Alterations of sarcolemmal phospholipase D and phosphatidate phosphohydrolase in congestive heart failure

Phospholipase D 2 (PLD2) is the major PLD isozyme associated with the cardiac sarcolemmal (SL) membrane. Hydrolysis of SL phosphatidylcholine (PC) by PLD2 produces phosphatidic acid (PA), which is then converted to 1,2 diacylglycerol (DAG) by the action of phosphatidate phosphohydrolase type 2 (PAP2). In view of the role of both PA and DAG in the regulation of Ca²⁺ movements and the association of abnormal Ca²⁺ homeostasis with congestive heart failure (CHF), we examined the status of both PLD2 and PAP2 in SL membranes in the infarcted heart upon occluding the left coronary artery in rats for 1, 2, 4, 8 and 16 weeks. A time-dependent increase in both SL PLD2 and PAP2 activities was observed in the non-infarcted left ventricular tissue following myocardial infarction (MI); however, the increase in PAP2 activity was greater than that in PLD2 activity. Furthermore, the contents of both PA and PC were reduced, whereas that of DAG was increased in the failing heart SL membrane. Treatment of the CHF animals with imidapril, an angiotensin-converting enzyme (ACE) inhibitor, attenuated the observed changes in heart function, SL PLD2 and PAP2 activities, as well as SL PA, PC and DAG contents. The results suggest that heart failure is associated with increased activities of both PLD2 and PAP2 in the SL membrane and the beneficial effect of imidapril on heart function may be due to its ability to prevent these changes in the phospholipid signaling molecules in the cardiac SL membrane.     

Sphingosine inhibits phosphatidate phosphohydrolase in human neutrophils by a protein kinase C-independent mechanism

Human neutrophils have been labeled in 1-O-alkyl-phosphatidylcholine with 3H in both the alkyl chain and the choline moiety. Upon stimulation of these labeled cells with formyl-Met-Leu-Phe, C5a, or phorbol 12-myristate 13-acetate, phospholipase D is activated to produce 1-O-[3H]alkylphosphatidic acid ([3H]alkyl-PA) and [3H]choline. The [3H]alkyl-PA is then dephosphorylated by phosphatidate phosphohydrolase (PPH) to produce 1-O-[3H]alkyldiglyceride ([3H]alkyl-DG). Sphingosine, a sphingoid base known to inhibit protein kinase C (PKC), causes a dose-dependent inhibition of [3H]alkyl-DG formation. This inhibition is accompanied by increased accumulation of [3H]alkyl-PA without alterations in [3H]choline formation. Studies using various other sphingoid bases demonstrate that a long hydrocarbon chain and an amino group are required for the inhibition of DG formation. These results suggest that sphingoid bases inhibit PPH activity without altering phospholipase D activation and that they exhibit a similar structure-activity relationship for both PPH and PKC. K252a, a PKC inhibitor which acts by competing for ATP binding sites, does not inhibit the formation of [3H]alkyl-DG, [3H]alkyl-PA, or [3H]choline at a concentration (3 microM) that completely blocks phorbol 12-myristate 13-acetate-induced protein phosphorylation. Moreover, in neutrophil homogenates, sphingosine but not octylamine, inhibits PPH activity in a dose-dependent manner. Thus sphingosine inhibits PPH activity by a PKC-independent mechanism, raising the possibility that sphingoid bases may play a role in regulating PPH-mediated lipid metabolism in stimulated cells.     

Differential changes in phospholipase D and phosphatidate phosphohydrolase activities in ischemia-reperfusion of rat heart

