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PubMed1997

Flavonoids as phospholipase A2 inhibitors: importance of their structure for selective inhibition of group II phospholipase A2.

The inhibitory effect of the plant flavonoid, rutin, on group I phospholipase A2 (PLA2-I) from porcine pancreas and Naja naja, and on group II phospholipase A2 (PLA2-II) from Vipera russelli and Crotalus atrox was investigated. Rutin efficiently inhibited PLA2-II from both Vipera russelli and Crotalus atrox but was only a weak inhibitor of PLA2-I from porcine pancreas and Naja naja. The lack of strong inhibition of pancreatic PLA2-I was not due to contaminating proteins in the enzyme preparation, since the same weak inhibition was obtained against pancreatic PLA2 purified to homogeneity as judged by two-dimensional gel electrophoresis. Rutin also efficiently inhibited human PLA2-II from synovial fluid but was only a weak inhibitor of human PLA2-I from pancreatic juice, suggesting that rutin is a selective PLA2-I from porcine pancreas. The results obtained indicate that the hydroxyl group in 5-position as well as the double bond and the double-bonded oxygen in the oxane ring are all important for the overall ability of flavonoids to inhibit PLA2 activity, and that the hydroxyl groups in 3'- and 4'- position are required for selective inhibition of PLA2-II.

Animals↗

PubMed1992

Differential pathways (phospholipase C and phospholipase D) of bradykinin-induced biphasic 1,2-diacylglycerol formation in non-transformed and K-ras-transformed NIH-3T3 fibroblasts. Involvement of intracellular Ca2+ oscillations in phosphatidylcholine breakdown.

Bradykinin (BK) induced a biphasic increase in 1,2-diacylglycerol (DAG) in both K-ras-transformed fibroblasts (DT) and the parent NIH-3T3 cells. The first phase was coincident with the increase in Ins(1,4,5)P3 resulting from PtdIns(4,5)P2 hydrolysis, and the second, sustained, phase was derived from phosphatidylcholine (PtdCho) hydrolysis. In NIH-3T3 cells, stimulation by BK induced greater production of choline than phosphocholine in [3H]choline-labelled cells and appreciable phosphatidylethanol (PtdEtOH) formation in [3H]myristic acid-labelled cells, suggesting that PtdCho was hydrolysed mainly by a phospholipase D (PLD) activity. Pretreatment with propranolol, an inhibitor of phosphatidate phosphohydrolase, markedly diminished the second DAG accumulation, supporting the above notion. In DT cells, BK induced predominantly phosphocholine generation and little PtdEtOH formation, indicating that the PtdCho hydrolysis was due to a phospholipase C (PLC) activity. The BK-induced oscillations in intracellular Ca2+ concentration ([Ca2+]i) observed in single DT cells [Fu, Sugimoto, Oki, Murakami, Okano & Nozawa (1991) FEBS Lett. 281, 263-266] were detected as a sustained [Ca2+]i elevation when assayed in a cell suspension. A receptor-operated Ca2+ channel blocker, SK&F 96365, suppressed both the BK-induced phosphocholine generation and the sustained [Ca2+]i elevation in a similar dose-dependent manner. These results thus suggested that oscillations in [Ca2+]i are involved in the activation of PtdCho-specific PLC in DT cells.

3T3 Cells↗

PubMed1997

Cytosolic phospholipase A2 and its mode of activation in human neutrophils by opsonized zymosan. Correlation between 42/44 kDa mitogen-activated protein kinase, cytosolic phospholipase A2 and NADPH oxidase.

