Independent PLA Research Articles

Identification of Diagnostic Metabolic Signatures in Thyroid Tumors Using Mass Spectrometry Imaging.

"Gray zone" thyroid follicular tumors are difficult to diagnose, especially when distinguishing between benign follicular thyroid adenoma (FTA) and malignant carcinoma (FTC). Thus, proper classification of thyroid follicular diseases may improve clinical prognosis. In this study, the diagnostic performance of metabolite enzymes was evaluated using imaging mass spectrometry to distinguish FTA from FTC and determine the association between metabolite enzyme expression with thyroid follicular borderline tumor diagnosis. Air flow-assisted desorption electrospray ionization mass spectrometry imaging (AFAIDESI-MSI) was used to build a classification model for thyroid follicular tumor characteristics among 24 samples. We analyzed metabolic enzyme marker expression in an independent validation set of 133 cases and further evaluated the potential biological behavior of 19 thyroid borderline lesions. Phospholipids and fatty acids (FAs) were more abundant in FTA than FTC (p < 0.001). The metabolic enzyme panel, which included FA synthase and Ca2+-independent PLA2, was further validated in follicular thyroid tumors. The marker combination showed optimal performance in the validation group (area under the ROC, sensitivity, and specificity: 73.6%, 82.1%, and 60.6%, respectively). The findings indicate that AFAIDESI-MSI, in combination with low metabolic enzyme expression, could play a role in the diagnosis of thyroid follicular borderline tumors for strict follow-up.

Destruction of polyunsaturated alkyl/acyl and alkenyl/acyl glycerophosphocholine of plasma lipoproteins during incubation with group V and X secretory phospholipase A2s

Plasma lipoproteins are carriers of various glycerophospholipids including diacyl, alkenyl/acyl, and alkyl/acyl glycerophosphocholines (GPCs), which become distributed among cells and tissues during metabolism. For metabolic function, these phospholipids require hydrolysis by phospholipases, but the responsible enzymes have not been identified. We had previously shown that after complete digestion of lipoprotein diacyl- and oxo-diacyl-GPCs, degradation of residual alkyl/acyl and alkenyl/acyl GPCs continues, despite the fact that ether lipids are resistant to hydrolysis by Ca2+ -activated secretory PLA2 s and require the presence of the Ca2+ -independent PLA2 . In the course of further investigation, we came across a report by Khaselev and Murphy in which the autoxidative degradation of plasmalogens in the presence of 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) proceeded beyond the formation of dihydroperoxides, hydroxides and epoxides, and led to an attack on the enyl bond of the plasmalogen, resulting in formation of 1-OH/2-20:4-GPC and 1-formyl/2-20:4-GPC. Our preliminary investigation indicated that lipoprotein 16:0p/20:4ω6-GPC yielded the same autoxidation products as those reported for synthetic 16:0p/20:4ω6-GPC in the presence of AAPH. Such autoxidative degradation of lipoprotein plasmalogens had not been previously reported with or without AAPH. Subsequent study led to the conclusion that this reaction was not limited to arachidonates, but extended to other polyunsaturated eicosanoids, docosanoids, and tetracosanoids, as well as oligounsaturated octadecanoids. These observations led to a hypothesis that the autoxidative cleavage of the lipoprotein plasmalogens proceeded under the influence of apo-protein-derived free radicals as intermediates of oxidative processes.

Moderate blood alcohol and brain neurovulnerability: Selective depletion of calcium-independent phospholipase A2, omega-3 docosahexaenoic acid, and its synaptamide derivative as a potential harbinger of deficits in anti-inflammatory reserve.