Phospholipase D (PLD2) produces phosphatidic acid (PA), which is converted to 1,2 diacylglycerol (DAG) by phosphatidate phosphohydrolase (PAP2). Since PA and DAG regulate Ca(2+) movements, we examined PLD2 and PAP2 in the sarcolemma (SL) and sarcoplasmic reticular (SR) membranes from hearts subjected to ischemia and reperfusion (I-R). Although SL and SR PLD2 activities were unaltered after 30 min ischemia, 5 min reperfusion resulted in a 36% increase in SL PLD2 activity, whereas 30 min reperfusion resulted in a 30% decrease in SL PLD2 activity, as compared to the control value. SR PLD2 activity was decreased (39%) after 5 min reperfusion, but returned to control levels after 30 min reperfusion. Ischemia for 60 min resulted in depressed SL and SR PLD2 activities, characterized with reduced V(max) and increased K(m) values, which were not reversed during reperfusion. Although the SL PAP2 activity was decreased (31%) during ischemia and at 30 min reperfusion (28%), the SR PAP2 activity was unchanged after 30 min ischemia, but was decreased after 5 min reperfusion (25%) and almost completely recovered after 30 min reperfusion. A 60 min period of ischemia followed by reperfusion caused an irreversible depression of SL and SR PAP2 activities. Our results indicate that I-R induced cardiac dysfunction is associated with subcellular changes in PLD2 and PAP2 activities.     

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.     

Complement receptor-mediated phagocytosis is associated with accumulation of phosphatidylcholine-derived diglyceride in human neutrophils. Involvement of phospholipase D and direct evidence for a positive feedback signal of protein kinase.

Complement receptor (CR)-mediated phagocytosis is associated with an increased accumulation of diglyceride (sn-1,2-diacylglycerol and/or 1-O-alkyl-2-acyl-glycerol) in human neutrophils. The C3bi-mediated increase in diglyceride (5-20 min) was only partially impaired when phosphoinositide-specific phospholipase C (PLC) activity was abolished by reduction of cytosolic free Ca2+. At an early time point (1 min), however, diglyceride production was barely detectable in control cells, whereas production was considerable in cells with a reduced cytosolic free Ca2+ concentration. C3bi stimulation of 32P-labeled neutrophils caused a rapid and significant breakdown of [32P]phosphatidylcholine (PC) which was not affected by inhibition of Ca(2+)-dependent phosphoinositide-specific PLC. Thus, PC hydrolysis could be involved in C3bi-induced diglyceride formation. Stimulation of cells labeled with [3H]1-O-alkyl-lyso-PC ([3H]alkyl-lyso-PC), resulted in an increased formation of [3H]1-O-alkyl-phosphatidic acid ([3H]alkyl-PA) and a later and slower formation of [3H]1-O-alkyl-diglyceride ([3H]alkyl-diglyceride); this suggests activation of phospholipase D (PLD). When these labeled cells were stimulated in the presence of 0.5% ethanol a marked accumulation of [3H]1-O-alkyl-phosphatidylethanol ([3H]alkyl-PEt) was observed in both controls and calcium-reduced cells, further strengthening the suggested involvement of PLD activity. In parallel with the sustained increase in diglyceride formation, CR-mediated phagocytosis was also associated with phosphorylation of a cellular protein kinase C substrate (MARCKS). Therefore it seems reasonable to suggest a causal relationship between C3bi-induced PLD activation, which results in diglyceride formation, and activation of protein kinase C. In electropermeabilized cells which were incapable of ingesting particles, C3bi particles were still able to activate PLD and induce formation of diglyceride. This signaling event must therefore be triggered by binding of particles to the cell and not by the engulfment process. Most importantly, introduction of the protein kinase C inhibitor peptides, PKC(19-36) and PKC(19-31), into these permeabilized cells resulted in a clear reduction of the C3bi-induced production of diglyceride, indicating that CR-mediated activation of protein kinase C directly triggers a positive feedback mechanism for additional diglyceride formation. Taken together, these data further clarify the mechanisms of CR-mediated diglyceride formation and give added support to the concept that protein kinase C plays an important role in the phagocytic process.     

Oleate stimulation of diacylglycerol formation from phosphatidylcholine through effects on phospholipase D and phosphatidate phosphohydrolase.

