Involvement of group VIA calcium-independent phospholipase A2 in macrophage engulfment of hydrogen peroxide-treated U937 cells

Hydrogen peroxide-induced apoptosis of U937 cells results in substantial hydrolysis of membrane phospholipids by calcium-independent group VIA phospholipase A(2) (iPLA(2)-VIA). However, abrogation of cellular iPLA(2)-VIA neither delays nor decreases apoptosis, suggesting that, beyond a mere destructive role, iPLA(2)-VIA may serve other specific roles. In this study, we report that phagocytosis of apoptosing U937 cells by macrophages is blunted if the cells are depleted of iPLA(2)-VIA by treatment with an inhibitor or an antisense oligonucleotide, and it is augmented by overexpression of iPLA(2)-VIA in the dying cells. Thus, the magnitude of macrophage phagocytosis correlates with the level of iPLA(2)-VIA activity of the dying cells. Eliminating by antisense oligonucleotide technology of cytosolic group IVA phospholipase A(2) does not attenuate phagocytosis of U937 dying cells by macrophages. Incubation of U937 cells with different fatty acids has no effect on either the extent of hydrogen peroxide-induced apoptosis or the degree of phagocytosis of the dying cells by macrophages. However, preincubation of the macrophages with lysophosphatidylcholine before exposing them to the dying cells blocks phagocytosis of the latter. These results indicate that formation of lysophosphatidylcholine by iPLA(2)-VIA in hydrogen peroxide-treated U937 cells to induce apoptosis directly contributes to their efficient clearance by macrophages.     

Involvement of calcium-independent phospholipase A2 in hydrogen peroxide-induced accumulation of free fatty acids in human U937 cells

Previous studies have demonstrated that U937 cells are able to mobilize arachidonic acid (AA) and synthesize prostaglandins in response to receptor-directed and soluble stimuli by a mechanism that involves the activation of Group IV cytosolic phospholipase A(2)alpha. In this paper we show that these cells also mobilize AA in response to an oxidative stress induced by H(2)O(2) through a mechanism that appears not to be mediated by cytosolic phospholipase A(2)alpha but by the calcium-independent Group VI phospholipase A(2) (iPLA(2)). This is supported by the following lines of evidence: (i) the response is essentially calcium-independent, (ii) it is inhibited by bromoenol lactone, and (iii) it is inhibited by an iPLA(2) antisense oligonucleotide. Enzyme assays conducted under a variety of conditions reveal that the specific activity of the iPLA(2) does not change as a result of H(2)O(2) exposure, which argues against the activation of a specific signaling cascade ending in the iPLA(2). Rather, the oxidant acts to perturb membrane homeostasis in a way that the enzyme susceptibility/accessibility to its substrate increases, and this results in altered fatty acid release. In support of this view, not only AA, but also other fatty acids, were found to be liberated in an iPLA(2)-dependent manner in the H(2)O(2)-treated cells. Collectively, these studies underscore the importance of the iPLA(2) in modulating homeostatic fatty acid deacylation reactions and document a potentially important route under pathophysiological conditions for increasing free fatty acid levels during oxidative stress.     

Blockade of arachidonic acid incorporation into phospholipids induces apoptosis in U937 promonocytic cells

Arachidonic acid (AA) participates in a reacylation/deacylation cycle of membrane phospholipids, the so-called Lands cycle, that serves to keep the concentration of this free fatty acid in cells at a very low level. To manipulate the intracellular AA level in U937 phagocytes, we have used several pharmacological strategies to interfere with the Lands cycle. We used inhibitors of the AA reacylation pathway, namely thimerosal and triacsin C, which block the conversion of AA into arachidonoyl-CoA, and a CoA-independent transacylase inhibitor that blocks the movement of AA within phospholipids. In addition, we used cells overexpressing group VIA phospholipase A(2), an enzyme with key roles in controlling basal fatty acid deacylation reactions in phagocytic cells. All of these different strategies resulted in the expected increase of cellular free AA but also in the induction of cell death by apoptosis. Moreover, when used in combination with any of the aforementioned drugs, AA itself was able to induce apoptosis at doses as low as 10 muM. Blocking cyclooxygenase or lipoxygenases had no effect on the induction of apoptosis by AA. Collectively, these results indicate that free AA levels within the cells may provide an important cellular signal for the onset of apoptosis and that perturbations of the mechanisms controlling AA reacylation, and hence free AA availability, may decisively affect cell survival.     