The role of cytosolic phospholipase A2 (cPLA2) and its mode of activation by opsonized zymosan (OZ) was studied in human neutrophils in comparison with activation by PMA. The activation of cPLA2 by 1 mg/ml OZ or 50 ng/ml PMA is evidenced by its translocation to the membrane fractions on stimulation. This translocation is consistent with dithiothreitol (DTT)-resistant phospholipase A2 (PLA2) activity detected in the membranes of activated cells. Neutrophils stimulated by either OZ or PMA exhibited an immediate stimulation of extracellular-signal-regulated kinases (ERKs). The inhibition of ERKs, DTT-resistant PLA2 and NADPH oxidase activities by the MAP kinase kinase inhibitor PD-98059 indicates that ERKs mediate the activation of cPLA2 and NADPH oxidase stimulated by either OZ or PMA. The protein kinase C (PKC) inhibitor GF-109203X inhibited epidermal growth factor receptor peptide kinase activity, the release of [3H]arachidonic acid, DTT-resistant PLA2 activity and superoxide generation induced by PMA, but did not inhibit any of these activities induced by OZ. PKC activity was similarly inhibited by GF-109203X in membrane fractions separated from neutrophils stimulated by either PMA or OZ. In the presence of the tyrosine kinase inhibit orgenistein, ERKs, PLA2 and NADPH oxidase activities were inhibited in cells stimulated by OZ, whereas they were hardly affected in cells stimulated by PMA. The results suggest that the activation of cPLA2 by PMA or OZ is mediated by ERKs. Whereas PMA stimulates ERKs activity through a PKC-dependent pathway, signal transduction stimulated by OZ involves tyrosine kinase activity leading to activation of ERKs via a PKC-independent pathway.

Calcium-Calmodulin-Dependent Protein Kinases↗

PubMed2000

Phospholipase C, protein kinase C, Ca(2+)/calmodulin-dependent protein kinase II, and tyrosine phosphorylation are involved in carbachol-induced phospholipase D activation in Chinese hamster ovary cells expressing muscarinic acetylcholine receptor of Caenorhabditis elegans.

Recently, we have isolated a cDNA encoding a muscarinic acetylcholine receptor (mAChR) from Caenorhabditis elegans. To investigate the regulation of phospholipase D (PLD) signaling via a muscarinic receptor, we generated stable transfected Chinese hamster ovary (CHO) cells that overexpress the mAChR of C. elegans (CHO-GAR-3). Carbachol (CCh) induced inositol phosphate formation and a significantly higher Ca(2+) elevation and stimulated PLD activity through the mAChR; this was insensitive to pertussis toxin, but its activity was abolished by the phospholipase C (PLC) inhibitor U73122. Western blot analysis revealed several apparent tyrosine-phosphorylated protein bands after CCh treatment. The CCh-induced PLD activation and tyrosine phosphorylation were significantly reduced by the protein kinase C (PKC) inhibitor calphostin C and down-regulation of PKC and the tyrosine kinase inhibitor genistein. Moreover, the Ca(2+)-calmodulin-dependent protein kinase II (CaM kinase II) inhibitor KN62, in addition to chelation of extracellular or intracellular Ca(2+) by EGTA and BAPTA/AM, abolished CCh-induced PLD activation and protein tyrosine phosphorylation. Taken together, these results suggest that the PLC/PKC-PLD pathway and the CaM kinase II/tyrosine kinase-PLD pathway are involved in the activation of PLD through mAChRs of C. elegans.

Animals↗

PubMed1998

Functional coupling between secretory phospholipase A2 and cyclooxygenase-2 and its regulation by cytosolic group IV phospholipase A2.

Secretory phospholipase A2 (sPLA2) is the major effector involved in arachidonic acid (AA) mobilization and prostaglandin E2 (PGE2) production during stimulation of P388D1 macrophages with the inflammatory stimuli bacterial lipopolysaccharide and platelet-activating factor. We herein demonstrate that PGE2 in stimulated P388D1 cells is accounted for by the inducible cyclooxygenase (COX)-2. COX-1, though present, appears not to participate significantly in stimulus-induced PGE2 production in P388D1 macrophages. Reconstitution experiments utilizing exogenous recombinant sPLA2 demonstrate that activation of the sPLA2 at the plasma membrane is highly dependent on previous activation of the cytosolic phospholipase A2 (cPLA2). Collectively these results demonstrate (i) that functional coupling exists between sPLA2 and COX-2 in activated cells, (ii) the critical role that cPLA2 plays in lipid mediator production, and (iii) that there is crosstalk between cPLA2 and sPLA2 in the cell.

