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Edward A Dennis

Publications and source records attributed to Edward A Dennis.

At least 19 recordsLinked to original sources
PubMed2006

Lipopolysaccharide-induced cyclooxygenase-2 expression in human U937 macrophages is phosphatidic acid phosphohydrolase-1-dependent.

Cyclooxygenase (COX) has two isoforms, COX-1 and -2, which catalyze the key step in the conversion of cellular arachidonic acid into prostaglandins. In recent years, interest in COX-2 has significantly increased since it has been a target for the development of specific non-steroidal anti-inflammatory drugs. We report that COX-2 expression is up-regulated in phorbol ester (phorbol myristate acetate, PMA)-differentiated human U937 macrophage-like cells stimulated with lipopolysaccharide (LPS), whereas COX-1 is not up-regulated. We show that the LPS-induced up-regulation of COX-2 depends on the activity of the Mg(+2)-dependent phosphatidic acid phosphohydrolase 1 (PAP-1). Inhibition of PAP-1 by bromoenol lactone, propranolol, or ethanol resulted in a decrease in LPS-induced COX-2 mRNA transcript production, COX-2 protein expression, and prostaglandin E(2) release from U937 macrophages. To ensure that these results did not arise because of PMA treatment of the U937 cells, similar experiments were conducted with the P388D(1) cell line, which does not require PMA differentiation. LPS increased the levels of endogenous cellular diacylglycerol (DAG) within 2 min of stimulation. This increase was observed to be sensitive to the PAP-1 inhibitors. Furthermore, phosphatidic acid phosphohydrolase activity assays showed that the bromoenol lactone-sensitive PAP-1 activity was translocated from the cytosolic fraction to the membrane fraction within 2 min of LPS exposure. Finally, DAG add-back experiments demonstrate that LPS-induced COX-2 expression is enhanced by the addition of exogenous DAG.

Animals↗
PubMed2006

The phospholipase A2 superfamily and its group numbering system.

The superfamily of phospholipase A(2) (PLA(2)) enzymes currently consists of 15 Groups and many subgroups and includes five distinct types of enzymes, namely the secreted PLA(2)s (sPLA(2)), the cytosolic PLA(2)s (cPLA(2)), the Ca(2+) independent PLA(2)s (iPLA(2)), the platelet-activating factor acetylhydrolases (PAF-AH), and the lysosomal PLA(2)s. In 1994, we established the systematic Group numbering system for these enzymes. Since then, the PLA(2) superfamily has grown continuously and over the intervening years has required several updates of this Group numbering system. Since our last update, a number of new PLA(2)s have been discovered and are now included. Additionally, tools for the investigation of PLA(2)s and approaches for distinguishing between the different Groups are described.

Animals↗
PubMed2006

Differential inhibition of group IVA and group VIA phospholipases A2 by 2-oxoamides.

Inhibitors of the Group IVA phospholipase A(2) (GIVA cPLA(2)) and GVIA iPLA(2) are useful tools for defining the roles of these enzymes in cellular signaling and inflammation. We have developed inhibitors of GVIA iPLA(2) building upon the 2-oxoamide backbone that are uncharged, containing ester groups. Although the most potent inhibitors of GVIA iPLA(2) also inhibited GIVA cPLA(2), there were three 2-oxoamide compounds that selectively and weakly inhibited GVIA iPLA(2). We further show that several potent 2-oxoamide inhibitors of GIVA cPLA(2) containing free carboxylic groups (Kokotos et al. J. Med. Chem. 2002, 45, 2891-2893) do not inhibit GVIA iPLA(2) and are, therefore, selective GIVA cPLA(2) inhibitors.

Animals↗
PubMed2006

Kdo2-Lipid A of Escherichia coli, a defined endotoxin that activates macrophages via TLR-4.

