA and alkyl-PEt formed during stimulation are very similar to that of th substrate alkyl-PC. These results demonstrate that, in C5a-stimulated neutrophils, alkyl-PA and alkyl-PEt are formed from alkyl-PC almost exclusively by PLD-catalyzed hydrolysis and transphosphatidylation, respectively. Upon C5a stimulation, neutrophils labeled with 3H and 32P also produce 1-O-[3H]alkyl-diglyceride [( 3H]alkyl-DG) and [32P]orthophosphate [( 32P]PO4), but not [32P]phosphocholine. [3H]Alkyl-DG and [32P]PO4 are formed in parallel, although temporally lagging behind alkyl-PA. Propranolol, a PA phosphohydrolase (PPH) inhibitor, decreases the formation of both [3H]alkyl-DG and [32P]PO4, although increasing alkyl-PA accumulation. These data support the conclusion that alkyl-DG is formed from alkyl-PC by the combined activities of PLD and PPH and not by phospholipase C (PLC). Furthermore, by using [3H]acyl-PC-labeled neutrophils, it is demonstrated that, like alkyl-PC, 1-acyl-PC is also degraded sequentially by PLD and PPH to 1-acyl-DG. Propranolol does not inhibit phosphoinositide-specific PLC and yet it causes almost complete inhibition of the total DG mass accumulation in C5a-stimulated neutrophils. We conclude that, in cytochalasin B-treated neutrophils stimulated with C5a, PLD-catalyzed hydrolysis of PC determines the levels of both PA and DG with potentially important ramifications for neutrophil-mediated defense functions.

Alkylation↗
PubMed1997

Erythromycin A-derived macrolides modify the functional activities of human neutrophils by altering the phospholipase D-phosphatidate phosphohydrolase transduction pathway: L-cladinose is involved both in alterations of neutrophil functions and modulation of this transductional pathway.

All erythromycin A derivatives, irrespective of the size of the lactone ring and the nature of the substituent, inhibit oxidant production by neutrophils and promote their degranulation. We demonstrate in this study that the L-cladinose at position 3 of the lactone ring is a key structure in the modulation of these two neutrophil functions, suggesting that this sugar (alone or combined with a lactone structure) interferes with cell target(s) involved in both oxidant production and exocytosis. Taking roxithromycin as an example of erythromycin A derivatives, we also found that these molecules interfered with the phospholipase D (PLD)-phosphatidate phosphohydrolase pathway in two ways. In nonstimulated neutrophils, roxithromycin and all L-cladinose-bearing molecules activated PLD, as reflected by 1-O-[3H]alkyl-2-acyl-phosphatidyl-ethanol production. In addition, these drugs induced an accumulation of 1-O-[3H]alkyl-2-acyl-phosphatidic acid (PA), but not 1-O-[3H]alkyl-2-acylglycerol. PA accumulation seems to be involved in the induction of exocytosis by macrolides, as the roxithromycin-induced release of granular enzymes was impaired strongly in the presence of ethanol. By contrast, in stimulated neutrophils, roxithromycin inhibited PLD activity and totally impaired 1-O-[3H]alkyl-2-acylglycerol production. The inhibition of diglyceride production by roxithromycin (not its descladinosyl derivative) could explain its inhibitory effect on oxidant production. The relevance of our data to the clinical situation, particularly the anti-inflammatory activity of these drugs, requires further investigation.

Diglycerides↗
PubMed1989

Second messenger function of phosphatidic acid in platelet activation.

Phosphatidic acid (PA) is synthesized as the result of the receptor-mediated response of platelets to physiologic agonists. The role of PA in platelet signal transduction, however, is largely unknown. We have examined the responses of platelets to 1-stearoyl-2-arachidonoyl phosphatidic acid (SAPA), the predominant molecular species of human platelet PA. SAPA alone causes platelet aggregation, and pretreatment of platelets with SAPA markedly enhances thrombin-induced aggregation and secretion. Addition of SAPA to intact human platelets causes rapid breakdown of phosphatidylinositol-4,5-bisphosphate (PIP2) and the generation of diacylglycerol and endogenous PA. These reactions are associated with mobilization of intracellular calcium and activation of protein kinase C. SAPA also stimulates the release of endogenous arachidonic acid and its conversion to thromboxane A2. Furthermore, platelet activation by SAPA is blocked by indomethacin, indicating that the actions of SAPA are mediated by cyclooxygenase products. These findings suggest that SAPA may play an important role as an endogenous positive feedback signal to amplify receptor-mediated activation of PIP2-specific phospholipase C in human platelets.