Repetitive, highly elevated blood alcohol (ethanol) concentrations (BACs) of 350 to 450mg/dl over several days cause brain neurodegeneration and coincident neuroinflammation in adult rats localized in the hippocampus (HC), temporal cortex (especially the entorhinal cortex; ECX), and olfactory bulb (OB). The profuse neuroinflammation involves microgliosis, increased proinflammatory cytokines, and elevations of Ca+2 -dependent phospholipase A2 (cPLA2) and secretory PLA2 (sPLA2), which both mobilize proinflammatory ω-6 arachidonic acid (ARA). In contrast, Ca+2 -independent PLA2 (iPLA2) and anti-inflammatory ω-3 docosahexaenoic acid (DHA), a polyunsaturated fatty acid regulated primarily by iPLA2, are diminished. Furthermore, supplemented DHA exerts neuroprotection. Given uncertainties about the possible effects of lower circulating BACs that are common occurring during short- term binges, we examined how moderate BACs affected the above inflammatory events, and the impact of supplemented DHA. Young adult male rats sustaining upper-moderate BACs (~150mg/dl) from once-daily alcohol intubations were sacrificed with appropriate controls after 1week. The HC, ECX and OB were quantitatively examined using immunoblotting, neurodegeneration staining, and lipidomics assays. Whereas neurodegeneration, increases in cPLA2 IVA, sPLA2 IIA, and ARA, and microglial activation were not detected, the HC and ECX regions demonstrated significantly reduced iPLA2 levels. Levels of DHA and synaptamide, its anti-inflammatory N-docosahexaenoylethanolamide derivative, also were lower in HC, and DHA supplementation prevented the iPLA2 decrements in HC. Additionally, adult mice maintaining upper-moderate BACs from limited alcohol binges had reduced midbrain iPLA2 levels. The apparently selective depletion by moderate BACs of the metabolically linked anti-inflammatory triad of hippocampal iPLA2, DHA, and synaptamide, and of iPLA2 in the ECX, potentially indicates an unappreciated deficit in brain anti-inflammatory reserve that may be a harbinger of regional neurovulnerability.

Reevaluating the pathogenicity of the variations c.439 G&gt;A and c.2132 C&gt;T in the PLA2G6 gene

The phospholipase A2 group VI (PLA2G6) gene encodes for a Ca2+-independent PLA2, which is localized in the cytosol, in the endoplasmic reticulum and in the mitochondrial membrane, plays a major role in phospholipid remodelling. Mutations within this gene have been reported to cause different phenotypes: infantile-onset neuroaxonal dystrophy associated with brain iron accumulation and adult-onset parkinsonian syndrome. In the present study, we analysed the PLA2G6 gene sequence in an asymptomatic young woman that was referred to our laboratory by a geneticist for an history of infantile neuroaxonal dystrophy in her little maternal cousin in whom the results of the genetical analysis were not available. We found two variants in the PLA2G6 gene (NM_003560.4, c.439 G>A and c.2132 C>T, p.Ala147Thr and p.Pro711Leu) previously reported as pathogenic. These results prompted us to perform a segregation analysis in the parents of this woman and we only found the presence of both variants in the asymptomatic 56-year-old patient's mother. Our molecular genetic testing clearly indicates that the c.439 G>A and c.2132 C>T variations identified in the PLA2G6 gene are positioned in cis and are not responsible for infantile neuroaxonal dystrophy which is an autosomal recessive disease.

CPLA2α-/- sympathetic neurons exhibit increased membrane excitability and loss of N-Type Ca2+ current inhibition by M1 muscarinic receptor signaling.

Group IVa cytosolic phospholipase A2 (cPLA2α) mediates GPCR-stimulated arachidonic acid (AA) release from phosphatidylinositol 4,5-bisphosphate (PIP2) located in plasma membranes. We previously found in superior cervical ganglion (SCG) neurons that PLA2 activity is required for voltage-independent N-type Ca2+ (N-) current inhibition by M1 muscarinic receptors (M1Rs). These findings are at odds with an alternative model, previously observed for M-current inhibition, where PIP2 dissociation from channels and subsequent metabolism by phospholipase C suffices for current inhibition. To resolve cPLA2α’s importance, we have investigated its role in mediating voltage-independent N-current inhibition (~40%) that follows application of the muscarinic agonist oxotremorine-M (Oxo-M). Preincubation with different cPLA2α antagonists or dialyzing cPLA2α antibodies into cells minimized N-current inhibition by Oxo-M, whereas antibodies to Ca2+-independent PLA2 had no effect. Taking a genetic approach, we found that SCG neurons from cPLA2α-/- mice exhibited little N-current inhibition by Oxo-M, confirming a role for cPLA2α. In contrast, cPLA2α antibodies or the absence of cPLA2α had no effect on voltage-dependent N-current inhibition by M2/M4Rs or on M-current inhibition by M1Rs. These findings document divergent M1R signaling mediating M-current and voltage-independent N-current inhibition. Moreover, these differences suggest that cPLA2α acts locally to metabolize PIP2 intimately associated with N- but not M-channels. To determine cPLA2α’s functional importance more globally, we examined action potential firing of cPLA2α+/+ and cPLA2α-/- SCG neurons, and found decreased latency to first firing and interspike interval resulting in a doubling of firing frequency in cPLA2α-/- neurons. These unanticipated findings identify cPLA2α as a tonic regulator of neuronal membrane excitability.