The LIPID MAPS Consortium (www.lipidmaps.org) is developing comprehensive procedures for identifying all lipids of the macrophage, following activation by endotoxin. The goal is to quantify temporal and spatial changes in lipids that occur with cellular metabolism and to develop bioinformatic approaches that establish dynamic lipid networks. To achieve these aims, an endotoxin of the highest possible analytical specification is crucial. We now report a large-scale preparation of 3-deoxy-D-manno-octulosonic acid (Kdo)(2)-Lipid A, a nearly homogeneous Re lipopolysaccharide (LPS) sub-structure with endotoxin activity equal to LPS. Kdo(2)-Lipid A was extracted from 2 kg cell paste of a heptose-deficient Escherichia coli mutant. It was purified by chromatography on silica, DEAE-cellulose, and C18 reverse-phase resin. Structure and purity were evaluated by electrospray ionization/mass spectrometry, liquid chromatography/mass spectrometry and (1)H-NMR. Its bioactivity was compared with LPS in RAW 264.7 cells and bone marrow macrophages from wild-type and toll-like receptor 4 (TLR-4)-deficient mice. Cytokine and eicosanoid production, in conjunction with gene expression profiling, were employed as readouts. Kdo(2)-Lipid A is comparable to LPS by these criteria. Its activity is reduced by >10(3) in cells from TLR-4-deficient mice. The purity of Kdo(2)-Lipid A should facilitate structural analysis of complexes with receptors like TLR-4/MD2.

Animals↗
PubMed2005

Systemic and intrathecal effects of a novel series of phospholipase A2 inhibitors on hyperalgesia and spinal prostaglandin E2 release.

Phospholipase A(2) (PLA(2)) forms are expressed in spinal cord, and inhibiting spinal PLA(2) induces a potent antihyperalgesia. Here, we examined the antihyperalgesic effects after systemic and i.t. delivery of four compounds constructed with a common motif consisting of a 2-oxoamide with a hydrocarbon tail and a four-carbon tether. These molecules were characterized for their ability to block group IVA calcium-dependent PLA(2) (cPLA(2)) and group VIA calcium-independent PLA(2) (iPLA(2)) in inhibition assays using human recombinant enzyme. The rank ordering of potency in blocking group IVA cPLA(2) was AX048 (ethyl 4-[(2-oxohexadecanoyl)amino]butanoate), AX006 (4-[(2-oxohexadecanoyl)amino]butanoic acid), and AX057 (tert-butyl 4-[(2-oxohexadecanoyl)amino]butanoate) > AX010 (methyl 4-[(2-oxohexadecanoyl)amino]butanoate) and for inhibiting group VIA iPLA(2) was AX048, AX057 > AX006, and AX010. No agent altered recombinant cyclooxygenase activity. In vivo, i.t. (30 mug) and systemic (0.2-3 mg/kg i.p.) AX048 blocked carrageenan hyperalgesia and after systemic delivery in a model of spinally mediated hyperalgesia induced by i.t. substance P (SP). The other agents were without activity. In rats prepared with lumbar i.t. loop dialysis catheters, SP evoked spinal prostaglandin E(2) (PGE(2)) release. AX048 alone inhibited PGE(2) release. Intrathecal SR141617, a cannabinoid CB1 inhibitor at doses that blocked the effects of i.t. anandamide had no effect upon i.t. AX048. These results suggest that AX048 is the first systemically bioavailable compound with a significant affinity for group IVA cPLA(2), which produces a potent antihyperalgesia. The other agents, although demonstrating enzymatic activity in cell-free assays, appear unable to gain access to the intracellular PLA(2) toward which their action is targeted.

Animals↗
PubMed2005

PGE2 release is independent of upregulation of Group V phospholipase A2 during long-term stimulation of P388D1 cells with LPS.

