Phosphatidylcholine-specific Phospholipase D Research Articles

ホスホリパーゼＤによる好中球機能の制御

Polymorphonuclear leukocytes (PMNs) adherent to fibrinogen exhibit a delay in onset of respiratory burst reaction and a biphasic formation of diradylglycerol (DRG) in response to formyl-Met-Leu-Phe. The H2O2 release coincides with the second phase of DRG formation by activation of phospholipase D (PLD) . Propranolol and ethanol abolish both H2O2 release and the second wave of DRG formation. PLD-mediated generation of [3H] -phosphatidylethanol from [3H] -phosphatidylcholine (PC) corresponds to the second phase of DRG formation. Intracellular calcium chelation blocks the H2O2 release as well as the second DRG formation. Addition of ionomycin and synthetic DRG to the calcium dampening PMNs stimulated them to release H2O2, showing the importance of DRG derived from calcium-dependent PLD activation.Accumulation of ceramide is observed in adherent PMNs when H2O2 release is terminated. The H2O2 release is suppressed by addition of exogenous free sphingoid amines and short chain ceramides. The inhibitory effects of N-acetyl-conjugated sphingols (C2 ceramides) on PC-specific PLD and phosphatidate phosphohydrolase are markedly different from those of related sphingoid bases. C2 ceramides demonstrate to inhibit oxidant release in stimulated PMNs. The effect of ceramide in respiratory burst is structurally specific, because either a 4, 5-trans double bond or 4-hydroxy group is required for the inhibition.The oxidant release in adherent human PMNs is stimulated or inhibited through PLD pathway, suggesting the regulatory role of PLD in the respiratory burst reaction.

Human ADP-ribosylation Factor-activated Phosphatidylcholine-specific Phospholipase D Defines a New and Highly Conserved Gene Family

Activation of phosphatidylcholine-specific phospholipase D (PLD) has been implicated as a critical step in numerous cellular pathways, including signal transduction, membrane trafficking, and the regulation of mitosis. We report here the identification of the first human PLD cDNA, which defines a new and highly conserved gene family. Characterization of recombinant human PLD1 reveals that it is membrane-associated, selective for phosphatidylcholine, stimulated by phosphatidylinositol 4,5-bisphosphate, activated by the monomeric G-protein ADP-ribosylation factor-1, and inhibited by oleate. PLD1 likely encodes the gene product responsible for the most widely studied endogenous PLD activity.

HDL3 stimulates multiple signaling pathways in human skin fibroblasts.

The influence of HDL3 on phospholipid breakdown was examined in human skin fibroblasts. HDL3 elicited phosphatidylcholine (PC) and phosphatidylinositol (PI) turnover and activated multiple phospholipases. In [14C]lyso-PC-labeled or [14C]choline (Cho)-labeled cells, a biphasic activation of PC-specific phospholipase D (PLD) with peak maxima 30 to 60 seconds and 5 to 7 minutes after stimulation with 20 micrograms/mL HDL3 was shown by (1) a 1.5- to 3-fold increase in Cho release, and (3) transphosphatidylation of PC to phosphatidylbutanol in the presence of 0.3% butanol. Activation of PC-specific PLD was paralleled by an activation of PC-specific phospholipase C (PLC). A significant increase in [14C]diacylglycerol (DG) was seen from 2 minutes after stimulation onward and remained for at least 2 hours. By means of butanol, the PA-phosphohydrolase (PPH) inhibitor propranolol, and the PC-PLC inhibitor D609, we demonstrated that the initial PC-derived DG formation occurred primarily by a coupled PLD/PPH pathway and that a major part of the sustained DG formation was derived directly from PC by PC-PLC. By down-regulating protein kinase C (PKC) we demonstrated that PKC activates PC-PLC and desensitizes PC-PLD at no longer incubation times. The sustained PC hydrolysis as well as HDL3-mediated PI turnover and PC resynthesis was observed on stimulation with 5 to 75 micrograms/mL HDL3, whereas the rapid activation of PC-PLD/PPH was detected only on stimulation with HDL3 at concentrations of between 10 and 75 micrograms/mL. Only the latter response could be mimicked by apolipoprotein A-I and apolipoprotein A-II proteoliposomes, and only this response was inducible by cholesterol loading. The HDL3-mediated second-messenger responses were inhibited by modification of HDL3 by tetranitromethane and could not be mimicked by protein-free liposomes. These data suggest that HDL3-induced cell signaling in human skin fibroblasts is mediated by specific protein-receptor interaction and that more than one agonist activity may be involved.