Specific enrichment of 2-arachidonoyl-lysophosphatidylcholine in carotid atheroma plaque from type 2 diabetic patients

Background and aimsDiabetic patients are at high risk of stroke and coronary artery disease. Recent data suggest that arachidonic acid metabolism is altered in diabetic conditions and that these alterations contribute to accelerated atherosclerosis. Little is known about how these alterations affect the metabolism and the proportions of different lipid species within the atherosclerotic plaque. The aim of our study was to perform a targeted lipidomic analysis of human atherosclerotic lesions, with a specific focus on PUFA-containing lipid species, to reveal differences in the fatty-acid composition of plaque in diabetic patients compared with non-diabetic controls. MethodsCarotid atheroma plaque samples were obtained from 31 diabetic and 48 non-diabetic patients undergoing carotid endarterectomy. Targeted lipidomic analysis was then performed to determine the fatty acid composition of major glycerophospholipids and cholesteryl ester species by liquid chromatography-tandem mass spectrometry. ResultsAtheroma plaques from diabetic patients were significantly enriched with 2-arachidonoyl-lysophosphatidylcholine (2-AA-LPC) (2.3 ± 0.8% Vs. 1.8 ± 0.6% p = 0.0002). Multivariable logistic regression showed that an increased 2-AA-LPC level was independently associated with diabetes. Finally, a positive relationship was found between 2-AA-LPC and HbA1c levels. Interestingly, endothelial lipase and calcium independent PLA2 gamma which could account for the production of 2-AA-LPC were detected in carotid plaques by immunohistochemistry. Conclusions2-AA-LPC stands at the crossroads of major metabolic pathways that lead to the synthesis of bioactive molecules such as AA-derived eicosanoids, 2-AA-lysophosphatidic acid and 2-AA-glycerol. 2-AA-LPC therefore appears to be a promising molecule to investigate in the context of diabetes.

Role of cytosolic and calcium independent phospholipases A2 in insulin secretion impairment of INS-1E cells infected by S. aureus

Cytosolic PLA2 (cPLA2) and Ca2+-independent PLA2 (iPLA2) play a significant role in insulin β-cells secretion. Bacterial infections may be responsible of the onset of diabetes. The mechanism by which Staphylococcus aureus infection of INS-1 cells alters glucose-induced insulin secretion has been examined. After acute infection, insulin secretion and PLA2 activities significantly increased. Moreover, increased expressions of phospho-cPLA2, phospho-PKCα and phospho-ERK 1/2 were observed. Chronic infection causes a decrease in insulin release and a significant increase of iPLA2 and COX-2 protein expression. Moreover, insulin secretion in infected cells could be restored using specific siRNAs against iPLA2 isoform and specific COX-2 inhibitor.

Group VIB calcium-independent phospholipase A2 (iPLA2γ) regulates platelet activation, hemostasis and thrombosis in mice.