Studies of the role of group VI phospholipase A2 in fatty acid incorporation, phospholipid remodeling, lysophosphatidylcholine generation, and secretagogue-induced arachidonic acid release in pancreatic islets and insulinoma cells.

An 84-kDa group VI phospholipase A2 (iPLA2) that does not require Ca2+ for catalysis has been cloned from Chinese hamster ovary cells, murine P388D1 cells, and pancreatic islet beta-cells. A housekeeping role for iPLA2 in generating lysophosphatidylcholine (LPC) acceptors for arachidonic acid incorporation into phosphatidylcholine (PC) has been proposed because iPLA2 inhibition reduces LPC levels and suppresses arachidonate incorporation and phospholipid remodeling in P388D1 cells. Because islet beta-cell phospholipids are enriched in arachidonate, we have examined the role of iPLA2 in arachidonate incorporation into islets and INS-1 insulinoma cells. Inhibition of iPLA2 with a bromoenol lactone (BEL) suicide substrate did not suppress and generally enhanced [3H]arachidonate incorporation into these cells in the presence or absence of extracellular calcium at varied time points and BEL concentrations. Arachidonate incorporation into islet phospholipids involved deacylation-reacylation and not de novo synthesis, as indicated by experiments with varied extracellular glucose concentrations and by examining [14C]glucose incorporation into phospholipids. BEL also inhibited islet cytosolic phosphatidate phosphohydrolase (PAPH), but the PAPH inhibitor propranolol did not affect arachidonate incorporation into islet or INS-1 cell phospholipids. Inhibition of islet iPLA2 did not alter the phospholipid head-group classes into which [3H]arachidonate was initially incorporated or its subsequent transfer from PC to other lipids. Electrospray ionization mass spectrometric measurements indicated that inhibition of INS-1 cell iPLA2 accelerated arachidonate incorporation into PC and that inhibition of islet iPLA2 reduced LPC levels by 25%, suggesting that LPC mass does not limit arachidonate incorporation into islet PC. Gas chromatography/mass spectrometry measurements indicated that BEL but not propranolol suppressed insulin secretagogue-induced hydrolysis of arachidonate from islet phospholipids. In islets and INS-1 cells, iPLA2 is thus not required for arachidonate incorporation or phospholipid remodeling and may play other roles in these cells.     

A requirement for calcium-independent phospholipase A2 in thrombin-induced arachidonic acid release and growth in vascular smooth muscle cells

Thrombin is a potent mitogen for vascular smooth muscle cells (VSMC). To understand its mitogenic signaling events, we have studied the role of calcium-independent phospholipase A2 (iPLA2). Without affecting its levels, thrombin increased iPLA2 activity in a time-dependent manner in VSMC. Thrombin also induced arachidonic acid release and DNA synthesis by about 2-fold as compared with control. Down-regulation of iPLA2 activity by its specific inhibitor, bromoenol lactone, or its expression by antisense oligonucleotides, significantly reduced thrombin-induced arachidonic acid release and DNA synthesis in VSMC. To learn the mechanism of thrombin-stimulated iPLA2 activity, we next tested the role of p38 MAPK. Thrombin stimulated p38 MAPK phosphorylation and activity in a time-dependent manner in VSMC. Inhibition of p38 MAPK activity by SB203580 and SB202190 resulted in decreased iPLA2 activity, arachidonic acid release, and DNA synthesis induced by thrombin in VSMC. Together, these results for the first time demonstrate that iPLA2 plays a role in thrombin-induced arachidonic acid release and growth in VSMC and that these responses are mediated by p38 MAPK.     