P388D1 cells release arachidonic acid (AA) and produce prostaglandin E2 (PGE2) upon long-term stimulation with lipopolysaccharide (LPS). The cytosolic Group IVA (GIVA) phospholipase A2 (PLA2) has been implicated in this pathway. LPS stimulation also results in increased expression and secretion of a secretory PLA2, specifically GV PLA2. To test whether GV PLA2 contributes to PGE2 production and whether GIVA PLA2 activation increases the expression of GV PLA2, we utilized the specific GIVA PLA2 inhibitor pyrrophenone and second generation antisense oligonucleotides (AS-ONs) designed to specifically inhibit expression and activity of GV PLA2. Treatment of P388D1 cells with antisense caused a marked decrease in basal GV PLA2 mRNA and prevented the LPS-induced increase in GV PLA2 mRNA. LPS-stimulated cells release active GV PLA2 into the medium, which is inhibited to background levels by antisense treatment. However, LPS-induced PGE2 release by antisense-treated cells and by control cells are not significantly different. Collectively, the results suggest that the upregulation of GV PLA2 during long-term LPS stimulation is not required for PGE2 production by P388D1 cells. Experiments employing pyrrophenone suggested that GIVA PLA2 is the dominant player involved in AA release, but it appears not to be involved in the regulation of LPS-induced expression of GV PLA2 or cyclooxygenase-2.

Animals↗
PubMed2005

Localization and functional interrelationships among cytosolic Group IV, secreted Group V, and Ca2+-independent Group VI phospholipase A2s in P388D1 macrophages using GFP/RFP constructs.

P388D(1) cells exposed to bacterial lipopolysaccharide (LPS) mobilize arachidonic acid (AA) for prostaglandin synthesis in two temporally distinct pathways. The "immediate pathway" is triggered within minutes by receptor agonists such as platelet-activating factor (PAF) but only if the cells have previously been primed with LPS for 1 h. The "delayed pathway" occurs in response to LPS alone over the course of several hours. We have now investigated the subcellular localization of both the Group IV cytosolic phospholipase A(2) (cPLA(2)) and the Group V secreted PLA(2) (sPLA(2)) during these two temporally distinct routes of AA release. We have prepared cells overexpressing fusion proteins of sPLA(2)-GFP and cPLA(2)-RFP. In the resting cells, cPLA(2)-RFP was uniformly located throughout the cytoplasm, and short-term treatment with LPS did not induce translocation to perinuclear and/or Golgi membranes. However, such a translocation occurred almost immediately after the addition of PAF to the cells. Long-term exposure of the cells to LPS led to the translocation of cPLA(2)-RFP to intracellular membranes after 3 h, and correlates with a significant release of AA in a cPLA(2)-dependent manner. At the same time period that the delayed association of cPLA(2) with perinuclear membranes is detected, an intense fluorescence arising from the sPLA(2)-GFP was found around the nucleus in the sPLA(2)-GFP stably transfected cells. In parallel with these changes, significant AA release was detected from the sPLA(2)-GFP transfectants in a cPLA(2)-dependent manner, which may reflect cross-talk between sPLA(2) and cPLA(2). The subcellular localization of the Group VIA Ca(2+)-independent PLA(2) (iPLA(2)) was also investigated. Cells overexpressing iPLA(2)-GFP showed no fluorescence changes under any activation condition. However, the iPLA(2)-GFP-expressing cells showed relatively high basal AA release, confirming a role for iPLA(2) in basal deacylation reactions. These new data illustrate the subcellular localization changes that accompany the distinct roles that each of the three kinds of PLA(2) present in P388D(1) macrophages play in AA mobilization.

Animals↗
PubMed2005

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.

Animals↗
PubMed2005

Molecular characterization of the lipopolysaccharide/platelet activating factor- and zymosan-induced pathways leading to prostaglandin production in P388D1 macrophages.

