Bromoenol Lactone Research Articles

Characterization of the lipolytic pathways that mediate free fatty acid release during Fas/CD95-induced apoptosis

We have undertaken a study to characterize the lipolytic pathway responsible for the generation of free fatty acids (FFA) during Fas/CD95-induced apoptosis in Jurkat cells. It was initially shown that the cellular lipid fraction that suffered the major quantitative decrease during Fas-induced apoptosis was that of phosphatidylcholine (PC). In addition, the secretion of palmitic acid-derived FFA was largely prevented by D609, an inhibitor of PC-specific phospholipase C (PC-PLC) and also by the diacylglycerol lipase (DAGL) inhibitor RHC-80267, suggesting that the secretion of these FFA during Fas-induced apoptosis is mediated by the generation of DAG by a PC-PLC activity and, sequentially, by a 1-DAGL activity which generates the FFA from its sn-1 position. The endocannabinoid 2-arachidonoyl glycerol (2-AG) should be generated as a sub-product of this pathway, but it did not accumulate inside the cells nor was secreted into the supernatant. Interestingly, the complete inhibition of free AA secretion during Fas-induced apoptosis was only achieved by using the AA trifluoromethylketone, which not only inhibits all types of phospholipase-A(2) (PLA(2)) activities, but also the described lytic activities on 2-AG. Using a combination of RHC-80267 and the iPLA(2)-specific inhibitor bromoenol lactone, it was shown that the DAGL pathway also cooperates with iPLA(2) in the generation of free arachidonate.

Calcium-independent phospholipase A2modulates cytosolic oxidant activity and contractile function in murine skeletal muscle cells

Phospholipase A2 (PLA2) activity supports production of reactive oxygen species (ROS) by mammalian cells. In skeletal muscle, endogenous ROS modulate the force of muscle contraction. We tested the hypothesis that skeletal muscle cells constitutively express the calcium-independent PLA2 (iPLA2) isoform and that iPLA2 modulates both cytosolic oxidant activity and contractile function. Experiments utilized differentiated C2C12 myotubes and a panel of striated muscles isolated from adult mice. Muscle preparations were processed for measurement of mRNA by real-time PCR, protein by immunoblot, cytosolic oxidant activity by the dichlorofluorescein oxidation assay, and contractile function by in vitro testing. We found that iPLA2 was constitutively expressed by all muscles tested (myotubes, diaphragm, soleus, extensor digitorum longus, gastrocnemius, heart) and that mRNA and protein levels were generally similar among muscles. Selective iPLA2 blockade by use of bromoenol lactone (10 microM) decreased cytosolic oxidant activity in myotubes and intact soleus muscle fibers. iPLA2 blockade also inhibited contractile function of unfatigued soleus muscles, shifting the force-frequency relationship rightward and depressing force production during acute fatigue. Each of these changes could be reproduced by selective depletion of superoxide anions using superoxide dismutase (1 kU/ml). These findings suggest that constitutively expressed iPLA2 modulates oxidant activity in skeletal muscle fibers by supporting ROS production, thereby influencing contractile properties and fatigue characteristics.

Late-Gestation Rat Myometrial Cells Express Multiple Isoforms of Phospholipase A2 That Mediate PCB 50-Induced Release of Arachidonic Acid with Coincident Prostaglandin Production

Previous reports have shown that ortho-substituted polychlorinated biphenyls (PCBs) are uterotonic and activate phospholipase A2 to release arachidonic acid (AA) from membrane phospholipids. AA serves as the precursor to various eicosanoids, which, in addition to AA itself, are capable of modulating uterine function. To examine whether PCBs stimulate phospholipase A2 (PLA2) to mobilize arachidonic acid from late-gestation rat uterus, primary cultures of gestation day 20 (gd20) rat myometrial cells (RMC) were labeled with 0.5 microCi 3H-AA prior to a 10-, 20-, or 30-min exposure to 2,2',4,6-tetrachlorobiphenyl (PCB 50) (1-50 microM) or 0.1% DMSO (solvent control). PCB 50 stimulated the release of 3H-AA from gd20 RMC in concentration- and time-dependent manners (p < 0.05). PCB 50 stimulation of RMC was attenuated with ethylene glycol bis(2-aminoethyl ether)-N,N,N'N'-tetraacetic acid (EGTA) and nifedipine, suggesting that AA release was dependent on the influx of extracellular calcium through L-type voltage-operated calcium channels. PCB 50-induced release of AA from RMC was also attenuated with the PLA2-specific inhibitors methyl arachidonyl fluorophosphonate (MAFP), bromoenol lactone (BEL), and manoalide (p < 0.05). Stimulation of PLA2 enzymes in response to PCB exposure occurred via p38 mitogen activated protein kinase (MAPK) activation as indicated by the significant attenuation of PCB 50-induced AA release from RMC in the presence of SB 202190. In addition to stimulating AA release, PCB 50 induced a significant production of prostaglandins from gd20 RMC compared with controls (p < 0.05). These results suggest that myometrial cells express multiple PLA2 isoforms that may serve as a target and effector for ortho-substituted PCB-mediated stimulation of uterine function through arachidonic acid and prostaglandin release.