In platelets, group IVA cytosolic phospholipase A2 (cPLA2α) has been implicated as a key regulator in the hydrolysis of platelet membrane phospholipids, leading to pro-thrombotic thromboxane A2 and anti-thrombotic 12-(S)-hydroxyeicosatetranoic acid production. However, studies using cPLA2α-deficient mice have indicated that other PLA2(s) may also be involved in the hydrolysis of platelet glycerophospholipids. In this study, we found that group VIB Ca2+-independent PLA2 (iPLA2γ)-deficient platelets showed decreases in adenosine diphosphate (ADP)-dependent aggregation and ADP- or collagen-dependent thromboxane A2 production. Electrospray ionization mass spectrometry analysis of platelet phospholipids revealed that fatty acyl compositions of ethanolamine plasmalogen and phosphatidylglycerol were altered in platelets from iPLA2γ-null mice. Furthermore, mice lacking iPLA2γ displayed prolonged bleeding times and were protected against pulmonary thromboembolism. These results suggest that iPLA2γ is an additional, long-sought-after PLA2 that hydrolyzes platelet membranes and facilitates platelet aggregation in response to ADP.

Serine hydrolase inhibitors block necrotic cell death by preventing calcium overload of the mitochondria and permeability transition pore formation (756.2)

Perturbation of calcium signaling occurs during cell injury and disease and promotes cell death. In mouse lung fibroblasts A23187 triggered mitochondrial permeability transition pore (MPTP) formation, necrotic cell death and LDH release that were blocked by cyclosporin A (CsA) and EGTA. LDH release temporally correlated with arachidonic acid release but did not involve cytosolic phospholipase A2 (cPLA2) or calcium independent PLA2. Surprisingly, release of arachidonic acid and LDH from cPLA2‐deficient fibroblasts was inhibited by the cPLA2 inhibitor pyrrophenone, and another serine hydrolase inhibitor KT195, by preventing mitochondrial calcium loading. Inhibitors of calmodulin‐dependent protein kinase II, a mitochondrial Ca2+ uniporter (MCU) regulator, also prevented MPTP formation and arachidonic acid release induced by A23187 and by H2O2. Pyrrophenone blocked MCU‐mediated mitochondrial calcium uptake in permeabilized fibroblasts but not in isolated mitochondria. Unlike pyrrophenone, the diacylglycerol analogue 1‐oleoyl‐2‐acetyl‐sn‐glycerol (OAG) and CsA blocked cell death and arachidonic acid release not by preventing mitochondrial calcium loading but by inhibiting MPTP formation. In fibroblasts stimulated with thapsigargin, which induces MPTP formation by a direct effect on mitochondria, LDH and arachidonic acid release were blocked by CsA and OAG but not by pyrrophenone or EGTA. Therefore serine hydrolase inhibitors prevent necrotic cell death by blocking mitochondrial calcium uptake but not fatty acid release that occurs by a novel pathway during MPTP formation. This work reveals the potential for development of small molecule cell permeable serine hydrolase inhibitors that block MCU‐mediated mitochondrial calcium overload, MPTP formation and necrotic cell death.Grant Funding Source: NIH HL34303, HL61378, HL50040, DA009789, DA033760, MH084512

Acceleration of the Development of Alzheimer’s Disease in Amyloid Beta-Infused Peroxiredoxin 6 Overexpression Transgenic Mice

The amyloid beta (Aβ) peptide in the brains of patients with Alzheimer's disease (AD) is cytotoxic to neurons and has a central role in the pathogenesis of the disease. Peroxiredoxin 6 (Prdx6) is an antioxidant protein and could act as a cytoprotective protein. However, the role of Prdx6 in neurodegenerative disease has not been studied. Thus, the roles and action mechanisms in the development of AD were examined. Aβ1-42-induced memory impairment in Prdx6 transgenic mice was worse than C57BL/6 mice, and the expression of amyloid precursor protein cleavage, C99, β-site APP-cleaving enzyme 1, inducible nitric oxide synthase, and cyclooxygenase-2 was greatly increased. In addition, the astrocytes and microglia cells of Aβ-infused Prdx6 transgenic mice were more activated, and Aβ also significantly increased lipid peroxidation and protein carbonyl levels, but decreased glutathione levels. Furthermore, we found that translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) to the nucleus was increased in Aβ-infused Prdx6 transgenic mice. These results suggest that the overexpression of Prdx6 could accelerate the development of AD through increased amyloidogenesis through independent PLA2 activation and Nrf2 transcription.