P388D1 cells release free arachidonic acid (AA) and prostaglandin E2 (PGE2) upon stimulation with platelet-activating factor (PAF) and zymosan. The response to PAF is dependent on priming of the cells with bacterial lipopolysaccharide (LPS). In the LPS/PAF pathway, both AA and PGE2 release are dependent on transcription and translation, whereas in the zymosan pathway the release of these compounds appears to be largely independent of these processes. Using quantitative real-time PCR, we analyzed the expression of mRNAs that encode proteins potentially responsible for the dependency of the LPS/PAF pathway on gene expression. These include all the phospholipases A2 (PLA2) that we detected in P388D1 cells, cyclooxygenases (COX), COX-1 and COX-2, the membrane-associated prostaglandin E synthase-1 (mPGES-1), the lipocalin-type prostaglandin D2 synthase (PGDS), hematopoietic PGDS and the subunit G(alpha i2) of heterotrimeric G-proteins. None of the mRNAs encoding PLA2s, PGDSs, or G(alpha i2) are substantially altered during LPS priming. However, cyclooxygenase-2 is up-regulated during LPS priming and after stimulation of the cells with zymosan. A modest but significant increase of mPGES-1 mRNA was also detected upon stimulation with zymosan. Thus, the dependency of the LPS/PAF-induced PGE2 production on gene expression can be attributed to the production of cyclooxygenase-2. The dependency of AA release on gene expression is not due to altered expression of any of the PLA2s. We suggest that an accessory regulatory protein affecting the release of AA must be responsible. Using HPLC we separated lipids that are secreted upon stimulation with LPS/PAF and zymosan and found that in both pathways PGD2 is the dominant prostaglandin produced and also detected PGE2, PGF(2alpha) and AA besides several unidentified compounds.

Animals↗
PubMed2005

A comprehensive classification system for lipids.

Lipids are produced, transported, and recognized by the concerted actions of numerous enzymes, binding proteins, and receptors. A comprehensive analysis of lipid molecules, "lipidomics," in the context of genomics and proteomics is crucial to understanding cellular physiology and pathology; consequently, lipid biology has become a major research target of the postgenomic revolution and systems biology. To facilitate international communication about lipids, a comprehensive classification of lipids with a common platform that is compatible with informatics requirements has been developed to deal with the massive amounts of data that will be generated by our lipid community. As an initial step in this development, we divide lipids into eight categories (fatty acyls, glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, prenol lipids, saccharolipids, and polyketides) containing distinct classes and subclasses of molecules, devise a common manner of representing the chemical structures of individual lipids and their derivatives, and provide a 12 digit identifier for each unique lipid molecule. The lipid classification scheme is chemically based and driven by the distinct hydrophobic and hydrophilic elements that compose the lipid. This structured vocabulary will facilitate the systematization of lipid biology and enable the cataloging of lipids and their properties in a way that is compatible with other macromolecular databases.

Database Management Systems↗
PubMed2005

Phosphocholine as a pattern recognition ligand for CD36.

We have previously shown that CD36 recognizes oxidation products of phospholipids on oxidized LDL (OxLDL) such as 1-palmitoyl-2-(5'-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC). The current study was designed to examine whether the phosphocholine (PC) headgroup in POVPC constitutes an obligatory binding target for CD36. To examine the contribution of PC in the binding of POVPC to CD36, we used well-defined synthetic oxidized phospholipids (OxPLs) cross-linked to BSA or to a hexapeptide. The OxPL adducts were then tested for their ability to bind to CD36-transfected cells and for their ability to inhibit OxLDL binding to CD36. Both POVPC-BSA and POVPC-peptide adducts were high-affinity ligands for CD36 and potent inhibitors of OxLDL binding. Enzymatic removal of the entire PC moiety of the POVPC-peptide, or of the choline headgroup alone, as well as substitution of the choline headgroup by ethanolamine abrogated the inhibitory activity of POVPC. Interestingly, PC by itself or cross-linked to BSA did not show any intrinsic competition activity. In conclusion, our data demonstrate that the PC headgroup of OxPL alone is sufficient for binding to CD36, but only if presented in the correct conformation as in OxPL of OxLDL or as in POVPC-peptide adducts.