A Soluble Protein Negatively Regulates Phospholipase D Activity. Partial Purification and Characterization

Phosphatidylcholine-specific phospholipase D (PLD) is an important signalling phospholipase in mammalian cells. Recently, PLD activity has been shown to be positively regulated by the GTP-binding protein ARF (ADP-ribosylating factor). In the present work, we document the presence of a factor negatively regulating PLD activity in bovine brain cytosol. The inhibitory factor is characterized as a large protein or a complex of proteins with a molecular mass higher than 300 kDa. Using permeabilized and pre-permeabilized HL-60 cells depleted of their cytosol, we demonstrate that the inhibitor acts on GTP[S]-stimulated PLD activity. This effect is immediate, persistent and dose dependent for GTP[S]-stimulated PLD. Different possibilities for a mechanism of action of the inhibitory factor on the regulation of GTP binding to ARF were investigated. This inhibitory factor is not the guanine-dissociating inhibitor (GDI) for the small G-binding proteins Rho (Rho-GDI), reported to be a PLD inhibitor, since specific antibodies against this protein did not recognize a protein in the peak containing the inhibitory factor for PLD activity. Furthermore, the inhibitory factor does not prevent the binding of GTP[S] to ARF in the presence of HL-60 membranes. This excludes its possible role as an inhibitor of an ARF/guanine exchange factor. The inhibitory factor not only inhibits a pathway of PLD through GTP[S] activation in particular of the small GTP-binding protein, ARF, but it also inhibits PLD activated via either protein kinase C (PKC) or tyrosine kinase activation. The inhibitory factor also decreases PLC activity and this effect seems to be secondary to the inhibition of PLD activity. We discuss a mechanism of action of the inhibitor on PLD and the importance of this enzyme activity for membrane traffic.

A Soluble Protein Negatively Regulates Phospholipase D Activity. Partial Purification and Characterization

Phosphatidylcholine-specific phospholipase D (PLD) is an important signalling phospholipase in mammalian cells. Recently, PLD activity has been shown to be positively regulated by the GTP-binding protein ARF (ADP-ribosylating factor). In the present work, we document the presence of a factor negatively regulating PLD activity in bovine brain cytosol. The inhibitory factor is characterized as a large protein or a complex of proteins with a molecular mass higher than 300 kDa. Using permeabilized and pre-permeabilized HL-60 cells depleted of their cytosol, we demonstrate that the inhibitor acts on GTP[S]-stimulated PLD activity. This effect is immediate, persistent and dose dependent for GTP[S]-stimulated PLD. Different possibilities for a mechanism of action of the inhibitory factor on the regulation of GTP binding of ARF were investigated. This inhibitory factor is not the guanine-dissociating inhibitor (GDI) for the small G-binding proteins Rho (Rho-GDI), reported to be a PLD inhibitor, since specific antibodies against this protein did not recognize a protein in the peak containing the inhibitory factor for PLD activity. Furthermore, the inhibitory factor does not prevent the binding of GTP[S] to ARF in the presence of HL-60 membranes. This excludes its possible role as an inhibitor of an ARF/guanine exchange factor. The inhibitory factor not only inhibits a pathway of PLD through GTP[S] activation in particular of the small GTP-binding protein, ARF, but it also inhibits PLD activated via either protein kinase C (PKC) or tyrosine kinase activation. The inhibitory factor also decreases PLC activity and this effect seems to be secondary to the inhibition of PLD activity. We discuss a mechanism of action of the inhibitor on PLD and the importance of this enzyme activity for membrane traffic.

Structure-activity relationship of the effect of cis-unsaturated fatty acids on heart sarcolemmal phospholipase D activity

This study examined the role of fatty acids on the phosphatidylcholine-specific phospholipase D (PLD) function of purified sarcolemmal (SL) membranes isolated from rat hearts. The enzyme's hydrolytic activity was determined by measuring [ 14C] phosphatidic acid formation from exogenous [ 14C] phosphatidylcholine (PtdCho) in the absence or presence of the sodium salts of various saturated or unsaturated long-chain fatty acids (FA). In certain experiments the enzyme was also assayed in the transphosphatidylation mode. Cisunsaturation and free carboxyl groups were structural prerequisites for the stimulatory effect exerted by FA on SL PLD. The most effective compounds were arachidonate and oleate, which maximally activated PLD at 4 and 5 mM concentration, respectively. To verify if a detergent-like mechanism was involved in PLD activation, anionic, zwitterionic and non-ionic detergents were used. Only anionic taurodeoxycholate had a slight effect, which was about 7% of that achieved by arachidonate or oleate. These results suggest that cis-unsaturated FA activate cardiac sarcolemmal PLD by a mechanism(s) which seems to be unrelated to non-specific perturbation of the membrane.