Hydrolysis of exogenous phosphatidylcholine (PtdCho) to 1,2-diacylglycerol by rat liver plasma membranes was stimulated by oleate concentrations as low as 0.1 mM. In the presence of 75 mM ethanol, the fatty acid also enhanced phosphatidylethanol (PtdEtOH) formation from PtdCho. These effects were also observed with linoleate and arachidonate, but not with saturated fatty acids or detergents, and were minimal in microsomes or mitochondria. Release of [3H]choline from exogenous Ptd[3H]Cho was stimulated by oleate, whereas phosphoryl[3H]choline formation was inhibited. Oleate and other unsaturated, but not saturated, fatty acids also stimulated the conversion of exogenous [14C]phosphatidic acid to [14C]diacylglycerol. These data are consistent with stimulatory effects of these fatty acids on both phospholipase D and phosphatidate phosphohydrolase in liver plasma membranes. The stimulatory effect of guanosine 5'-O-[3-thio]triphosphate) (20 microM) on PtdEtOH and diacylglycerol formation from PtdCho was enhanced by low concentrations of oleate. Phospholipase A2 also stimulated PtdEtOH and diacylglycerol formation from exogenous PtdCho. It is proposed that unsaturated fatty acids may play a physiological role in the regulation of diacylglycerol production through activation of phospholipase D and phosphatidate phosphohydrolase.     

Sequential actions of phospholipase D and phosphatidic acid phosphohydrolase 2b generate diglyceride in mammalian cells

Phosphatidylcholine (PC) is a major source of lipid-derived second messenger molecules that function as both intracellular and extracellular signals. PC-specific phospholipase D (PLD) and phosphatidic acid phosphohydrolase (PAP) are two pivotal enzymes in this signaling system, and they act in series to generate the biologically active lipids phosphatidic acid (PA) and diglyceride. The identity of the PAP enzyme involved in PLD-mediated signal transduction is unclear. We provide the first evidence for a functional role of a type 2 PAP, PAP2b, in the metabolism of PLD-generated PA. Our data indicate that PAP2b localizes to regions of the cell in which PC hydrolysis by PLD is taking place. Using a newly developed PAP2b-specific antibody, we have characterized the expression, posttranslational modification, and localization of endogenous PAP2b. Glycosylation and localization of PAP2b appear to be cell type and tissue specific. Biochemical fractionation and immunoprecipitation analyses revealed that PAP2b and PLD2 activities are present in caveolin-1-enriched detergent-resistant membrane microdomains. We found that PLD2 and PAP2b act sequentially to generate diglyceride within this specialized membrane compartment. The unique lipid composition of these membranes may provide a selective environment for the regulation and actions of enzymes involved in signaling through PC hydrolysis.     

Contribution of mitogen-activated protein kinase to stimulation of phospholipase D by the chemotactic peptide fMet-Leu-Phe in human neutrophils.

Phospholipase D (PLD) plays an important role in signaling through phosphatidylcholine (PC) and in the production of superoxide (respiratory burst) by polymorphonuclear leukocytes (PMN) stimulated by the chemoattractant fMet-Leu-Phe (fMLP). However, the regulation of PLD activity by protein kinases is not fully understood. In the present study, we have used a mitogen-activated protein (MAP) kinase inhibitor (PD 98059) to investigate a possible connection between extracellular signal-regulated kinase (ERK) and PLD activity and respiratory burst. Using a range of concentrations (3-20 microM) which inhibit ERK activity, PD 98059 inhibited PLD activity induced by fMLP in cytochalasin B-primed PMN, as assessed by production-tritiated phosphatidylethanol (PEt), phosphatidic acid (PA), and hydrolysis of PC. However, the inhibition was partial (approximately 50%), while inhibition of PC hydrolysis was almost complete, suggesting a concomitant inhibition of PLA2 activity. In addition, PD 98059 reduced fMLP-induced respiratory burst by 50%, an effect which was correlated with PLD inhibition of PLD (r = 0.981, P < 0.01), and neither did PD 98059 inhibit the PLD activity and respiratory burst induced by PKC upon its direct activation by phorbol myristate acetate. These data provide the first evidence for implication of the ERK cascade in the stimulation of PLD through Gi signaling. They further indicate that PLD stimulation by fMLP receptors occurs through two pathways, dependent and independent on MAP kinase, the former pathway being linked to superoxide production.     

The measurement of phosphatidate phosphohydrolase in human amniotic fluid.