Group IV cytosolic phospholipase A2 mediates arachidonic acid release in H9c2 rat cardiomyocyte cells in response to hydrogen peroxide

Damaging reactive oxygen species are released during episodes of ischemia and reperfusion. Some cellular adaptive responses are triggered to protect the injured organ, while other cascades are triggered which potentiate the damage. In these studies, we demonstrate that rat cardiomyocte H9c2 cells release arachidonic acid in response to hydrogen peroxide. In H9c2 cells, arachidonic acid release is attenuated by methyl arachidonyl fluorophosphonate (MAFP) and pyrrophenone, indicating that a phospholipase A2 Group IV enzyme mediates arachidonic acid mobilization. Moreover, hydrogen peroxide alters the cellular morphology of the H9c2 cells, causing drastic cell shrinkage. Because MAFP and pyrrophenone prevent the morphological alterations caused by hydrogen peroxide, these studies show that phospholipase A2 Group IV activity is likely integrally involved in the damage initiated by hydrogen peroxide.     

Cellular regulation and proposed biological functions of group VIA calcium-independent phospholipase A2 in activated cells

Mammalian cells contain several calcium-independent phospholipase A2 (PLA2) enzymes. The best studied of them is the so-called Group VIA PLA2 (iPLA2-VIA), which is an 85-88 kDa enzyme with unique structural features among the PLA2 superfamily of enzymes, and has been found to play a key role in homeostatic membrane phospholipid metabolism in various cell types. Growing evidence suggests that, in addition to its homeostatic function, iPLA2-VIA may also play distinct roles in cellular signaling. This review focuses on the biochemical mechanisms that regulate the activity of iPLA2-VIA in activated cells, and the biological functions proposed for this enzyme during stimulus-response coupling.     

Involvement of hydrogen peroxide in mistletoe lectin-II-induced apoptosis of myeloleukemic U937 cells

Mistletoe lectin-II, a major component of Korean mistletoe (Viscum album var. coloratum) induces apoptotic death in cancer cells. In this study, we demonstrated that lectin-II induced the generation of pro-oxidants and thus resulted in the apoptotic death of human myeloleukemic U937 cells. We observed that lectin-II-induced apoptotic death was inhibited by antioxidants including reduced glutathione (GSH), N-acetylcysteine (NAC), ebselen, mnTBP, catalase and pyrrolidine dithiocarbamate (PDTC). GSH and NAC also abolished the apoptotic DNA ladder pattern fragmentation of U937 cells after lectin-II stimulation. Obviously, lectin-II treatment of cells resulted in a remarkable generation of intracellular hydrogen peroxide (H2O2) as an early event, which was monitored fluorimetrically using scopoletin-horse radish peroxidase (HRP) assay and peroxide-sensitive fluorescent probe, DCF-DA. In addition, antioxidants inhibited the activation of c-Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK) as well as cytosolic release of cytochrome c by mistletoe lectin-II. Moreover, lectin-II-induced activation of caspase-9 and 3-like protease and cleavage of poly(ADP-ribose) polymerase (PARP) were inhibited by pretreatment of cells with thiol antioxidants, GSH and NAC. Taken together, these results suggest that Korean mistletoe lectin-II is a strong inducer of pro-oxidant generation such as H2O2, which mediates the JNK/SAPK activation, cytochrome c release, activation of caspase-9 and caspase 3-like protease, and PARP cleavage in human myeloleukemic U937 cells.     

A calcium-independent phospholipase activity insensitive to bromoenol lactone mediates arachidonic acid release by lindane in rat myometrial cells.