Adenosine Triphosphate↗
PubMed1989

Phosphatidate phosphohydrolase in purified rat brain myelin.

Highly purified myelin from rat brain stem has been shown to contain phosphatidate phosphohydrolase, an enzyme which converts phosphatidate to diacylglycerol. The high levels relative to cytosol and microsomes (17% and 22%, respectively) tended to preclude contamination by these fractions as the source of activity. Additional evidence came from study of repeated purification, mixing experiments, and washing of the myelin with salt and detergent. We conclude that this enzyme, in addition to being widely distributed in other subcellular fractions, is intrinsic to the myelin membrane. Through its activity it generates a key substrate for the cytidine (Kennedy) pathway which was previously shown to occur in this membrane.

Animals↗
PubMed1995

Selective inhibition of phosphatidylinositol 3-kinase by phosphatidic acid and related lipids.

Activation of phosphatidylinositol 3-kinase (PI 3-kinase) is necessary for stimulation of cell division and inhibition of apoptosis in several cell types. We report that a synthetic phosphonolipid, 4-(hexadecyloxy)-3-(S)-methoxybutyl phosphonic acid (PoA), as well as the naturally occurring lipids, phosphatidic acid and lyso-phosphatidic acid, are potent and specific inhibitors of PI 3-kinase. The IC50's for inhibition using phosphatidylinositol as substrate ranged from 10-20 microM. PoA is also the putative primary intracellular metabolite following phospholipase D hydrolysis of the anti-tumour ether lipid, 2'-(trimethylammonio) ethyl-4-(hexadecyloxy)-3-(S)-methoxybutanephosphonate. These results suggests that inhibition of PI 3-kinase following metabolic degradation of ether lipids by phospholipase D may contribute to the cytotoxicity of these compounds. The sensitivity of PI 3-kinase to PA and lyso-PA could imply cross-talk between the phospholipase D and PI 3-kinase signal transduction pathways in vivo.

Animals↗
PubMed2000

Cloning, expression, and chromosomal localization of a mouse gene homologous to the germ cell migration regulator wunen and to type 2 phosphatidic acid phosphatases.

The wunen gene of Drosophila melanogaster encodes a multipass membrane-spanning protein that negatively regulates primordial germ cell migration. Here we describe the cloning of a mouse gene that encodes a protein homologous to wunen and to the Type 2 phosphatidic acid phosphatases. This gene encodes a 251-amino-acid protein that most closely resembles the human Type 2 phosphatidic acid phosphatase PAP-2c. Northern blot analysis revealed the presence of a single 1.9-kb Ppap2c transcript. The Ppap2c gene was localized to the central portion of mouse Chromosome 10 by interspecific backcross analysis.

Amino Acid Sequence↗
PubMed1998

Primed neutrophils require phosphatidic acid for maximal receptor-activated elastase release.

BACKGROUND: Priming of neutrophils (PMNs) for protease release is believed to be central to the pathogenesis of PMN-mediated tissue injury observed in ARDS/MOF. Defining the intracellular signaling pathways involved with this excessive protease release may aid in establishing future therapies for ARDS. Phospholipase D (PLD) production of phosphatidic acid (PA) is thought to be pivotal in reactive oxygen species generation but its role in degranulation (i. e., protease release) remains unclear. Our hypothesis was that primed neutrophils require PLD production of PA for maximal activated release of elastase. METHODS: Isolated human PMNs were incubated with a well described antagonist of PA production, ethanol (ETOH, 100-1000 mg/dL), and then primed (PAF, 200 nM) followed by activation (fMLP, 1 microM). To mimic fMLP receptor-dependent activation, PMNs were primed and then directly activated with exogenous dioctanoyl l-alpha-phosphatidic acid (PA8, 0.5-200 microM). To confirm the importance of PA in elastase release, PA8 was given to primed-activated PMNs after ethanol pretreatment in an attempt to recover the maximal response. Elastase release was measured by the cleavage of AAPV-pNA. RESULTS: PA blockade with ETOH attenuated PAF-primed/fMLP-activated PMN elastase release in a dose-dependent manner. Exogenous PA8 reproduced maximally primed-activated PMN elastase release, and furthermore, PA8 was able to restore maximal elastase release following ethanol attenuation. CONCLUSIONS: Elastase release from PAF-primed/ fMLP-activated neutrophils is dependent on PA production. Thus, PA production appears to be involved in both oxidant-dependent and independent mechanisms of neutrophil cytotoxicity and may be a potential therapeutic target in the treatment of hyperinflammatory diseases such as ARDS/MOF.