Phosphatidate phosphohydrolase (EC 3.1.3.4) activity can be found in late gestational human amniotic fluid and is thought to originate in type II alveolar cells of the fetal lungs where it plays an important role in lung surfactant synthesis. In the present study, phosphatidate phosphohydrolase activity was detected and characterized in a 105 000 X g pellet of amniotic fluid using either [32P]phosphatidate or a water-soluble analog, 1-O-hexadecyl-rac-[2-(3)H]glycerol 3-phosphate as substrate. With either substrate, enzyme activity was optimal at pH 6.0. The soluble analog was hydrolyzed with a Km value of 163 micrometer and a V of 30 nmole/min per mg of protein, and offered several advantages over phosphatidate as a substrate for assaying phosphatidate phosphohydrolase in amniotic fluid. Using the synthetic analog, phosphatidate phosphohydrolase activity was measured in the 700 X g supernatant fraction of 30 human amniocentesis samples and compared with another index of fetal lung maturity, the phosphatidylcholine/sphingomyelin ratio. The results suggest that the new phosphohydrolase assay may be clinically useful in the assessment of fetal lung development.     

Phosphatidylcholine hydrolysis stimulated by phorbol myristate acetate is mediated principally by phospholipase D in endothelial cells

The mechanism of phosphatidylcholine (PC) degradation stimulated by phorbol myristate acetate (PMA) was investigated in bovine pulmonary artery endothelial cells prelabeled with [methyl-3H]choline ([3H]choline) or [9,10-3H]myristic acid ([3H]myristic acid). Both labels were selectively incorporated into PC, and addition of PMA stimulated comparable losses of 3H from PC in cells prelabeled with [3H]choline or [3H]myristate. In cells prelabeled with [3H]choline, the loss of 3H from PC correlated with a rapid increase in intracellular free [3H]choline. The increase in intracellular [3H]choline stimulated by PMA was not preceded by an increase in any other 3H-labeled PC degradation product. PMA did not stimulate the formation of PC deacylation products in cells prelabeled with [3H]choline. In permeabilized cells prelabeled with [3H]choline, PMA stimulated the formation of [3H]choline but not [3H]phosphocholine. In intact cells prelabeled with [3H]myristate, the loss of 3H from PC induced by PMA correlated with the formation of [3H]phosphatidic acid ([3H]PA) and [3H]diacylglycerol. In the presence of ethanol, PMA stimulated the formation of [3H]phosphatidylethanol ([3H]PEt) at the expense of [3H]PA. The time-course of [3H]PEt formation was similar to the time-course of intracellular [3H]choline formation in cells stimulated with PMA. These data taken together support the notion that PC degradation in endothelial cells stimulated with PMA is mediated principally by phospholipase D. PC breakdown via phospholipase D was not observed in cells treated with phorbol esters incapable of interacting with protein kinase C. Activation of phospholipase D by phorbol esters was inhibited by long-term pretreatment of cells with PMA to down-regulate protein kinase C and by pretreatment of the cells with staurosporine. These data support the notion that activation of phospholipase D by phorbol esters is dependent upon protein kinase C.     

Regulation of cyclooxygenase-2 expression by phosphatidate phosphohydrolase in human amnionic WISH cells.

Prostaglandins are known to play a key role in the initiation of labor in humans, but the mechanisms governing their synthesis in amnion are largely unknown. In this study, we have examined the regulatory pathways for prostaglandin E(2) (PGE(2)) production during protein kinase C-dependent activation of human WISH cells. In these cells, PGE(2) synthesis appears to be limited not by free arachidonic acid availability but by the expression levels of cyclooxygenase-2 (COX-2). Concomitant with the cells being able to synthesize and secrete PGE(2), we detected significant elevations of both COX-2 protein and mRNA levels. Specific inhibition of COX-2 by NS-398 totally ablated PGE(2) synthesis. All of these responses were found to be strikingly dependent on an active phosphatidate phosphohydrolase 1 (PAP-1). Inhibition of PAP-1 activity by three different strategies (i.e. use of bromoenol lactone, propranolol, and ethanol) resulted in inhibition of COX-2 expression and hence of PGE(2) production. These data unveil a novel signaling mechanism for the regulation of PGE(2) production via regulation of COX-2 expression and implicate phosphatidate phosphohydrolase 1 as a key regulatory component of eicosanoid metabolic pathways in the amnion.     