Arachidonic acid release is an important regulatory component of uterine contraction and parturition, and previous studies showed that lindane stimulates arachidonic acid release from myometrium. The present study partially characterized the enzyme activity responsible for lindane-induced arachidonic acid release in myometrial cells. Lindane released arachidonic acid from cultured rat myometrial cells in concentration- and time-dependent manners. This release was primarily from phosphatidylcholine and phosphatidylinositol, and was independent of intracellular and extracellular calcium. In cells prelabeled with [3H]arachidonic acid, 85% of radiolabel was recovered as free arachidonate and only 5% was recovered as eicosanoids. Pretreatment with the antioxidants Cu, Zn-superoxide dismutase, alpha-tocopherol or Trolox did not significantly modify lindane-induced arachidonic acid release. Pretreatment of cells with the phosphatidylcholine-specific phospholipase C inhibitor D609, phosphatidylinositol-specific phospholipase C inhibitor ET-18-OCH3, or an interrupter of the phospholipase D pathway (ethanol) did not suppress lindane-induced arachidonic acid release. Although these results are consistent with calcium-independent phospholipase A2 activation by lindane, the calcium-independent phospholipase A2 inhibitor bromoenol lactone failed to inhibit lindane-induced arachidonic acid release in myometrial cells, even though bromoenol lactone effectively blocked arachidonic acid release in neutrophils. These results suggest that myometrial cells express a novel, previously unidentified phospholipase that is arachidonate-specific, calcium-independent, insensitive to bromoenol lactone, insensitive to reactive oxygen species activation, shows substrate preference for phosphatidylcholine and phosphatidylinositol, and is stimulated by lindane. Moreover, the data show that the overwhelming majority of arachidonic acid released remains as arachidonate, but that lindane does not significantly inhibit metabolism of arachidonate to eicosanoids.     

Dexamethasone-induced stimulation of arachidonic acid release by U937 cells grown in defined medium

Since the presence of serum in culture media has been shown to alter prostaglandin production, as well as to interfere with the action of anti-inflammatory drugs, we have studied the effect of dexamethasone, a potent steroidal anti-inflammatory drug, on the metabolism of arachidonic acid by human monocyte-like cells (U937) grown in a fully defined medium. Under these culture conditions, dexamethasone (10(-6) M, 24 h) induced a marked stimulation of the release of unmetabolized arachidonic acid into the culture medium. The steroid also induced an inhibition of cell proliferation which became significant only after 48 h of treatment. The accumulation of arachidonic acid in the medium after steroid treatment was associated with a significant inhibition of cell acyltransferase activity, suggesting that steroids may also act upon arachidonic acid metabolism at sites other than those of phospholipase activity.     

Smooth muscle cell arachidonic acid release, migration, and proliferation are markedly attenuated in mice null for calcium-independent phospholipase A2beta

Pharmacologic evidence suggests that the lipid products generated by one or more calcium-independent phospholipases A(2) (iPLA(2)s) participate in the regulation of vascular tone through smooth muscle cell (SMC) Ca(2+) signaling and the release of arachidonic acid. However, the recent identification of new members of the iPLA(2) family, each inhibitable by (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one, has rendered definitive identification of the specific enzyme(s) mediating these processes difficult. Accordingly, we used iPLA(2)beta(-/-) mice to demonstrate that iPLA(2)beta is responsible for the majority of thapsigargin and ionophore (A23187)-induced arachidonic acid release from SMCs. Both thapsigargin and A23187 stimulated robust [(3)H]arachidonate (AA) release from wild-type aortic SMCs that was dramatically attenuated in iPLA(2)beta(-/-) mice (>80% reduction at 5 min; p < 0.01). Moreover, iPLA(2)beta(-/-) mice displayed defects in SMC Ca(2+) homeostasis and decreased SMC migration and proliferation in a model of vascular injury. Ca(2+)-store depletion resulted in the rapid entry of external Ca(2+) into wild-type aortic SMCs that was significantly slower in iPLA(2)beta-null cells (p < 0.01). Furthermore, SMCs from iPLA(2)beta-null mesenteric arterial explants demonstrated decreased proliferation and migration. The defects in migration and proliferation in iPLA(2)beta-null SMCs were restored by 2 mum AA. Remarkably, the cyclooxygenase-2-specific inhibitor, NS-398, prevented AA-induced rescue of SMC migration and proliferation in iPLA(2)beta(-/-) mice. Moreover, PGE(2) alone rescued proliferation and migration in iPLA(2)beta(-/-) mice. We conclude that iPLA(2)beta is an important mediator of AA release and prostaglandin E(2) production in SMCs, modulating vascular tone, cellular signaling, proliferation, and migration.     