Humans↗
PubMed1998

Structural-functional relationships in pig heart AMP-deaminase in the presence of ATP, orthophosphate, and phosphatidate bilayers.

The secondary structure of pig heart AMP-deaminase (AMP-d) in the absence and in the presence of orthophosphate or dioleoyl phosphatidic acid (DOPA) or ATP was investigated by FT-IR spectroscopy. While the latter substance activates the enzyme, orthophosphate is a well-known negative allosteric effector and DOPA exerts a noncompetitive inhibition on AMP-deaminase. Small changes in the secondary structure of AMP-d were induced by the above mentioned substances. Only DOPA reduced the thermal stability of AMP-d and avoided protein intermolecular interactions suggesting structural-functional relationships in AMP-d in the presence of the above substances and a possible role of phosphatidic acid in the subtle regulation of AMP-d activity by temporary binding of the enzyme to cellular membranes.

AMP Deaminase↗
PubMed1990

Diffusion of Mn2+ ions into liposomes mediated by phosphatidate and monitored by the activation of an encapsulated enzymatic system.

Transbilayer diffusion of Mn2+ ions occurred in liposomes formed from dipalmitoyl-phosphatidylcholine or egg-yolk phosphatidylcholine and egg-yolk phosphatidate (molar ratio 2:1) containing DNA and DNase I within their aqueous compartments. Cation diffusion was demonstrated by the hydrolytic activity of DNase I, activated by the Mn2+ ions that diffused into the vesicles, and this was confirmed by light scattering. Phosphatidate, a cone-shaped lipid which has been synthesized under simulated prebiotic conditions, was necessary for cation diffusion across the liposome membranes. Such liposomes represent a simple precellular system that interchanges cations with the surroundings and provides a microenvironment for enzymatic reactions, as evidenced by the hydrolysis of DNA by DNase I inside these closed lipid compartments.

Cations↗
PubMedDate not supplied

Plasticity of cholinoreceptors of neurons of the common snail after effects on inositol-1,4,5-triphosphate- and CA(2+)-dependant mobilization of stored CA2+ and the level of phosphatidic acid.

The influences on the depth of extinction of the inward current induced by acetylcholine (the ACh-current) of a number of compounds affecting the mobilization of stored Ca2+ and the intracellular level of Ca(2+)-mobilizing second messengers, namely, inositol-1,4,5-trisphosphate (IP3), inositol hexakisphosphate, TMB-8 (an inhibitor of (IP3)-dependent Ca2+ mobilization), tetracaine (an inhibitor of Ca(2+)-dependant mobilization of Ca2+), as well as phospholipase D, which leads to the formation of phosphatidic acid through the hydrolysis of phosphatidylcholine, were investigated in identified RPa3 and LPa3 neurons of the common snail using the two-electrode voltage clamp technique for the recording of the potential on the membrane. The participation of IP3, of IP3-dependant, and Ca(2+)-dependant mobilized intracellular Ca2+, as well as phosphatidic acid in the regulation of the plasticity of the cholinoreceptors of the neurons was demonstrated.

Acetylcholine↗
PubMed2002

Regulation of phosphatidic acid phosphohydrolase 1 by fatty acids.