Selective Activation by Atrial Natriuretic Factor of Phosphatidylcholine-specific Phospholipase Activities in Purified Heart Muscle Plasma Membranes

We have characterized a membrane-bound phosphatidylcholine (PC) specific phospholipase C (PC-PLC) in plasma membranes from rat cardiac muscle, and have investigated the role of PC-PLC and PC-specific phospholipase D (PC-PLD) activities in the mechanism of action of atrial natriuretic factor (ANF). In purified sarcolemma, ANF stimulated over a wide range of concentrations with a maximum at 10-11 M the hydrolysis of phosphatidylcholine through PC-PLD giving phosphatidate and choline, whereas higher concentrations of ANF (10-10 M) preferentially stimulated PC breakdown through PC-PLC to form diacylglycerol and phosphocholine. To confirm the involvement of the PC-PLD in the mechanism of ANF action, we measured the transphosphatidylation reaction, a specific assay for this phospholipase which in the presence of ethanol catalyses the phosphatidylethanol formation from PC. ANF stimulated phosphatidylethanol formation with the same dose-response behavior as phosphatidate formation. The significant diacylglycerol increase at 10-10 M ANF, in the presence of propranolol, a potent inhibitor of phosphatidate phosphatase which can hydrolyse phosphatidate to give diacylglycerol, suggested a direct involvement of PC-PLC. The use of GTPγ-S, a non hydrolysable analog of GTP, and of pertussis toxin showed the involvement of a pertussis toxin insensitive G protein in PC-PLC mediated ANF signal transduction. We suggest a differential effect of ANF on PC breakdown by phospholipases C and D depending on the concentration of the peptide.

IL-1 beta amplifies bradykinin-induced prostaglandin E2 production via a phospholipase D-linked mechanism.

The proinflammatory cytokine IL-1 was shown to increase the responsiveness of synovial cells to the potent inflammatory peptide bradykinin (BK). We have investigated the biochemical events linked to this amplifying action of IL-1. Stimulation of synoviocytes with only BK elicited a rapid increase in inositol phosphates and a concomitant accumulation of diacylglycerol (DAG), monoacylglycerol, and free arachidonic acid (AA). In contrast, IL-1 did not stimulate any of these events. Thus, BK can induce AA release via the hydrolysis of phosphatidylinositols by a phospholipase C (PLC). BK also activated a phospholipase D (PLD) to cleave phosphatidylcholine (PC), because it caused an increase in phosphatidic acid (PA) content and a sustained DAG formation, which both were inhibited by ethanol in [3H]myristic acid-labeled cells. Moreover, the addition of ethanol diverted PLD into the formation of phosphatidylethanol (PEt) thus inhibiting the amounts of PA and DAG formed. Priming of synovial cells with rIL-1 beta 24 h before exposure to BK in the presence of ethanol further enhanced the BK-induced formation of PEt. Conversely, preincubation with IL-1 did not influence the BK-induced PLC activation nor did it alter the liberation of AA. Finally, we demonstrated that the IL-1-mediated amplification of PGE2 release in response to BK was reduced by the presence of ethanol in the culture medium, suggesting that part of the synergistic action of IL-1 and BK on prostanoid production was dependent on the activation of the PC-specific PLD pathway.

Temporal regulation of the IgE-dependent 1,2-diacylglycerol production by tyrosine kinase activation in a rat (RBL 2H3) mast-cell line.

We explored the possible role of tyrosine kinases in the IgE-dependent regulation of 1,2-diacylglycerol (DAG) production in RBL 2H3 cells. When triggered via their high-affinity IgE receptors (Fc epsilon RI), there was a rapid phosphorylation of tyrosine residues on a number of proteins. The phosphorylation of these proteins and ultimately histamine release were inhibited in a concentration-dependent manner by the tyrosine kinase inhibitor, tyrphostin. In cells labelled with [3H]myristic acid, we observed a characteristic biphasic increase in [3H]DAG production. In the presence of tyrosine kinase inhibitor, the initial increase in DAG was still observed, but the secondary increase, which was dependent on phosphatidylcholine-specific phospholipase D (PC-PLD) activation, was completely abolished. Tyrphostin significantly inhibited IgE-dependent activation of PC-PLD, suggesting that PC-PLD activation was regulated by tyrosine phosphorylation. Furthermore, when proteins from RBL 2H3 cells were immunoprecipitated with an anti-phosphotyrosine antibody, PC-PLD activity was recovered from the immunoprecipitated fraction. These results demonstrate that the secondary, but not the initial, phase of 1,2-DAG production in response to Fc epsilon RI aggregation is regulated by the initial activation of tyrosine kinases and that PC-PLD may be regulated directly by this mechanism.