Animals↗

PubMed1996

Phosphatidylinositol phospholipase C is activated allosterically by the aminoglycoside G418. 2-deoxy-2-fluoro-scyllo-inositol-1-O-dodecylphosphonate and its analogs inhibit glycosylphosphatidylinositol phospholipase C.

Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus is inhibited by myo-inositol-1-O-dodecylphosphonate (Ins-1-O-dodecylphosphonate) (Morris, J. C., Ping-Sheng, L., Shen, T. Y., and Mensa-Wilmot, K.(1995) J. Biol. Chem. 270, 2517-2524). A set of novel fluorinated 2-deoxy-Ins-1-O-dodecylphosphonates were tested against PI-PLC, with potent competitive inhibition by 2-deoxy-2-fluoro-scyllo-Ins-1-O-dodecylphosphonate (VP-616L) (Xi(50) = 0.09). 2-Deoxy-2-fluoro-myo-Ins-1-O-dodecylphosphonate and 2-deoxy-2,2-difluoro-myo-Ins-1-O-dodecylphosphonate were 8.3-fold and 4.8-fold less effective, respectively, than VP-616L. Methyl 2-deoxy-2,2-difluoro-myo-Ins-1-O-dodecylphosphonate was inactive. Also, a hundredfold less PI-PLC is required to cleave a glycosylphosphatidylinositol (GPI) than is needed to cleave PI. Implied in these observations are the following: (i) in powerful inhibitors an active site residue probably interacts with the equatorially oriented fluoro substituent; (ii) substrate recognition requires a negative charge on the phosphoryl at the Ins-1 position, and (iii) a GPI is better substrate than PI, for PI-PLC. Aminoglycoside antibiotics kanamycin A, gentamycin, and G418 stimulated PI-PLC cleavage of the GPI anchor of variant surface glycoprotein (VSG) from Trypanosoma brucei 2- to 4-fold. G418, which appears to act on the enzyme.substrate complex, increased kcat and Km 6.4-fold and 9.9-fold, respectively. PI-PLC was activated by G418 even in the presence of the inhibitor VP-616L. In control experiments, the lectin concanavalin A (ConA), which probably acts by substrate sequestration, inhibited both PI-PLC (Xi(50) = 0.00025) and GPI-specific phospholipase D (Xi(50) = 0.00018). G418 failed to activate PI-PLC when ConA was present. These observations indicate that G418 is an allosteric activator of Bacillus cereus PI-PLC. Since G418 stimulates a purified enzyme that is not involved in aminoglycoside metabolism, we propose that binding of aminoglycosides to cellular proteins could contribute to the development of the nephrotoxicity associated with the use of these aminoglycoside antibiotics.

Allosteric Regulation↗

PubMed1997

Prostaglandin E2 amplifies cytosolic phospholipase A2- and cyclooxygenase-2-dependent delayed prostaglandin E2 generation in mouse osteoblastic cells. Enhancement by secretory phospholipase A2.

We used the MC3T3-E1 cell line, which originates from C57BL/6J mouse that is genetically type IIA secretory phospholipase A2 (sPLA2)-deficient, to reveal the type IIA sPLA2-independent route of the prostanglandin (PG) biosynthetic pathway. Kinetic and pharmacological studies showed that delayed PGE2 generation by this cell line in response to interleukin (IL)-1beta and tumor necrosis factor alpha (TNFalpha) was dependent upon cytosolic phospholipase A2 (cPLA2) and cyclooxygenase (COX)-2. Expression of these two enzymes was reduced by cPLA2 or COX-2 inhibitors and restored by adding exogenous arachidonic acid or PGE2, indicating that PGE2 produced by these cells acted as an autocrine amplifier of delayed PGE2 generation through enhanced cPLA2 and COX-2 expression. Exogenous addition or enforced expression of type IIA sPLA2 significantly increased IL-1beta/TNFalpha-initiated PGE2 generation, which was accompanied by increased expression of both cPLA2 and COX-2 and suppressed by inhibitors of these enzymes. Thus, our results revealed a particular cross-talk between the two PLA2 enzymes and COX-2 for delayed PGE2 biosynthesis by a type IIA sPLA2-deficient cell line. cPLA2 is responsible for initiating COX-2-dependent delayed PGE2 generation, and sPLA2, if introduced, enhances PGE2 generation by increasing cPLA2 and COX-2 expression via endogenous PGE2.