In the starved state and during metabolic stress, free fatty acids (FFA) are the principal hepatic energy supply, undergoing beta-oxidation. Accordingly, it appears paradoxical that FFA have been reported to increase the liver's esterification capacity by translocating the rate-limiting enzyme phosphatidic acid phosphohydrolase (PAP-1) from the cytosol to the endoplasmic reticulum. We have therefore investigated the regulation of rat liver PAP-1. Oleic acid inhibited PAP activity in all subcellular fractions, with PAP-1 activity in cytosol being the most sensitive. Inhibition was also observed with oleoyl-CoA, linoleate, and palmitate. Fatty acids and their derivatives show detergent effects at high concentrations, and such effects can lead to enzyme inhibition. Inhibition by oleate, however, was reversed by phosphatidic acid and albumin and exhibited sigmoidal kinetics. These results demonstrate that PAP-1 is reversibly inhibited by FFA and their CoA esters, which may play a role in directing hepatic FFA to beta-oxidation during times of increased energy demand.

Acyl Coenzyme A↗
PubMed1986

A procedure for the preparation of [32P]phosphatidic acid.

The phosphorylation procedure of F. Cramer, W. Rittersdorf, and W. Bohm [(1961) Chem. Ber. 654, 180] using bis(triethylammonium) phosphate and trichloroacetonitrile was shown to be effective in the synthesis of [32P]phosphatidic acid. From diacylglyceride and 0.5 mCi H(3)32PO4, 25-50 microCi of labeled material (sp act = 1 mCi/mumol) can be prepared in 2 h. The product was shown to be radiochemically pure by both TLC and HPLC. L- and DL-[32P]dipalmitoyl phosphatidic acid prepared using this procedure were shown to be hydrolyzed by rat liver microsomes at approximately the same rates.

Animals↗
PubMed1991

Assays for investigations of signal transduction mechanisms involving phospholipase D: mass measurements of phosphatidate, phosphatidylethanol, and diacylglycerol in cultured cells.

With the intent of achieving a better understanding of agonist-induced phospholipase D activity, we have developed simple, rapid assays for quantitating the mass of phosphatidate, phosphatidylethanol, and diacylglycerol. Crude lipid extracts of cultured cells are used; preliminary sample cleanup or derivatization procedures are not necessary. The assays resolve the particular lipids by short-bed/continuous-development thin-layer chromatography. Quantitative assessments are derived from photodensitometric analysis of charred lipid spots. The assays may be employed for as little as 45 pmol of diacylglycerol and 50 pmol of phosphatidate or phosphatidylethanol. The newly developed assays are compared to other procedures for quantitating lipid mediators. The utility of the assays is illustrated in experiments that use a variety of cultured cells to demonstrate the agonist activation of the phospholipase D pathway. In addition, experiments designed to screen various agonists for signal-response coupling to phospholipase D are described.

Absorptiometry, Photon↗
PubMed1991

Sequential degradation of choline phosphoglycerides by phospholipase D and phosphatidate phosphohydrolase in dibutyryl cAMP-differentiated U937 cells.

Dibutyryl-cAMP-differentiated U937 cells incorporate alkyllyso-sn-glycero-3-[32P]phosphocholine (alkyllyso-[32P]GPC) into cellular alkylacyl-sn-glycero-3-phosphocholine (alkylacyl-GPC). Upon stimulation with fMet-Leu-Phe (fMLP), recombinant C5a, or phorbol 12-myristate 13-acetate (PMA), these cells produce alkylacyl-sn-glycero-3-[32P]phosphate (alkylacyl-[32P]GP). In the presence of ethanol (0.5%), alkylacyl-sn-glycero-3-[32P]phosphoethanol (alkylacyl-[32P]GPet) is also formed with a concomitant reduction in alkylacyl-[32P]GP accumulation. Because cellular ATP is not labeled with 32P, alkylacyl-[32P]GP and alkylacyl-[32P]GPet must be formed by phospholipase D (PLD)-catalyzed hydrolysis and transphosphatidylation, respectively. Activation by receptor agonists, but not by PMA, requires extracellular Ca2+ and is augmented by cytochalasin B pretreatment. Upon stimulation, dibutryl cAMP-differentiated U937 cells labeled with alkylacyl-[32P]GPC produce [32P]PO4 but not [32P]phosphocholine. Furthermore, when these cells were labeled in alkylacyl-GPC by incubation with [3H]alkyllyso-GPC and then stimulated, [3H]alkylacyl-glycerol ([3H]alkylacyl-Gro) is produced with a time-course similar to that of [32P]PO4 formation and coincident with the decline in alkylacyl-GP accumulation. These results demonstrate that alkylacyl-GP formed by PLD is dephosphorylated by phosphatidate phosphohydrolase to produce PO4 and alkylacyl-Gro. Upon stimulation with fMLP or C5a, U937 cells labeled in diacyl-sn-glycero-3-phosphocholine (diacyl-GPC) by incubation with [3H]acyllyso-GPC generate [3H]diacyl-GP, [3H]diacyl-GPEt, and [3H]diacyl-Gro with kinetics similar to those for the generation of the [3H]alkyl products. Thus, in differentiated U937 cells stimulated with receptor agonists, both alkylacyl-GPC and diacyl-GPC are sequentially metabolized by PLD and phosphatidate phosphohydrolase.