P2‐097: Inhibition of PLA2 inactivates GSK3B via Akt and CAMKII in primary cultures of hippocampal neurons

Glycogen synthase kinase 3-beta (GSK3B) is overactive in Alzheimer's disease (AD), favoring the hyperphosphorylation of microtubule-associated protein Tau and the formation of neurofibrillary tangles. For this reason, GSK3B inhibition may be considered as a candidate disease-modifying approach in AD. In addition to the direct enzymatic inhibition (eg. by lithium), GSK3B can be also indirectly inactivated by phosphorylation at the Ser9 residue. This is a downstream effect of several intracellular cascades, including Akt and CaMKII signaling. Phospholipase A 2 (PLA 2) are key enzymes in membrane homeostasis and cell signaling. Reduced PLA 2 activity has been demonstrated in cerebral and peripheral tissues of patients with AD, and recent studies suggested that PLA 2 is further implicated in GSK3B homeostasis. The objective of this study is to evaluate the effect of the inhibition of PLA 2 on the GSK3B activity and to determine whether this effect involves CAMKII and Akt. Primary cultures of cortical and hippocampal neurons started from embryonic rats (E18) were treated after 4 four days in culture with different concentrations (5-250 μM) of methyl-aracdonyl-fluorophosphonate (MAFP), which is an irreversible, dual inhibitor of calcium-dependent and -independent cytosolic PLA 2. Treatment effects were evaluated by Western-blot, addressing the expression of total- and phosphorylated (Ser9) GSK3B, phosphorylated Akt (Ser473), and total CAMKII at protein level. The inhibition of GSK3B is a downstream effect of cytosolic PLA 2 inhibition, which apparently involves the activation of Akt and CAMKII. This effect seems to be specific to hippocampal neurons.

The P2Y2/Src/p38/COX-2 pathway is involved in the resistance to ursolic acid-induced apoptosis in colorectal and prostate cancer cells

One of the hallmarks of cancer is resistance to apoptosis. Elucidating the mechanisms of how cancer cells evade or delay apoptosis should lead to novel therapeutic strategies. Previously, we showed that HT-29 colorectal cancer cells undergoing apoptosis overexpressed cyclooxygenase-2 (COX-2), in a p38 dependent pathway, to delay ursolic acid-induced apoptosis. Here, we focused on elucidating the upstream signaling pathways regulating this resistance mechanism. The role of ATP as an extracellular signaling molecule took a long time to be accepted. In recent years, ATP and its analogs, via the activation of specific purinergic receptors, have been implicated in many biological processes including cell proliferation, differentiation and apoptosis. In the present report, we have demonstrated a novel role involving purinergic receptors and particularly the P2Y(2) receptor in resistance to ursolic acid-induced apoptosis in both colorectal HT-29 and prostate DU145 cancer cells. We found that ursolic acid induced an increase in intracellular ATP and P2Y(2) transcript levels. Upon activation, P2Y(2) activated Src which in turn phosphorylated p38 leading to COX-2 overexpression which induced resistance to apoptosis in both HT-29 and DU145 cells. Furthermore, Ca(2+)-independent PLA(2) (iPLA(2)) and Ca(2+)-dependent secretory PLA(2) (sPLA(2)) were responsible for arachidonic acid release, the substrate of COX-2. Our findings document that apoptosis triggering was dependent on protein kinase C (PKC) activation in both cell lines after ursolic acid treatment.

Analysis of Two Major Intracellular Phospholipases A2 (PLA2) in Mast Cells Reveals Crucial Contribution of Cytosolic PLA2α, Not Ca2+-independent PLA2β, to Lipid Mobilization in Proximal Mast Cells and Distal Fibroblasts