Animals↗

PubMed1998

Replacements of single basic amino acids in the pleckstrin homology domain of phospholipase C-delta1 alter the ligand binding, phospholipase activity, and interaction with the plasma membrane.

The pleckstrin homology (PH) domain of phosphatidylinositol-specific phospholipase C-delta1 (PLC-delta1) binds to both D-myo-inositol 1,4, 5-trisphosphate (Ins(1,4,5)P3) and phosphatidylinositol 4, 5-bisphosphate (PtdIns(4,5)P2) with high affinities. We have previously identified a region rich in basic amino acids within the PH domain critical for ligand binding (Yagisawa, H., Hirata, M., Kanematsu, T., Watanabe, Y., Ozaki, S., Sakuma, K., Tanaka, H., Yabuta, N., Kamata, H., Hirata, H., and Nojima, H. (1994) J. Biol. Chem. 269, 20179-20188; Hirata, M., Kanematsu, T., Sakuma, K., Koga, T., Watanabe, Y., Ozaki, S., and Yagisawa, H. (1994) Biochem. Biophys. Res. Commun. 205, 1563-1571). To investigate the role of these basic residues, we have performed site-directed mutagenesis replacing each of the basic amino acid in the N-terminal 60 residues of PLC-delta1 (Lys24, Lys30, Lys32, Arg37, Arg38, Arg40, Lys43, Lys49, Arg56, Lys57, and Arg60) with a neutral or an acidic amino acid. The effects of these mutations on the PH domain ligand binding properties and their consequence for substrate hydrolysis and membrane interactions of PLC-delta1 were analyzed using several assay systems. Analysis of [3H]-Ins(1,4,5)P3 binding, measurement of the binding affinities, and measurements of phospholipase activity using PtdIns(4,5)P2-containing phospholipid vesicles, demonstrated that residues Lys30, Lys32, Arg37, Arg38, Arg40, and Lys57 were required for these PLC-delta1 functions; in comparison, other mutations resulted in a moderate reduction. A subset of selected mutations was further analyzed for the enzyme activity toward substrate present in cellular membranes of permeabilized cells and for interaction with the plasma membrane after microinjection. These experiments demonstrated that mutations affecting ligand binding and PtdIns(4,5)P2 hydrolysis in phospholipid vesicles also resulted in reduction in the hydrolysis of cellular polyphosphoinositides and loss of membrane attachment. All residues (with the exception of the K43E substitution) found to be critical for the analyzed PLC-delta1 functions are present at the surface of the PH domain shown to contain the Ins(1,4,5)P3 binding pocket.

Amino Acid Sequence↗

PubMed2003

Phospholipase activity of phospholipase C-gamma1 is required for nerve growth factor-regulated MAP kinase signaling cascade in PC12 cells.

Phospholipase C-gamma1 (PLC-gamma1) hydrolyzes phosphatidylinositol 4,5-bisphosphate to the second messengers inositol 1,4,5-trisphosphate and diacylglycerol (DAG). PLC-gamma1 is implicated in a variety of cellular signalings and processes including mitogenesis and calcium entry. However, numerous studies demonstrate that the lipase activity is not required for PLC-gamma1 to mediate these events. Here, we report that the phospholipase activity of PLC-gamma1 plays an essential role in nerve growth factor (NGF)-triggered Raf/MEK/MAPK pathway activation in PC12 cells. Employing PC12 cells stably transfected with an inducible form of wild-type PLC-gamma1 or lipase inactive PLC-gamma1 with histidine 335 mutated into glutamine in the catalytic domain, we show that NGF provokes robust activation of MAP kinase in wild-type but not in lipase inactive cells. Both Ras/C-Raf/MEK1 and Rap1/B-Raf/MEK1 pathways are intact in the wild-type cells. By contrast, these signaling cascades are diminished in the mutant cells. Pretreatment with cell permeable DAG analog 1-oleyl-2-acetylglycerol rescues the MAP kinase pathway activation in the mutant cells. These observations indicate that the lipase activity of PLC-gamma1 mediates NGF-regulated MAPK signaling upstream of Ras/Rap1 activation probably through second messenger DAG-activated Ras and Rap-GEFs.