Interaction between Phosphoinositide-Phospholipase C and Phosphatidylcholine- Phospholipase D in Plasma Membranes of Retinal Capillary Pericytes

The existence of phosphatidylcholine-specific phospholipase D (PC-PLD) was demonstrated in the isolated plasma membranes of retinal capillary pericytes. We determined PC-PLD activity, taking advantage of the transphosphatidylation activity of PC-PLD in the presence of ethanol, with phosphatidylethanol (PEt) formation. In the membrane environment, both phosphoinositide-specific phospholipase C (PI-PLC) and PC-PLD were activated by GTP gamma S. Neomycin, an inhibitor of PI-PLC, at a concentration (500 microM) capable of inhibiting 90% of the PI-PLC activity, only slightly inhibited the basal level of PC-PLD, but significantly decreased the GTP gamma S-activated PC-PLD by 66%. These findings indicate that the inhibitory effects of neomycin on PC hydrolysis may exert through the regulatory mechanisms of G proteins. Exogenous phosphatidic acid, a product of PC-PLD, stimulated, whereas PC, the substrate of PC-PLD, inhibited PI-PLC. These data implicate one mechanism by which PC-PLD may modulate PI-PLC activity through changing the phospholipid composition in the membrane, specifically the ratio of PA/PC. These findings are useful for the further studies on the communication between membrane-associated PI-PLC- and PC-PLD-mediated signal transduction pathways.

Endothelin activation of phospholipase D: dual modulation by protein kinase C and Ca2+.

Previous work from this laboratory has identified an endothelin (ET) type A (ETA) receptor on cultured rat renal medullary interstitial cells (RMIC), coupled to phosphatidylinositol-specific phospholipase C (PI-PLC), dihydropyridine-insensitive receptor-operated Ca2+ channels, and phospholipase A2. The current studies explored a role for ET stimulation of phosphatidylcholine-specific phospholipase D (PC-PLD) in intracellular signaling of this cell type. ET stimulated PLD activation, as measured by phosphatidic acid (PA) or phosphatidylethanol (PEt) accumulation, in a time- and concentration-dependent manner. Inhibition of diacylglycerol (DAG) kinase by ethylene glycol dioctanoate or 6-(2)4-[(4-fluorophenyl)-phenylmethylene]-1-piperadinyl]ethy l-7-methyl-5H - thiaxolo-[3,2-alpyrimidin]-5-one (R 59022) failed to blunt PA accumulation, indicating that PLD, and not DAG, was the source of PA. Inhibition of PA phosphohydrolase (PAP) by propranolol increased late accumulation of PA, suggesting that the prevailing metabolic flow was in the direction of PA to DAG. Phorbol 12-myristate 13-acetate (PMA) augmented ET-evoked PEt accumulation, whereas downregulation of protein kinase C (PKC) obviated agonist-induced PEt production. PMA augmentation of PLD activity proceeded independent of cytosolic free Ca2+ concentration. Ca2+ derived from either intracellular or extracellular sources enhanced ET-related PEt accumulation but was without effect in PKC-downregulated cells. Collectively, these observations indicate that ET stimulates PLD production in RMIC. PKC is the major regulator of this process, with Ca2+ playing a secondary, modulatory role. In addition, these data suggest that PC-PLD is coupled to the ETA receptor.

Phosphatidylcholine-specific phospholipase D-derived 1,2-diacylglycerol does not initiate protein kinase C activation in the RBL 2H3 mast-cell line.