Phosphorylation of AMPA receptor subunits is differentially regulated by phospholipase A 2 inhibitors

Our laboratory recently discovered that the phosphorylation of subunits forming the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) subtype of glutamate receptors is regulated by constitutive phospholipase A 2 (PLA 2) activity in rat brain sections. In the present investigation, arachidonyl trifluoromethyl ketone (AACOCF3) and bromoenol lactone (BEL) were used to compare the influence of calcium-dependent (cPLA 2) and calcium-independent (iPLA 2) enzymes on phosphorylation of AMPA and N-methyl- d-aspartate (NMDA) subtypes of glutamate receptors. Incubation of rat brain sections with 3 μM BEL enhanced phosphorylation on the serine (Ser) 831 residue of the AMPA receptor GluR1 subunit in synaptosomal P2 fractions, whereas AACOCF3 at the same concentration resulted in increased phosphorylation on residues Ser880/891 of GluR2/3 subunits. These effects were restricted to the AMPA receptor subtype as no changes in phosphorylation were elicited on the NMDA receptor NR1 subunit. The effects of BEL and AACOCF3 were not occluded during blockade of protein phosphatases since AMPA receptor phosphorylation was still apparent in the presence of okadaic acid, indicating that the PLA 2 inhibitor-induced increase in AMPA receptor phosphorylation does not rely on a decrease in dephosphorylation reactions. However, pretreatment of rat brain sections with a cell-permeable protein kinase C (PKC) inhibitor prevented BEL- and AACOCF3-induced phosphorylation on the Ser831 and Ser880/891 sites of GluR1 and GluR2/3 subunits, respectively. These results suggest that constitutive cPLA 2 and iPLA 2 systems may differentially influence AMPA receptor properties and function in the rat brain through mechanisms involving PKC activity.

Potential role for mast cell tryptase in recruitment of inflammatory cells to endothelium

Recent research suggests that activation of protease-activated receptors (PARs) on the surface of endothelial and epithelial cells may play a role in general mechanisms of inflammation. We hypothesized that mast cell tryptase activation of endothelial cell PAR-2 is coupled to increased calcium-independent PLA2 (iPLA2) activity and increased platelet-activating factor (PAF) production that may play a role in inflammatory cell recruitment at sites of vascular injury. Stimulation of human coronary artery endothelial cells (HCAEC) with 20 ng/ml tryptase increased iPLA2 activity, arachidonic acid release, and PAF production. These tryptase-stimulated responses were inhibited by pretreatment with the iPLA2-selective inhibitor bromoenol lactone (BEL; 5 microM, 10 min). Similar patterns of increased iPLA2 activity and PAF production were also seen when HCAEC were treated with SLIGKV, which represents the tethered ligand sequence for the human PAR-2 once the receptor is cleaved by tryptase. Tryptase stimulation also increased cell surface expression of P-selectin, decreased electrical resistance, and increased neutrophil adherence to the endothelial cell monolayer. The tryptase-stimulated increases in both cell surface P-selectin expression and neutrophil adhesion were also inhibited with BEL pretreatment. We conclude that tryptase stimulation of HCAEC contributes importantly to early inflammatory events after vascular injury by activation of iPLA2, leading to arachidonic acid release, PAF production, cell surface P-selectin expression, and increased neutrophil adherence.