Resveratrol inhibits the formation of phosphatidic acid and diglyceride in chemotactic peptide- or phorbol ester-stimulated human neutrophils

Resveratrol (trans-3,5,4'-trihydroxystilbene, Res) is a naturally occurring antioxidant found in grape berry skins and red wine. It has anti-inflammatory effects. In this study, we examined the effect of Res on the formation of phosphatidic acid (PA) and diglyceride (DG), in human neutrophils stimulated by formyl-methionyl-leucyl-phenylalanine (fMLP) or by phorbol 12-myristate 13-acetate (PMA). We measured the masses of PA and DG by using a nonradioactive method. Our results showed that Res inhibited the formation of PA in a concentration dependent manner with an IC(50) value of 42.4 and 60.9 microM in fMLP- and PMA-stimulated cells, respectively. Res also suppressed the formation of phosphatidylethanol (PEt), thereby implying inhibition of phospholipase D (PLD) activity. In addition, Res inhibited the formation of both diacylglycerol (DAG) and ether-linked acylglycerol (EAG) induced by fMLP and by PMA. Our results suggest that Res inhibition of PLD activity may contribute to its anti-inflammatory effects.     

Platelet-activating factor mediates phosphatidylcholine hydrolysis by phospholipase D in human endometrium.

Human preimplantation embryos and endometrium secrete platelet-activating factor (PAF). The mechanism of phosphatidylcholine (PC) degradation stimulated by PAF was investigated in endometrial explants prelabeled with [methyl-3H]choline or preincubated with [3H]butan-1-ol. Analysis of the water-soluble metabolites of PAF-induced PC hydrolysis in secretory endometrium demonstrated that the stimulated generation of [3H]choline ([3H]Cho) precedes that of [3H]choline phosphate ([3H]ChoP) and [3H]glycerophosphocholine ([3H]GPC). Within 30 sec there was a rapid rise in PAF-induced [3H]Cho generation and by 2 min this had increased to 59.9% +/- 10.6% (p less than 0.02), with no effect upon [3H]ChoP and [3H]GPC during this period. Both [3H]GPC and [3H]ChoP, however, were increased at a later time point. The slower [3H]ChoP generation may suggest that PC-specific phospholipase C activation as well as delayed [3H]GPC rise may be due to PC-specific phospholipase A2 and lysophospholipase activation. Phospholipase D activity was confirmed by the incorporation of high-specific-activity [3H]butan-1-ol into [3H]phosphatidylbutanol ([3H]PBut). The rapid generation of [3H]PBut, which paralleled the rise in intracellular [3H]Cho, strongly suggests that PC breakdown is catalyzed by the phospholipase D pathway. It is proposed that PAF induces PC hydrolysis as a consequence of an early phospholipase D-catalyzed breakdown of PC in human secretory endometrium. This may be an alternative source for prostaglandin synthesis and an important pathway essential for long-term activation of local cellular events at the time of implantation.     

v-Src induces elevated levels of diglyceride by stimulation of phosphatidylcholine hydrolysis.

When Rat-1 cells bearing the ts LA29 mutant of Rous sarcoma virus (Rat1 LA29) are shifted from restrictive to permissive temperature, the pp60v-Src tyrosine kinase is activated and there is an increase in the cellular level of sn1,2-diacylglycerol (DRG) within 30 min which is not accompanied by increased inositol phospholipid hydrolysis. Temperature shift also increases the hydrolysis of phosphatidylcholine (PC), as determined by an increase in the generation of water soluble choline metabolites. Transphosphatidylation studies have shown that this occurs at least in part via a phospholipase D (PLD) catalysed pathway.     

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.     