Mast cells release a variety of mediators, including arachidonic acid (AA) metabolites, to regulate allergy, inflammation, and host defense, and their differentiation and maturation within extravascular microenvironments depend on the stromal cytokine stem cell factor. Mouse mast cells express two major intracellular phospholipases A(2) (PLA(2)s), namely group IVA cytosolic PLA(2) (cPLA(2)α) and group VIA Ca(2+)-independent PLA(2) (iPLA(2)β), and the role of cPLA(2)α in eicosanoid synthesis by mast cells has been well documented. Lipidomic analyses of mouse bone marrow-derived mast cells (BMMCs) lacking cPLA(2)α (Pla2g4a(-/-)) or iPLA(2)β (Pla2g6(-/-)) revealed that phospholipids with AA were selectively hydrolyzed by cPLA(2)α, not by iPLA(2)β, during FcεRI-mediated activation and even during fibroblast-dependent maturation. Neither FcεRI-dependent effector functions nor maturation-driven phospholipid remodeling was impaired in Pla2g6(-/-) BMMCs. Although BMMCs did not produce prostaglandin E(2) (PGE(2)), the AA released by cPLA(2)α from BMMCs during maturation was converted to PGE(2) by microsomal PGE synthase-1 (mPGES-1) in cocultured fibroblasts, and accordingly, Pla2g4a(-/-) BMMCs promoted microenvironmental PGE(2) synthesis less efficiently than wild-type BMMCs both in vitro and in vivo. Mice deficient in mPGES-1 (Ptges(-/-)) had an augmented local anaphylactic response. These results suggest that cPLA(2)α in mast cells is functionally coupled, through the AA transfer mechanism, with stromal mPGES-1 to provide anti-anaphylactic PGE(2). Although iPLA(2)β is partially responsible for PGE(2) production by macrophages and dendritic cells, it is dispensable for mast cell maturation and function.

Delayed production of arachidonic acid contributes to the delay of proteinase‐activated receptor‐1 (PAR1)‐triggered prostaglandin E2 release in rat gastric epithelial RGM1 cells

Proteinase-activated receptor-1 (PAR1), upon activation, exerts prostanoid-dependent gastroprotection, and increases prostaglandin E(2) (PGE(2)) release through cyclooxygenase-2 (COX-2) upregulation in rat gastric mucosal epithelial RGM1 cells. However, there is a big time lag between the PAR1-triggered PGE(2) release and COX-2 upregulation in RGM1 cells; that is, the former event takes 18 h to occur, while the latter rapidly develops and reaches a plateau in 6 h. The present study thus aimed at clarifying mechanisms for the delay of PGE(2) release after PAR1 activation in RGM1 cells. Although a PAR1-activating peptide, TFLLR-NH(2), alone caused PGE(2) release at 18 h, but not 6 h, TFLLR-NH(2) in combination with arachidonic acid dramatically enhanced PGE(2) release even for 1-6 h. TFLLR-NH(2) plus linoleic acid caused a similar rapid response. CP-24879, a Δ(5)/Δ(6)-desaturase inhibitor, abolished the PGE(2) release induced by TFLLR-NH(2) plus linoleic acid, but not by TFLLR-NH(2) alone. The TFLLR-NH(2)-induced PGE(2) release was not affected by inhibitors of cytosolic phospholipase A(2) (cPLA(2)), Ca(2+)-independent PLA(2) (cPLA(2)) or secretory PLA(2) (sPLA(2)), but was abolished by their mixture or a pan-PLA(2) inhibitor. Among PLA(2) isozymes, mRNA of group IIA sPLA(2) (sPLA(2)-IIA) was upregulated following PAR1 stimulation for 6-18 h, whereas protein levels of PGE synthases were unchanged. These data suggest that the delay of PGE(2) release after COX-2 upregulation triggered by PAR1 is due to the poor supply of free arachidonic acid at the early stage in RGM1 cells, and that plural isozymes of PLA(2) including sPLA(2)-IIA may complementarily contribute to the liberation of free arachidonic acid.

Extracellular-derived calcium does not initiate in vivo neurotransmission involving docosahexaenoic acid