Animals↗

PubMed2006

Highly selective hydrolysis of fatty acyl-CoAs by calcium-independent phospholipase A2beta. Enzyme autoacylation and acyl-CoA-mediated reversal of calmodulin inhibition of phospholipase A2 activity.

Calcium-independent phospholipase A2beta (iPLA2beta) participates in numerous diverse cellular processes, such as arachidonic acid release, insulin secretion, calcium signaling, and apoptosis. Herein, we demonstrate the highly selective iPLA2beta-catalyzed hydrolysis of saturated long-chain fatty acyl-CoAs (palmitoyl-CoA approximately myristoyl-CoA >> stearoyl-CoA >> oleoyl-CoA approximately = arachidonoyl-CoA) present either as monomers in solution or guests in host membrane bilayers. Site-directed mutagenesis of the iPLA2beta catalytic serine (S465A) completely abolished acyl-CoA thioesterase activity, demonstrating that Ser-465 catalyzes both phospholipid and acyl-CoA hydrolysis. Remarkably, incubation of iPLA2beta with oleoyl-CoA, but not other long-chain acyl-CoAs, resulted in robust stoichiometric covalent acylation of the enzyme. Moreover, S465A mutagenesis or pretreatment of wild-type iPLA2beta with (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one unexpectedly increased acylation of the enzyme, indicating the presence of a second reactive nucleophilic residue that participates in the formation of the fatty acyl-iPLA2beta adduct. Radiolabeling of intact Sf9 cells expressing iPLA2beta with [3H]oleic acid demonstrated oleoylation of the membrane-associated enzyme. Partial trypsinolysis of oleoylated iPLA2beta and matrix-assisted laser desorption ionization mass spectrometry analysis localized the acylation site to a hydrophobic 25-kDa fragment (residues approximately 400-600) spanning the active site to the calmodulin binding domain. Intriguingly, calmodulin-Ca2+ blocked acylation of iPLA2beta by oleoyl-CoA. Remarkably, the addition of low micromolar concentrations (5 microM) of oleoyl-CoA resulted in reversal of calmodulin-mediated inhibition of iPLA2 beta phospholipase A2 activity. These results collectively identify the molecular species-specific acyl-CoA thioesterase activity of iPLA2beta, demonstrate the presence of a second active site that mediates iPLA2beta autoacylation, and identify long-chain acyl-CoAs as potential candidates mediating calcium influx factor activity.

Acyl Coenzyme A↗

PubMed1998

Epidermal growth factor stimulates phospholipase D independent of phospholipase C, protein kinase C or phosphatidylinositol-3 kinase activation in immortalized rabbit corneal epithelial cells.