Animals↗
PubMed1979

Cooperative lipid-protein interaction. Effect of pH and ionic strength on polymyxin binding to phosphatidic acid membranes.

The binding of polymyxin-B to charged dipalmitoyl phosphatidic acid membranes has been studied as function of the external pH and of the ionic strength of the buffer solution. The phase transition curves were obtained by measuring the fluorescence depolarization of diphenyl hexatriene incorporated into the membrane with temperature. The molecular process of polymyxin binding was elucidated: 1. At an ionic strength of I greater than or equal to 0.1 mol/l a three step phase transition curve is found. A high-temperature step corresponds to the non-bound lipid. A lowered phase transition concerns to protein-bound lipid domains. This again is splitted into two steps. An inner core of the domain is characterized by a lipid-protein complex which is stabilized through hydrophobic and electrostatic interactions between polymyxin and the charged lipid. This core is surrounded by an outer belt of only hydrophobically bound molecules. This part shows a lower phase transition temperature than the inner core. 2. The binding curves of polymyxin to phosphatidic acid membranes depend strongly on the ionic strength of the water phase. The cooperativity of the binding process increases with increasing ionic strength and reaches a constant value at I greater than 0.2 mol/l. The maximum fraction of bound lipid decreases with increasing ionic strength. 3. The pH of the water phase strongly influences the cooperative binding process. At pH 6 a loss of cooperativity is observed at low ionic strength. Increasing the ion concentration to I = 0.3 mol/l recuperates the cooperativity of the binding process. At pH 3.0 no cooperative binding is obtained even at high ionic strength.

Hydrogen-Ion Concentration↗
PubMed1979

Influence of pH on phosphatidic acid multilayers. A rippled structure at high pH values.

The influence of pH on the structure of 1,2-(ditetradecyl)-phosphatidic acid was investigated by differential scanning calorimetry and freeze-fracture electron microscopy. At pH 13.5--14 (2.6 M K+), where phosphatidic acid has two negative charges, calorimetric scans show a small transition (pretransition) below the main phase transition temperature. Freeze-fracture studies of the same dispersions reveal regular band patterns (so-called ripples) in the plane of the bilayers, when the lipid is quenched from below the main phase transition temperature. This rippled structure is similar to the well-known rippled structure of phosphatidylcholines.

Calorimetry, Differential Scanning↗
PubMed1980

Fusion kinetics of phosphatidylcholine-phosphatidic acid mixed lipids vesicles: a proton nuclear magnetic resonance study.

The kinetics of Ca2+-induced fusion of phosphatidylcholine-phosphatidic acid vesicles has been studied using the dependence of proton nuclear magnetic resonance linewidths on vesicle size. The linewidth of the lipid acyl chain methylene resonance has been shown to be sensitive to changes in vesicle size but insensitive to vesicle aggregation. For vesicle systems with the same lipid composition, the linewidth increases in a linear fashion with vesicle radius over the range 125--300 A. This dependence has been used to determine quantitatively fusion rates and the dependence of such rates of Ca2+ as well as an vesicle concentration. For vesicle concentrations in the range of 3 . 10(-6)--10(-5) M and Ca2+ concentration at a level approaching 1 : 1 with respect to phosphatidic acid, the initial fusion rates have been found to be fast, with half-times of 1--10 min. An order of reaction of 2.7 with respect to vesicle concentration has been observed. Mechanisms of vesicle fusion are discussed in view of these observations.

Calcium↗