The expression and function of a group VIA calcium-independent phospholipase A2 (iPLA2beta) in beta-cells

Many cells express a Group VIA phospholipase A2, designated iPLA2beta, that does not require calcium for activation, is stimulated by ATP, and is sensitive to inhibition by a bromoenol lactone suicide substrate (BEL). Studies in various cell systems have led to the suggestion that iPLA2beta has a role in phospholipid remodeling, signal transduction, cell proliferation, and apoptosis. We have found that pancreatic islets, beta-cells, and glucose-responsive insulinoma cells express an iPLA2beta that participates in glucose-stimulated insulin secretion but is not involved in membrane phospholipid remodeling. Additionally, recent studies reveal that iPLA2beta is involved in pathways that contribute to beta-cell proliferation and apoptosis, and that various phospholipid-derived mediators are involved in these processes. Detailed characterization of the enzyme suggests that the beta-cells express multiple isoforms of iPLA2beta, and we hypothesize that these participate in different cellular functions.     

Dual stimulation of phospholipase activity in human monocytes. Role of calcium-dependent and calcium-independent pathways in arachidonic acid release and eicosanoid formation

Human peripheral blood monocytes, prelabeled with [3H]arachidonic acid (AA), release labeled eicosanoids in response to soluble or particulate stimuli. Treatment with 12-O-tetradecanoate phorbol-13 acetate (20 nM), calcium ionophores, A23187 (2 microM) or ionomycin (1 microM), or serum-treated zymosan (300 micrograms) resulted in production of cyclooxygenase (CO) metabolites, 6-keto-PG-F1 alpha, thromboxane-B2, PGE2, PGF2 alpha, PGD2, PGB2, 12-L-hydroxy-5,8,10-heptadecatrienoic acid; 15-lipoxygenase products, including 15-hydroxyeicosatetraenoic acid (HETE); and unmetabolized AA. Labeled 5-lipoxygenase (LO) products, 5-HETE, and leukotriene-B4 were detected only after exposure to ionophore or serum-treated zymosan. The calcium dependence of 5-LO activation was confirmed in experiments where calcium was omitted from the incubation medium, and EGTA (0.5 mM) was added, as well as by direct measurement of increased intracellular calcium in phagocytosing monocytes. Combined or sequential treatment with two stimuli increased the release of unmetabolized AA without a commensurate augmentation of labeled metabolites, indicating that release of CO and LO metabolites does not necessarily reflect the extent of phospholipase activation. Quantitation of individual eicosanoids by RIA confirmed results by using radionuclides. These studies show the following. Activation of human monocyte phospholipase may be regulated by at least two pathways, one "12-O-tetradecanoate phorbol-13 acetate-like," which is largely independent of calcium, and another which is mediated by increased intracellular Ca2+ ("ionophore-like"). "Physiologic" stimulation of monocyte arachidonate release, such as that seen accompanying phagocytosis of opsonized particles, may occur via either a calcium-sensitive or calcium-insensitive pathway or both. Calcium may regulate eicosanoid formation at the level of phospholipase or 5-LO. Free AA, CO products, and 12- or 15-LO products are ordinarily released after phagocytosis, but leukotriene-B4, 5-HETE, or other 5-LO metabolites are produced only under conditions where calcium concentrations are optimal.     

Intracellular actions of group IIA secreted phospholipase A2 and group IVA cytosolic phospholipase A2 contribute to arachidonic acid release and prostaglandin production in rat gastric mucosal cells and transfected human embryonic kidney cells

Gastric epithelial cells liberate prostaglandin E(2) in response to cytokines as part of the process of healing of gastric lesions. Treatment of the rat gastric epithelial cell line RGM1 with transforming growth factor-alpha and interleukin-1beta leads to synergistic release of arachidonate and production of prostaglandin E(2). Results with highly specific and potent phospholipase A(2) inhibitors and with small interfering RNA show that cytosolic phospholipase A(2)-alpha and group IIA secreted phospholipase A(2) contribute to arachidonate release from cytokine-stimulated RGM1 cells. In the late phase of arachidonate release, group IIA secreted phospholipase A(2) is induced (detected at the mRNA and protein levels), and the action of cytosolic phospholipase A(2)-alpha is required for this induction. Results with RGM1 cells and group IIA secreted phospholipase A(2)-transfected HEK293 cells show that the group IIA phospholipase acts prior to externalization from the cells. RGM1 cells also express group XIIA secreted phospholipase A(2), but this enzyme is not regulated by cytokines nor does it contribute to arachidonate release. The other eight secreted phospholipases A(2) were not detected in RGM1 cells at the mRNA level. These results clearly show that cytosolic and group IIA secreted phospholipases A(2) work together to liberate arachidonate from RGM1 cell phospholipids in response to cytokines.     