Animals↗
PubMed2005

Synthesis and activity of 2-oxoamides containing long chain beta-amino acids.

2-Oxoamides based on long chain beta-amino acids were synthesized. 1-Benzyl substituted long chain amines, needed for such synthesis, were synthesized starting from Boc-phenylalaninol. The oxidative conversion of a phenyl group to a carboxyl group was used as the key transformation synthetic step. The compounds synthesized were studied for their activity against GIVA PLA(2), and were proven to be weak inhibitors.

Amino Acids↗
PubMed2005

Distinguishing phospholipase A2 types in biological samples by employing group-specific assays in the presence of inhibitors.

This manuscript reviews and updates radiolabel-based enzyme assays designed to distinguish the activity of phospholipase A2 (PLA2) types in biological samples. This approach should be useful in lipidomics studies. The assays were originally designed to differentiate between Group IVA cytosolic PLA2 (GIVA cPLA2), Group VIA calcium-independent PLA2 (GVIA iPLA2), Group IIA secreted PLA2 (GIIA sPLA2) and Group V secreted PLA2 (GV sPLA2). The specificity of these assays has now been confirmed using purified, recombinant human PLA2s and the utility of these assays is demonstrated with rat spinal cord homogenate as an example of a biological tissue sample of interest to the neuroscience community. Modifications to the original assays by the addition of group-specific inhibitors are presented to ensure the specificity of the assays and to further differentiate between recently identified PLA2s. Specific tests are suggested to confirm the specificity of each assay. Additionally, it was discovered that one commonly used GIVA cPLA2/GVIA iPLA2 inhibitor, methyl arachidonyl fluorophosphonate (MAFP) from one commercial source, was found to inhibit GIIA sPLA2 and GV sPLA2, but not GIVA cPLA2, presumably due to oxidation of the compound during shipment, resulting in a different molecule with altered specificity.

Animals↗
PubMed2005

Spinal phospholipase A2 in inflammatory hyperalgesia: role of group IVA cPLA2.

Current work has shown the importance of spinal cyclooxygenase (COX) products in facilitatory processes leading to tissue injury induced hyperalgesia. This cascade must originate with free arachidonic acid (AA) released by the activity of spinal phospholipase A2s (PLA2). In the present work, we studied the role of PLA2s in spinal sensitization. We first demonstrate the presence of constitutive mRNA in the spinal cord for PLA2 Groups IB, IIA, IIC, IVA, V and VI by reverse transcription-polymerase chain reaction (RT-PCR) and sequencing. Using quantitative-PCR, we found that Group IVA cPLA2 and Group VI iPLA2 are the predominant PLA2 messages in the spinal cord. Western blotting and activity assays specific for Group IVA cPLA2 and Group VI iPLA2 verified the presence of these enzymes. PLA2 activity in spinal cord homogenates was suppressed by methyl arachidonyl fluorophosphonate (MAFP) and arachidonyl trifluoromethylketone (AACOCF3), mixed inhibitors of Group IVA cPLA2 and Group VI iPLA2 as well as by bromoenol lactone (BEL), a Group VI iPLA2 inhibitor. The spinal expression of PLA2 mRNA or protein was not altered in the face of peripheral inflammation. Secondly, we showed that intrathecal (i.t.) administration of MAFP and AACOCF3, but not BEL, dose-dependently prevented thermal hyperalgesia induced by intraplantar carrageenan as well as formalin-induced flinching. Finally, i.t. injection of AACOCF3, at antihyperalgesic doses, decreased the release of prostaglandin E2 (PGE2) into spinal dialysate evoked by i.t. NMDA, while i.t. injection of BEL had no effect. Taken together, this work points to a role for constitutive Group IVA cPLA2 in spinal nociceptive processing.