PURPOSE: Activation of phospholipase D (PLD) is believed to be an important signaling pathway involved in cell growth and differentiation in several tissues, in response to a variety of mitogens. The aim of the present study was to investigate the effect of epidermal growth factor (EGF) on PLD activity in rabbit corneal epithelial cells (RCEC). We have also examined whether the EGF effect is dependent on concurrent activation of phospholipase C (PLC), protein kinase C (PKC) or phosphatidylinositol 3-kinase (PI-3-kinase) in these cells. METHODS: RCEC, immortalized with adenovirus SV-40, were cultured until they became confluent. The cells were labeled with [3H]myristic acid and incubated with or without EGF or other agents for specified time intervals. PLD activity was measured by quantifying [3H]phosphatidylethanol in cells incubated in the presence of ethanol. PLC activity was determined by measuring the radioactivity in inositol trisphosphate in myo[3H]inositol-labeled RCEC. PI 3-kinase activity was assessed by measuring the production of PIP3 in 32P-labeled cells. RESULTS: Addition of EGF to RCEC stimulated PLD activity in a time- and dose-dependent manner. The maximal effect was observed with 150 ng/ml EGF and at 10 min of incubation. The PLD activity was also stimulated when phorbol myristate acetate (PMA) was added to the cells. Treatment of the cells with EGF stimulated PLC activity which was inhibited by U73122, a PLC inhibitor. Under the same experimental conditions, the inhibitor had no effect on EGF-stimulated PLD activity. Down-regulation of PKC or treatment of the cells with RO31-8220, a PKC inhibitor, inhibited the PMA- but not EGF-stimulated PLD activity. Incubation of the cells with wortmannin, a PI 3-kinase inhibitor, abolished the EGF-stimulated PI 3-kinase activity, but potentiated the EGF-stimulated PLD activity. The EGF effect was inhibited by treatment of the cells with tyrphostin B42, a receptor tyrosine kinase inhibitor. CONCLUSIONS: These results indicate that EGF stimulates PLD activity in RCEC by a mechanism that involves tyrosine phosphorylation of a protein(s) in the cascade of biochemical reactions initiated by EGF-receptor interaction, and it is not dependent on concurrent activation of PKC, PLC, or PI 3-kinase in these cells.

Animals↗

PubMed1991

Assessment of receptor-dependent activation of phosphatidylcholine hydrolysis by both phospholipase D and phospholipase C.

Enhancement of cellular phospholipase D (PLD)-1 and phospholipase C (PLC)-mediated hydrolysis of endogenous phosphatidylcholine (PC) during receptor-mediated cell activation has received increasing attention inasmuch as both enzymes can result in the formation of 1,2-diacylglycerol (DAG). The activities of PLD and PLC were examined in purified mast cells by quantitating the mass of the water-soluble hydrolysis products choline and phosphorylcholine, respectively. Using an assay based on choline kinase-mediated phosphorylation of choline that is capable of measuring choline and phosphorylcholine in the low picomole range, we quantitated the masses of both cell-associated and extracellular choline and phosphorylcholine. Activating mast cells by crosslinking its immunoglobulin E receptor (Fc epsilon-RI) resulted in an increase in cellular choline from 13.1 +/- 1.2 pmol/10(6) mast cells (mean +/- SE in unstimulated cells) to levels 5- to 10-fold higher, peaking 20 s after stimulation and rapidly returning toward baseline. The increase in cellular choline mass paralleled the increase in labeled phosphatidic acid accumulation detected in stimulated cells prelabeled with [3H]palmitic acid and preceded the increase in labeled DAG. Although intracellular phosphorylcholine levels were approximately 15-fold greater than choline in unstimulated cells (182 +/- 19 pmol/10(6) mast cells), stimulation resulted in a significant fall in phosphorylcholine levels shortly after stimulation. Pulse chase experiments demonstrated that the receptor-dependent increase in intracellular choline and the fall in phosphorylcholine were not due to hydrolysis of intracellular phosphorylcholine and suggested a receptor-dependent increase in PC resynthesis. When the extracellular medium was examined for the presence of water-soluble products of PC hydrolysis, receptor-dependent increases in the mass of both choline and phosphorylcholine were observed. Labeling studies demonstrated that these extracellular increases were not the result of leakage of these compounds from the cytosol. Taken together, these data lend support for a quantitatively greater role for receptor-mediated PC-PLD compared with PC-PLC during activation of mast cells.

Antigens, Differentiation, B-Lymphocyte↗

PubMed2002

Phospholipase C-gamma1: a phospholipase and guanine nucleotide exchange factor.