NF-kappa B mediates the adaptation of human U937 cells to hydrogen peroxide

Low doses of oxidative stress can induce cellular resistance to subsequent higher doses of the same stress. By using human U937 leukemia cells, we previously demonstrated that H(2)O(2) can induce such an adaptive response without elevating the cellular capacity to degrade H(2)O(2), and were able to confer the cells a cross-resistance to an H(2)O(2)-independent lethal stimulus, C(2)-ceramide. In this study, it was found that the adaptation is accompanied by the translocation of cytoplasmic NF-kappa B to the nuclei. This event was promoted or abolished when either IKK alpha or a dominant negative mutant of I kappa B, respectively, was overexpressed. The overexpression of IKK alpha also resulted in the suppression of H(2)O(2)-induced cell death and DNA fragmentation, whereas these events were accelerated by the expression of the I kappa B mutant. The protective effect of IKK alpha was accompanied neither by an elevation of protein levels of various antioxidant enzymes such as catalase, superoxide dismutase, and glutathione peroxidase, nor by an increase in the cellular capacity to consume H(2)O(2). Moreover, the overexpression of IKK alpha resulted in an enhancement of H(2)O(2)-induced resistance to C(2)-ceramide. The overall data suggest that NF-kappa B mediates the H(2)O(2) adaptation induced in a manner independent of H(2)O(2)-degrading activity.     

Myeloid- and hepatocyte-specific deletion of group VIA calcium-independent phospholipase A2 leads to dichotomous opposing phenotypes during MCD diet-induced NASH

Polymorphisms of phospholipase A2VIA (iPLA2β or PLA2G6) are associated with body weights and blood C-reactive protein. The role of iPLA2β/PLA2G6 in non-alcoholic steatohepatitis (NASH) is still elusive because female iPla2β-null mice showed attenuated hepatic steatosis but exacerbated hepatic fibrosis after feeding with methionine- and choline-deficient diet (MCDD). Herein, female mice with myeloid- (MPla2g6^?/?) and hepatocyte- (LPla2g6^?/?) specific PLA2G6 deletion were generated and phenotyped after MCDD feeding. Without any effects on hepatic steatosis, MCDD-fed MPla2g6^?/? mice showed further exaggeration of liver inflammation and fibrosis as well as elevation of plasma TNFα, CCL2, and circulating monocytes. Bone-marrow-derived macrophages (BMDMs) from MPla2g6^?/? mice displayed upregulation of PPARγ and CEBPα proteins, and elevated release of IL6 and CXCL1 under LPS stimulation. LPS-stimulated BMDMs from MCDD-fed MPla2g6^?/? mice showed suppressed expression of M1 Tnfa and Il6, but marked upregulation of M2 Arg1, Chil3, IL10, and IL13 as well as chemokine receptors Ccr2 and Ccr5. This in vitro shift was associated with exaggeration of hepatic M1/M2 cytokines, chemokines/chemokine receptors, and fibrosis genes. Contrarily, MCDD-fed LPla2g6^?/? mice showed a complete protection which was associated with upregulation of Ppara/PPARα and attenuated expression of Pparg/PPARγ, fatty-acid uptake, triglyceride synthesis, and de novo lipogenesis genes. Interestingly, LPla2g6^?/? mice fed with chow or MCDD displayed an attenuation of blood monocytes and elevation of anti-inflammatory lipoxin A4 in plasma and liver. Thus, PLA2G6 inactivation specifically in myeloid cells and hepatocytes led to opposing phenotypes in female mice undergoing NASH. Hepatocyte-specific PLA2G6 inhibitors may be further developed for treatment of this disease.     