Animals↗
PubMed2004

Inhibition of group IVA cytosolic phospholipase A2 by novel 2-oxoamides in vitro, in cells, and in vivo.

The Group IVA cytosolic phospholipase A(2) (GIVA PLA(2)) is a particularly attractive target for drug development because it is the rate-limiting provider of proinflammatory mediators. We previously reported the discovery of novel 2-oxoamides that inhibit GIVA PLA(2) [Kokotos, G.; et al. J. Med. Chem. 2002, 45, 2891-2893]. In the present work, we have further explored this class of inhibitors and found that the 2-oxoamide functionality is more potent when it contains a long 2-oxoacyl residue and a free carboxy group. Long-chain 2-oxoamides based on gamma-aminobutyric acid and gamma-norleucine are potent inhibitors of GIVA PLA(2). Such inhibitors act through a fast and reversible mode of inhibition in vitro, are able to block the production of arachidonic acid and prostaglandin E(2) in cells, and demonstrate potent in vivo anti-inflammatory and analgesic activity.

Amides↗
PubMed2004

Phospholipid binding and the activation of group IA secreted phospholipase A2.

Equilibrium dialysis was used to study the binding of two nonhydrolyzable, short chain phospholipid analogues to the secreted group IA phospholipase A(2) (PLA(2)), which has been shown to contain several phospholipid binding sites that dramatically affect activity. This study provides new insight into how these activations occur. One analogue contained a phosphorylethanolamine (DiC(6)SNPE) headgroup, while the other contained a phosphorylcholine (DiC(6)SNPC) headgroup. Using phospholipase D, we incorporated tritium into each analogue. No binding of DiC(6)SNPE to PLA(2) was observed under submicellar conditions. Addition of submicellar amounts of Triton X-100 resulted in a linear nonsaturating response to lipid concentration, suggestive of premicellar aggregation of the DiC(6)SNPE with Triton X-100 and PLA(2). Binding of DiC(6)SNPE when presented as Triton X-100 mixed micelles saturated at 0.93 binding sites per PLA(2) with a K(D) of 38 microM. Addition of sphingomyelin, a potent activator of PLA(2) hydrolysis of phosphorylethanolamine containing compounds, resulted in a 13-fold decrease in the K(D), to 2.8 microM. This suggests that changes in the catalytic site binding affinity contribute to "phosphatidylcholine activation". Binding of DiC(6)SNPC with 2.0 mM Triton X-100 showed positive cooperativity (Hill coefficient of 1.7), which saturated at 2.0 binding sites per PLA(2). No binding of either analogue was observed when the catalytic site was alkylated with p-bromophenacyl bromide. Since p-bromophenacyl bromide does not physically block the phosphatidylcholine activator site, this indicates that the two phosphatidylcholine binding sites interact. The binding studies show that DiC(6)SNPC binds cooperatively to two sites on group IA PLA(2), while DiC(6)SNPE binds to only one site.

Acetophenones↗
PubMed2004

The ABC's of Group IV cytosolic phospholipase A2.

The three known human Group IV phospholipase A2 (PLA2) paralogs, Group IVA, IVB and IVC, were overexpressed in Sf9 insect cells using the baculovirus expression system. An endogenous, calcium-independent PLA2 activity was identified in the insect cell lysates, which can be inhibited by bromoenol lactone (BEL). The Group IV PLA2 enzymes were characterized in overexpressing insect cell lysates in the presence of BEL, enabling their differentiation from the endogenous PLA2 activity. Group IVC PLA2 was found to have significant lysophospholipase activity, while Group IVB PLA2 did not. Of the three paralogs, only the Group IVA PLA2 shows enhanced activity in the presence of PIP2, which enables its differential detection in cell homogenates. RT-PCR was used to demonstrate the presence of all three enzymes in human U937 and human WISH cells, while only Group IVA and Group IVB PLA2 were detected in murine P388D1 cells and human astrocytes at the mRNA level.

Animals↗