Although phospholipase C-gamma (PLC-gamma) participates in cellular mitogenesis, evidence indicates that the catalytic activity of PLC-gamma (to hydrolyze certain phosphoinositides) is nonessential to the process. So how is it that PLC-gamma is necessary but its lipase activity is not? Recently published results from Snyder and colleagues describe the ability of PLC-gamma to facilitate guanine nucleotide exchange for the recently identified nucleus-localized GTPase PIKE, which acts to enhance the enzymatic activity of phosphatidylinositol 3'-kinase (PI3K). The authors contend that the SH3 domain, rather than the catalytic domain, of PLC-gamma is required for aiding PIKE, and furthermore, that the mitogenic activity of PLC-gamma depends not on its phospholipase activity, but rather on its interaction with PIKE. Wang and Moran examine the results and piece together a picture of how PLC-gamma cooperates with PIKE.

Animals↗

PubMed1994

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.

Animals↗

PubMed2004

v-Src accelerates spontaneous motility via phosphoinositide 3-kinase, phospholipase C and phospholipase D, but abrogates chemotaxis in Rat-1 and MDCK cells.

In Rat-1 fibroblasts, v-Src causes a profound remodelling of cortical actin cytoskeleton. This transformation includes membrane ruffling, a hallmark of the leading edge in migrating cells, and results from activation of phosphoinositide 3-kinase (PI 3-kinase), phospholipase C (PLC) and phospholipase D (PLD). We therefore reexamined whether motility is constitutively triggered by v-Src and studied whether this response is controlled by the same signalling pathway. The study was performed using Rat-1/tsLA29 and MDCK/tsLA31 cells, each harbouring a different thermosensitive v-Src kinase, active at 34 degrees C but inactivated at 40 degrees C. In both cell lines, overnight v-Src activation induced transformation and accelerated spontaneous motility by approximately twofold, as evidenced by wound-healing assay and by single-cell track, time-lapse recording in Dunn chambers. Inhibitors of PI 3-kinase, PLC and PLD selectively abrogated acceleration of motility by v-Src. Since mechanisms that co-ordinate spontaneous, as distinct from oriented, cell migration are separable, we further analysed in Dunn chambers chemotactic response of Rat-1/tsLA29 cells to PDGF and of MDCK/tsLA31 cells to EGF. In both cases, v-Src decreased the steady-state level of growth factor receptors at the cell surface twofold, and abrogated movement directionality at comparable level of occupancy as in non-transformed cells. The burst of pinocytosis in response to growth factors was also abolished by v-Src. Altogether, these results indicate that v-Src triggers motility in a PI 3-kinase-, PLC- and PLD-dependent manner, but abrogates directionality by suppressing polarised signalling downstream of growth factor receptors.

Actins↗

PubMed1999

The role of membrane structure in activation of mitochondrial phospholipases. 2. Mechanism of crambin action on the activities of mitochondrial membrane phospholipases and microviscosity.

Effects of crambin on the phospholipase activity, lipid peroxidation and structure of mitochondrial membranes have been investigated. Crambin has been shown to inhibit lipid peroxidation and phospholipase activity induced by Ca2+ and freezing-thawing of mitochondria. As shown by ESR studies, these effects are based on the ability of crambin to cause changes in mitochondrial membrane structure.

Animals↗

PubMed1992

Thrombin stimulation of human endothelial cell phospholipase D activity. Regulation by phospholipase C, protein kinase C, and cyclic adenosine 3'5'-monophosphate.