Role of secretory and cytosolic phospholipase A(2) enzymes in lysophosphatidylcholine-stimulated monocyte arachidonic acid release

To determine if lysophosphatidylcholine (lysoPC) is able to induce proinflammatory changes in monocytes, its ability to stimulate arachidonic acid (AA) release, a product of phospholipase A2 (PLA(2)) activity, has been analyzed. LysoPC increased AA release in THP-1 and Mono Mac6 cells in a time- and concentration-dependent manner. The monocytes expressed both secretory and cytosolic PLA(2) enzymes and AA release was strongly reduced by cellular pretreatment with different PLA(2) inhibitors and by pertussis toxin, an inhibitor of G(i)-protein activation. This indicates that both cytosolic and secretory PLA(2) enzymes regulate specific lysoPC receptor-induced AA release, suggesting lysoPC participation in monocyte proinflammatory activation.     

Activation of the cytosolic calcium-independent phospholipase A2 β isoform contributes to TRPC6 externalization via release of arachidonic acid

During vascular interventions, oxidized low-density lipoprotein and lysophosphatidylcholine (lysoPC) accumulate at the site of arterial injury, inhibiting endothelial cell (EC) migration and arterial healing. LysoPC activates canonical transient receptor potential 6 (TRPC6) channels, leading to a prolonged increase in intracellular calcium ion concentration that inhibits EC migration. However, an initial increase in intracellular calcium ion concentration is required to activate TRPC6, and this mechanism remains elusive. We hypothesized that lysoPC activates the lipid-cleaving enzyme phospholipase A₂ (PLA₂), which releases arachidonic acid (AA) from the cellular membrane to open arachidonate-regulated calcium channels, allowing calcium influx that promotes externalization and activation of TRPC6 channels. The focus of this study was to identify the roles of calcium-dependent and/or calcium-independent PLA₂ in lysoPC-induced TRPC6 externalization. We show that lysoPC induced PLA₂ enzymatic activity and caused AA release in bovine aortic ECs. To identify the specific subgroup and the isoform(s) of PLA₂ involved in lysoPC-induced TRPC6 activation, transient knockdown studies were performed in the human endothelial cell line EA.hy926 using siRNA to inhibit the expression of genes encoding cPLA₂α, cPLA₂γ, iPLA₂β, or iPLA₂γ. Downregulation of the β isoform of iPLA₂ blocked lysoPC-induced release of AA from EC membranes and TRPC6 externalization, as well as preserved EC migration in the presence of lysoPC. We propose that blocking TRPC6 activation and promoting endothelial healing could improve the outcomes for patients undergoing cardiovascular interventions.     

Vitamin E up-regulates arachidonic acid release and phospholipase A2 in megakaryocytes

The alteration in calcium transport in the liver nuclei of rats orally administered carbon tetrachloride (CCl4) was investigated. Rats received a single oral administration of CCl₄(5, 10, and 25%, 1.0ml/100 g body weight), and 5, 24 and 48 h later the animals were sacrificed. The administration of CCl₄ (25%) caused a remarkable elevetion of calcium content in the liver tissues and the nuclei of rats. Liver nuclear Ca2+-ATPase activity was markedly decreased by CCl₄ (25%) administration. The presence of dibutyryl cyclic AMP(10^-4 and 10^-3 M) or inositol 1,4,5-trisphosphate (10^-6 and 10^-5 M) in the enzyme reaction mixture caused a significant decrease in Ca²⁺-ATPase activity in the liver nuclei obtained from normal rat, while the enzyme activity was significantly increased by calmodulin (1.0 and 2.0 g/ml). These signaling factor's effects were completely impaired in the liver nuclei obtained from CCl₄ (25%)-administered rats. DNA fragmentation in the liver nuclei obtained from CCl₄ -administered rats was significantly decreased by the presence of EGTA (2 mM) in the reaction mixture, suggesting that the endogenous calcium activates nuclear DNA fragmentation. The present study demonstrates that calcium transport system in the liver nuclei is impaired by liver injury with CCl₄ administration in rats.