In vitro studies show that docosahexaenoic acid (DHA) can be released from membrane phospholipid by Ca(2+)-independent phospholipase A(2) (iPLA(2)), Ca(2+)-independent plasmalogen PLA(2) or secretory PLA(2 (sPLA2)), but not by Ca(2+)-dependent cytosolic PLA(2) (cPLA2), which selectively releases arachidonic acid (AA). Since glutamatergic NMDA (N-methyl-D-aspartate) receptor activation allows extracellular Ca(2+) into cells, we hypothesized that brain DHA signaling would not be altered in rats given NMDA, to the extent that in vivo signaling was mediated by Ca(2+)-independent mechanisms. Isotonic saline, a subconvulsive dose of NMDA (25 mg/kg), MK-801, or MK-801 followed by NMDA was administered i.p. to unanesthetized rats. Radiolabeled DHA or AA was infused intravenously and their brain incorporation coefficients k*, measures of signaling, were imaged with quantitative autoradiography. NMDA or MK-801 compared with saline did not alter k* for DHA in any of 81 brain regions examined, whereas NMDA produced widespread and significant increments in k* for AA. In conclusion, in vivo brain DHA but not AA signaling via NMDA receptors is independent of extracellular Ca(2+) and of cPLA(2). DHA signaling may be mediated by iPLA(2), plasmalogen PLA(2), or other enzymes insensitive to low concentrations of Ca(2+). Greater AA than DHA release during glutamate-induced excitotoxicity could cause brain cell damage.

Phospholipase A2 and its molecular mechanism after spinal cord injury.

Phospholipases A(2) (PLA(2)s) are a diverse family of lipolytic enzymes which hydrolyze the acyl bond at the sn-2 position of glycerophospholipids to produce free fatty acids and lysophospholipids. These products are precursors of bioactive eicosanoids and platelet-activating factor which have been implicated in pathological states of numerous acute and chronic neurological disorders. To date, more than 27 isoforms of PLA(2) have been found in the mammalian system which can be classified into four major categories: secretory PLA(2), cytosolic PLA(2), Ca(2+)-independent PLA(2), and platelet-activating factor acetylhydrolases. Multiple isoforms of PLA(2) are found in the mammalian spinal cord. Under physiological conditions, PLA(2)s are involved in diverse cellular responses, including phospholipid digestion and metabolism, host defense, and signal transduction. However, under pathological situations, increased PLA(2) activity, excessive production of free fatty acids and their metabolites may lead to the loss of membrane integrity, inflammation, oxidative stress, and subsequent neuronal injury. There is emerging evidence that PLA(2) plays a key role in the secondary injury process after traumatic spinal cord injury. This review outlines the current knowledge of the PLA(2) in the spinal cord with an emphasis being placed on the possible roles of PLA(2) in mediating the secondary SCI.

Phospholipase A2-derived lysophosphatidylcholine triggers Ca2+ entry in dystrophic skeletal muscle fibers

Duchenne muscular dystrophy is an inherited disease caused by the absence of dystrophin, a structural protein normally located under the sarcolemma of skeletal muscle fibers. Muscle degeneration occurring in this disease is thought to be partly caused by increased Ca2+ entry through sarcolemmal cationic channels. Using the Mn2+ quench method, we show here that Mn2+ entry triggered by Ca2+ store depletion but not basal Mn2+ entry relies on Ca2+-independent PLA2 (iPLA2) activity in dystrophic fibers isolated from a murine model of Duchenne muscular dystrophy, the mdx5cv mouse. iPLA2 was found to be localized in the vicinity of the sarcolemma and consistently, the iPLA2 lipid product lysophosphatidylcholine was found to trigger Ca2+ entry through sarcolemmal channels, suggesting that it acts as an intracellular messenger responsible for store-operated channels opening in dystrophic fibers. Our results suggest that inhibition of iPLA2 and lysophospholipid production may be of interest to reduce Ca2+ entry and subsequent degeneration of dystrophic muscle.

The tumor suppressor gene H-Rev107 functions as a novel Ca2+-independent cytosolic phospholipase A1/2 of the thiol hydrolase type