The activation of membrane-bound phospholipase D (PLD) resulting in the generation of phosphatidic acid (PA) is increasingly recognized as an integral event in the initiation of a variety of cellular responses. We explored whether alpha-thrombin is a physiologic agonist for PLD activation in human umbilical vein endothelial cells (HUVEC). HUVEC monolayers were labeled with [32Pi] and PLD activity determined by formation of the PLD metabolite [32P] phosphatidylethanol (PEt) in the presence of 5 g/L ethanol by thin-layer chromatography. alpha-Thrombin rapidly (1 minute) increased PA and PEt formation in a dose-dependent manner (10(-6) to 10(-10)) with maximal PLD stimulation achieved with 10 nmol/L alpha-thrombin producing a threefold to fourfold increase in PA and a sixfold to eightfold increase in PEt over controls at 15 minutes. Esterolytically active zeta-thrombin (10 nmol/L) and gamma-thrombin (1 mumol/L), but not inactive DIP-alpha-thrombin (1 mumol/L) also increased PLD activity. The role of Ca2+ flux in human endothelial cell PLD activation was investigated and PEt formation was significantly enhanced by Ca2+ ionophores A23187 and ionomycin (1 mumol/L, three-fold to fourfold increase in PEt). Alpha-Thrombin-stimulated PEt formation was abolished (greater than 90% inhibition) with chelation of intracellular calcium (Ca2+i) by pretreatment with BAPTA-AM (25 mumol/L, 30 minutes) but only mildly attenuated (30% inhibition) by removal of extracellular calcium (Ca2+E) with EGTA (5 mmol/L). The protein kinase C (PKC) inhibitor staurosporine reduced alpha-thrombin-induced PEt formation in a dose-dependent manner (10 mumol/L, 78% inhibition) and PKC downregulation with chronic PMA treatment (18 hours) also resulted in marked inhibition of alpha-thrombin-induced PEt formation. Neither pertussis nor botulinum C bacterial toxins significantly altered alpha-thrombin-induced PLD responses. In contrast, similar pretreatment with cholera toxin (1 microgram/mL, 60 minutes) consistently augmented alpha-thrombin-stimulated PLD activity by 50% to 90%. Comparable results were observed with agents which increased cAMP such as forskolin, 8-bromo cAMP, or dibutyryl cAMP and cholera toxin augmentation was abolished by 2-dideoxyadenosine, a competitive inhibitor of adenylyl cyclase activity. These studies demonstrate that alpha-thrombin is a potent stimulus for human PLD-mediated PA formation and that cyclic adenosine nucleotides modulate agonist-induced cellular PLD activity. In this model of PLD activation, alpha-thrombin receptor occupancy leads to the breakdown of phosphatidylinositol 4,5-bisphosphate catalyzed by phospholipase C producing the Ca2+ secretagogue IP3 and DAG.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenylate Cyclase Toxin↗

PubMed1992

IL-1 increases phospholipase A2 activity, expression of phospholipase A2-activating protein, and release of linoleic acid from the murine T helper cell line EL-4.

The early events in IL-1-mediated activation of T cells were investigated in the murine T cell line, EL-4. Treatment of EL-4 cells with human rIL-1 beta resulted in a rapid increase in phospholipase A2 (PLA2) activity. PLA2 activity increased approximately fivefold within 4 min after exposure to IL-1. Synthesis of the phospholipase A2- activating protein (PLAP) and its mRNA were also increased within 4 min of IL-1 treatment and preceded the increase in PLA2 enzyme activity. The increases in PLA2 activity and PLAP protein and mRNA levels were all transient and declined to baseline within 10 min after the addition of IL-1. The changes in levels of PLAP as a function of time after IL-1 treatment were consistent with PLAP playing an important role in the regulation of PLA2 activity in this system. The consequence of the elevated PLA2 activity was examined by analysis of the fatty acids released from IL-1-treated cells. There was a 20-fold increase in the release of radioactivity from [14C]-linoleic acid labeled cells whereas there was very little change in the release of radioactivity from [14C]-arachidonic acid labeled cells in response to the addition of IL-1. The radioactivity released from [14C]-linoleic acid labeled cells was analyzed by HPLC; no conversion of radiolabeled linoleic into arachidonic acid was observed. In EL-4 cells, IL-1 potentiates PMA-mediated release of IL-2 at suboptimal concentrations of PMA. Linoleic acid also augmented PMA-induced IL-2 release from the EL-4 cells. This fatty acid was more than 10 times more effective than arachidonic acid in this regard. Furthermore, the addition of exogenous PLAP to EL-4 cells could substitute for IL-1 in the stimulation of IL-2 release. These results suggest that the IL-1 effects on T cells may be mediated at least in part through increased PLA2 activity due to increased synthesis of PLAP. Furthermore, the release of the unsaturated fatty acid linoleic acid or its metabolites may be of functional importance in IL-1-mediated IL-2 production by EL-4 cells.

Animals↗