We examined the role of phosphatidylcholine-specific phospholipase D (PC-PLD) in the IgE-dependent activation of protein kinase C (PKC) in RBL 2H3 cells (a model for mast-cell function). Cells were sensitized with mouse monoclonal anti-trinitrophenol (TNP) IgE (0.5 micrograms/ml) and were then triggered with an optimal concentration (10 ng/ml) of TNP-ovalbumin conjugate (TNP-OVA). This resulted in an immediate biphasic increase in the production of 1,2-diacylglycerol (DAG) and activation of PKC. The initial increase in DAG production reached a peak within 30 s, and the second phase reached a plateau within 5 min after stimulation. TNP-OVA-induced PC-PLD activation followed the initial increase in DAG formation in response to IgE-receptor cross-bridging, but coincided with the second peak. Phosphatidic acid (PA), derived from the PC-PLD pathway, is metabolized to DAG by the action of PA phosphohydrolase (PAPase). Propranolol (0.3 mM), which inhibits PAPase, blocked the IgE-dependent increase in DAG, activation of PKC, and subsequently degranulation. The PKC inhibitor staurosporine (0.1 microM) inhibited the second, but not first, peak of DAG accumulation, reversed PKC translocation after 10 min and inhibited subsequent mediator release. Taken together, these results demonstrate that PC-PLD does not initiate, but may play a latent role in, IgE-dependent DAG production, PKC activation and mediator release from RBL 2H3 cells.

Differential effects of propranolol on responses to receptor-dependent and receptor-independent stimuli in human neutrophils

Recent evidence suggests that the hydrolysis of phosphatidylcholine (PC) by phospholipase D (PLD) may mediate superoxide anion (O 2 −) production in human neutrophils. To define the role of the PC-specific PLD products phosphatidic acid (PA) and diacylglycerol (DAG) in O 2 − production in response to agonists which activate the PLD pathway, we blocked the metabolism of PA to DAG with propranolol, an inhibitor of PA phosphohydrolase. Propranolol (150 μM) enhanced the production of O 2 − in response to the receptor agonists n-formyl-methionyl-leucyl-phenylalanine (FMLP, 292 ± 94% of controls), platelet-activating factor (PAF, 932 ± 215%) and leukotriene B 4 (LTB 4, 1305 ± 475%). In the presence of propranolol, total O 2 − production in response to PAF and LTB 4, which are potent priming stimuli but very weak direct agonists, was similar to that obtained with FMLP. In contrast, responses to receptor-independent agonists phorbol myristate acetate (PMA) and ionomycin were inhibited (81 ± 8% and 87 ± 5% inhibition, respectively). The effects of propranolol were demonstrable in the absence of cellular calcium and were shared by both stereoisomers of the drug. These data are consistent with the hypothesis that PA produced through the hydrolysis of PC by PLD is an important mediator of O 2 − production in response to receptor-dependent agonists. However, the inhibitory effects of propranolol on receptor-independent stimuli suggest that PA generated through the PLD pathway plays a different role in the signal transduction mechanisms of these agonists or that propranolol may have additional effects beyond inhibition of PA phosphohydrolase.

Phospholipase D in homogenates from HL-60 granulocytes: Implications of calcium and G protein control

Occupancy of chemotactic peptide receptors leads to rapid initiation of phospholipase D (PLD) activity in intact dimethylsulfoxide-differentiated HL-60 granulocytes (Pai, J.-K, Siegel, M.I., Egan, R.W., and Billah, M.M. (1988) J. Biol. Chem. 263 , 12472). To gain further insight into the activation mechanisms, PLD has been studied in cell lysates from HL-60 granulocytes, using 1-0-alkyl-2-oleoyl-[ 32P]phosphatidylcholine (alkyl-[ 32P]PC), 1-0-[ 3H]alkyl-2-oleoyl-phosphatidylcholine ([ 3H]alkyl-PC) and [ 14C]arachidonyl-phospholipids as substrates. In the presence of Ca 2+ and GTP γS, post-nuclear homogenates degrade alkyl-[ 32P]PC to produce 1-0-alkyl-[ 32P]phosphatidic acid (alkyl-[ 32P]-PA), and in the presence of ethanol, also 1-0-alkyl-[ 32P]phosphatidylethanol (alkyl-[ 32P]PEt). By comparing the 3H/ 32P ratios of PA and PEt to that of PC, it is concluded that PA and PEt are formed exclusively by a PLD that catalyzes both hydrolysis and transphosphatidylation between PC and ethanol. Furthermore, PC containing either ester- or ether-linkage at the sn -1 position is degraded in preference to phosphatidylethanolamine and phosphatidylinositol by PLD in HL-60 cell homogenates. It is concluded that HL-60 granulocytes contain a PC-specific PLD that requires both Ca 2+ and GTP for activation.