Diethylstilbestrol inhibits phospholipase D activity and degranulation by stimulated human neutrophils

In the present study the effects of diethylstilbestrol on phospholipase D activity and degranulation by human neutrophils were examined. Diethylstilbestrol is a synthetic estrogen and has structural similarity to resveratrol. Resveratrol is a natural polyphenolic antioxidant and has been shown to inhibit the activity of phospholipase D in stimulated neutrophils. Phospholipase D catalyzes the hydrolysis of phosphatidylcholine to yield phosphatidic acid and choline. It also catalyzes the transfer of the phosphatidyl group to ethanol forming phosphatidylethanol at the expense of phosphatidic acid. Phospholipase D activation is associated with degranulation by neutrophils stimulated with chemotactic peptide, formyl-methionyl-leucyl-phenylalanine. The results show that diethylstilbestrol at 100 microM induced a complete inhibition of phosphatidic acid formation in neutrophils, the latter activated by chemotactic peptide. In the presence of ethanol, diethylstilbestrol dose dependently reduced phosphatidylethanol formation induced by chemotactic peptide or by phorbol 12-myristate 13-acetate, indicative of diethylstilbestyrol inhibition of phospholipase D activity. The results also demonstrate that diethylstilbestrol inhibited degranulation by chemotactic peptide-stimulated neutrophils. In comparison to resveratrol, diethylstilbestrol exhibits a stronger inhibition on PA formation, phospholipase D activity and degranulation. These findings suggest that diethylstilbestrol-like resveratrol, may have anti-inflammatory effect in vitro.     

Epidermal growth factor-induced hydrolysis of phosphatidylcholine by phospholipase D and phospholipase C in human dermal fibroblasts

The enzymatic pathways for formation of 1,2-diradylglyceride in response to epidermal growth factor in human dermal fibroblasts have been investigated. 1,2-Diradylglyceride mass was elevated 2-fold within one minute of addition of EGF. Maximal accumulation (4-fold) occurred at 5 minutes. Since both diacyl and ether-linked diglyceride species occur naturally and may accumulate following agonist activation, we developed a novel method to determine separately the alterations in diacyl and ether-linked diglycerides following stimulation of fibroblasts with EGF. Utilizing this method, it was found that approximately 80% of the total cellular 1,2-diradylglyceride was diacyl, the remaining 20% being ether-linked. Addition of EGF caused accumulation of 1,2-diacylglyceride without alteration in the level of ether-linked diglyceride. Thus, the observed induction of 1,2-diradylglyceride by EGF was due exclusively to increased formation of 1,2-diacylglyceride. In cells labelled with [3H]choline, the water soluble phosphatidylcholine hydrolysis products, phosphorylcholine and choline, were increased 2-fold within 5 minutes of addition of EGF. No hydrolysis of phosphatidylethanolamine, phosphatidylserine, or phosphatidylinositol was observed. Quantitation by radiolabel and mass revealed equivalent elevations in phosphorylcholine and choline, suggesting stimulation of both phospholipase C and phospholipase D activities. To identify the presence of EGF-induced phospholipase D activity, cells were labelled with exogenous [3H]1-0-hexadecyl, 2-acyl phosphatidylcholine and its conversion to phosphatidic acid in response to EGF determined. Radiolabelled phosphatidic acid was detectable in 15 seconds after addition of EGF and was maximal (3-fold) at 30 seconds. Consistent with the presence of EGF-induced phospholipase D activity, treatment of cells with EGF, in the presence of [14C]ethanol, resulted in the rapid formation of [14C]phosphatidylethanol, the product of phospholipase D-catalyzed transphosphatidylation. The formation of phosphatidylethanol, which competes for the formation of phosphatidic acid by phospholipase D, did not diminish the induction of 1,2-diglyceride by EGF. These data suggest that the phosphatidic acid formed by phospholipase D-catalyzed hydrolysis of phosphatidylcholine is not a major precursor of the observed increased 1,2-diglyceride. Thus, the induction of 1,2-diacylglycerol by EGF may occur primarily via phospholipase C-catalyzed hydrolysis of phosphatidylcholine.     

Interleukin-1 rapidly stimulates lysophosphatidate acyltransferase and phosphatidate phosphohydrolase activities in human mesangial cells.