H-Rev107 is a protein that was previously cloned as a negative regulator of proto-oncogene Ras and classified as a class II tumor suppressor. Its structural similarity to lecithin retinol acyltransferase and Ca2+-independent phosphatidylethanolamine (PE) N-acyltransferase led us to analyze H-Rev107 as an enzyme involved in phospholipid metabolism. Here, we show that recombinant H-Rev107s from rat, human, and mouse possess phospholipase (PL) A1 or A2 activity toward phosphatidylcholine (PC). Further examination with purified recombinant protein revealed that H-Rev107 functions as a cytosolic Ca2+-independent PLA(1/2) for PC and PE with higher PLA1 activity than PLA2 activity. Dithiothreitol and iodoacetic acid exhibited stimulatory and inhibitory effects, respectively. Histidine-21 and cysteine-111 of rat H-Rev107 were presumed to form a catalytic dyad based on database analysis, and their single mutants were totally inactive. These results suggested that H-Rev107 is a hydrolase of the thiol type. The N-terminal proline-rich and C-terminal hydrophobic domains of H-Rev107 were earlier reported to be responsible for the regulation of cell proliferation. Analysis of deletion mutants indicated that these domains are also catalytically essential, suggesting relevance of the catalytic activity to the anti-proliferative activity.

Cloning of a novel acidic phospholipase A2 from the venom gland of Crotalus durissus cascavella (Brazilian northeastern rattlesnake)

The phospholipase A2 superfamily encompasses 15 groups that are classified into: secreted PLA2 (sPLA2); cytosolic PLA2 (cPLA2); Ca2+-independent intracellular PLA2 (iPLA2); platelet-activating factor acetylhydrolase (PAF-AH); and lysosomal PLA2. Currently, approximately 700 PLA2 sequences are known, of which 200 are obtained from the venom gland of Crotalinae snakes. However, thus far, little information is available on cloning, purification and structural characterization of PLA2 from Crotalus durisssus cascavela venom gland. In the present work, we report the molecular cloning of a novel svPLA2 from C. d. cascavella (Cdc), a predominant rattlesnake subspecies in northeastern Brazil. The Cdc svPLA2 cDNA precursor is 689 nucleotides long and encodes a protein of 138 amino acid residues, with a calculated molecular mass of approximately 13,847 Da and an estimated isoelectric point of 5.14. Phylogenetic analysis of Crotalinae PLA2 reveals that Cdc PLA2 clustered with other acidic type IIA PLA2 homologues is also present in the venom of North American rattlesnakes. Hitherto, this study presents a novel PLA2 cDNA precursor from C. d. cascavella and data reported herein will be useful for further steps in svPLA2 purification and analysis.

ATP-induced apoptosis involves a Ca 2+-independent phospholipase A 2 and 5-lipoxygenase in macrophages

Macrophages express P2X 7 and other nucleotide (P2) receptors, and display the phenomena of extracellular ATP (ATP e)-induced P2X 7-dependent membrane permeabilization and cell death by apoptosis and necrosis. P2X 7 receptors also cooperate with toll-like receptors (TLRs) to induce inflammasome activation and IL-1β secretion. We investigated signaling pathways involved in the induction of cell death by ATP e in intraperitoneal murine macrophages. Apoptosis (hypodiploid nuclei) and necrosis (LDH release) were detected 6 h after an induction period of 20 min in the presence of ATP. Apoptosis was blocked by caspase 3 and caspase 9 inhibitors and by cyclosporin A. The MAPK inhibitors PD-98059, SB-203580 and SB-202190 provoked no significant effect on apoptosis, but SB-203580 blocked LDH release. Neither apoptosis nor necrosis was inhibited when both intra- and extracellular Ca 2+ were chelated during the induction period. Mepacrine, a generic PLA 2 inhibitor and BEL, an inhibitor of Ca 2+-independent PLA 2 (iPLA 2) blocked apoptosis, while pBPB and AACOOPF 3, inhibitors of secretory and Ca 2+-dependent PLA 2 respectively, had no significant effect. Cycloxygenase inhibitors had no effect on apoptosis, while the inhibitors of lipoxygenase (LOX) and leukotriene biosynthesis nordihydroguaiaretic acid (NDGA), zileuton, AA-861, and MK-886 significantly decreased apoptosis. Neither NDGA nor MK-886 blocked apoptosis of 5-LOX −/− macrophages. CP-105696 and MK-571, antagonists of leukotriene receptors, had no significant effect on apoptosis. None of the inhibitors of PLA 2 and LOX/leukotriene pathway had a significant inhibitory effect on LDH release. Our results indicate that a Ca 2+-independent step involving an iPLA 2 and 5-LOX are involved in the triggering of apoptosis but not necrosis by P2X 7 in macrophages.