Phosphatidic acid (PA) is a cytokine in a variety of cell types, and an intermediary in cell activation. It is produced from membrane phospholipids by either lysophosphatidate acyl-CoA:acyltransferase (lyso-PA AT) or phospholipase D. Interleukin-1 (IL-1) stimulation of human mesangial cells (HMC) induced activation of lyso-PA AT, and synthesis of new PA species with significant increase in PA mass. These PA species were enriched in long-chain unsaturated acyl side chains (C18:1, C18:2, C20:5, and C22:6) in both the sn-2 and sn-1 positions, and stimulated the action of the lyso-PA AT as a positive feedback mechanism. Gas-liquid chromatography and mass spectrometry demonstrate that the acyl composition of phosphatidic acid does not resemble that of the major phospholipid fractions of this preparation and therefore is not the product of phospholipase D. The PA species were rapidly converted to 1,2-sn-diacylglycerols by phosphatidate phosphohydrolase, which also was activated by IL-1 via a separate mechanism involving a pertussis-sensitive G-protein. The activities of lyso-PA AT and phosphatidate phosphohydrolase were associated with plasma membrane enriched and refined microsomal fractions. IL-1 stimulation of a murine T cell (thymoma) line, EL-4, also caused stimulation of lyso-PA AT, resulting in PA formation. EL-4 mutants with defective IL-1 receptors did not demonstrate stimulation of lyso-PA AT, showing the necessity of intact IL-1 receptors for activation of this enzyme. We conclude that PA is a significant signaling intermediary for IL-1 via activation of lyso-PA AT and a G-protein, which activates phosphatidate phosphohydrolase. This system suggests a novel mechanism whereby a low intensity signal may be translated into cellular activation.     

Involvement of phospholipase D-related signal transduction in chemical-induced programmed cell death in tomato cell cultures

Phospholipase D (PLD) and its product phosphatidic acid (PA) are incorporated in a complex metabolic network in which the individual PLD isoforms are suggested to regulate specific developmental and stress responses, including plant programmed cell death (PCD). Despite the accumulating knowledge, the mechanisms through which PLD/PA operate during PCD are still poorly understood. In this work, the role of PLDα1 in PCD and the associated caspase-like proteolysis, ethylene and hydrogen peroxide (H₂O₂) synthesis in tomato suspension cells was studied. Wild-type (WT) and PLDα1-silenced cell lines were exposed to the cell death-inducing chemicals camptothecin (CPT), fumonisin B1 (FB1) and CdSO₄. A range of caspase inhibitors effectively suppressed CPT-induced PCD in WT cells, but failed to alleviate cell death in PLDα1-deficient cells. Compared to WT, in CPT-treated PLDα1 mutant cells, reduced cell death and decreased production of H₂O₂ were observed. Application of ethylene significantly enhanced CPT-induced cell death both in WT and PLDα1 mutants. Treatments with the PA derivative lyso-phosphatidic acid and mastoparan (agonist of PLD/PLC signalling downstream of G proteins) caused severe cell death. Inhibitors, specific to PLD and PLC, remarkably decreased the chemical-induced cell death. Taken together with our previous findings, the results suggest that PLDα1 contributes to caspase-like-dependent cell death possibly communicated through PA, reactive oxygen species and ethylene. The dead cells expressed morphological features of PCD such as protoplast shrinkage and nucleus compaction. The presented findings reveal novel elements of PLD/PA-mediated cell death response and suggest that PLDα1 is an important factor in chemical-induced PCD signal transduction.     

Thyroxine signal transduction in liver cells involves phospholipase C and phospholipase D activation. Genomic independent action of thyroid hormone

Background  

Numerous investigations demonstrate a novel role of thyroid hormone as a modulator of signal transduction. Protein kinase C (PKC) is critical to the mechanism by which thyroid hormones potentiate both the antiviral and immunomodulatory actions of IFNγ in different cells and regulate the exchange of signalling phospholipids in hepatocytes. Because nothing is known about accumulation of PKC modulator - diacylglycerol in cells treated with T₄, we examined the nongenomic effect of thyroid hormones on DAG formation and phospholipase activation in liver cells.