Genetic and Pharmacologic Evidence That Calcium-independent Phospholipase A2β Regulates Virus-induced Inducible Nitric-oxide Synthase Expression by Macrophages

Recent evidence supports a regulatory role for the calcium-independent phospholipase A2 (iPLA2) in the antiviral response of inducible nitric-oxide synthase (iNOS) expression by macrophages. Because two mammalian isoforms of iPLA2 (iPLA2beta and iPLA2gamma) have been cloned and characterized, the aim of this study was to identify the specific isoform(s) in macrophages that regulates the expression of iNOS in response to virus infection. Bromoenol lactone (BEL), a suicide substrate inhibitor of iPLA2, inhibits the activity of both isoforms at low micromolar concentrations. However, the R- and S-enantiomers of BEL display approximately 10-fold greater potency for inhibition of the enzymatic activity of iPLA2gamma and iPLA2beta, respectively. In this study, we show that the iPLA2beta-selective (S)-BEL inhibits encephalomyocarditis virus (EMCV)-induced iNOS expression, nitric oxide production, and iPLA2 enzymatic activity in macrophages in a concentration-related manner that closely resembles the inhibitory properties of racemic BEL. cAMP response element-binding protein (CREB) is one downstream target of iPLA2 that is required for the transcriptional activation of iNOS in response to virus infection, and consistent with the effects of BEL enantiomers on iNOS expression, (S)-BEL more effectively inhibits EMCV-induced CREB phosphorylation than (R)-BEL in macrophages. Using macrophages isolated from iPLA2beta-null mice, virus infection fails to stimulate iNOS mRNA accumulation and protein expression, thus providing genetic evidence that iPLA2beta is required for EMCV-induced iNOS expression. These findings provide evidence for a signaling role for iPLA2beta in virus-induced iNOS expression by macrophages.

2n-fatty acids from phosphatidylcholine label sphingolipids—A novel role of phospholipase A 2?

In order to find out whether there is a phospholipase A 2 (PLA 2)-mediated link between glycerophospholipids and sphingolipids, L929 cells were labeled with 1n-palmitoyl-2n-[1- 14C]palmitoyl phosphatidylcholine for 16–18 h or 90 min. After labeling for 16–18 h, 14C-sphingomyelin (SM), 14C-ceramide and 14C-sphingosine were demonstrated on autoradiograms of thin layer chromatograms of untreated or mildly hydrolyzed lipid extracts in different chromatographic systems. Strong hydrolysis of labeled SM proved that both possible moieties of SM, sphingosine and acyl moiety, had been labeled. The identity of SM and its enzymatic degradation product, ceramide, was verified by mass spectrometry. The label in SM-derived ceramide was demonstrated on an autoradiogram after thin layer chromatography. The inhibitor of (dihydro)ceramide synthase fumonisin B1 suppressed the label in sphingolipids significantly during 16–18 h (ceramide and SM), as well as during 90-min labeling (SM). The presence of inhibitors of PLA 2 (bromoenol lactone, aristolochic acid and quinacrine dihydrochloride) diminished the label in SM significantly during the 90-min labeling. These results demonstrate a close metabolic relationship between glycerophospholipids and sphingolipids and give evidence for a novel role of PLA 2.

Synthesis of Iodoenol Lactone Derivatives from the Baylis—Hillman Adducts Using Iodolactonization Protocol.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Inhibition of calcium-independent phospholipase A2 activity in rat hippocampus impairs acquisition of short- and long-term memory

Phospholipase A(2) (PLA(2)) is a family of enzymes that cleave membrane phospholipids generating important lipid mediators in signal transduction. In rat hippocampal slices, both intracellular cytosolic Ca(2+)-dependent PLA(2) (cPLA(2)) and Ca(2+)-independent PLA(2) (iPLA(2)) have been implicated in mechanisms of synaptic plasticity underlying memory processes. In mice, intraperitoneal injections of a selective iPLA(2) inhibitor impaired spatial learning. Accordingly, reduced cPLA(2) and iPLA(2) activities were found in postmortem hippocampus of patients with Alzheimer's disease. This study investigates the effects of injections of PLA(2) inhibitors directly into rat hippocampus on the acquisition of short-term (STM) and long-term memory (LTM) of a one-trial step-down inhibitory avoidance (IA) task. Wistar rats were bilaterally implanted with cannulae in the CA1 region of the dorsal hippocampus. After surgery, the rats received bilateral injections of a vehicle, or of dual cPLA(2) and iPLA(2) inhibitors (MAFP or PACOCF(3)), or a selective iPLA(2) inhibitor (bromoenol lactone) before training in IA. The animals were tested 1.5 h (for STM) and 24 h (for LTM) after training. Significant inhibition of iPLA(2) activity in rat hippocampus impaired acquisition of STM and LTM. Memory impairment did not result from neuronal death after iPLA(2) inhibition. Moreover, IA training per se increased significantly hippocampal PLA(2) activity. The present results suggest a functional effect of hippocampal PLA(2) on the neurochemistry of memory acquisition and support the hypothesis that reduced PLA(2) activity may contribute to memory impairment in Alzheimer's disease.

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.

Chymotrypsin‐like proteases contribute to human monocytic THP‐1 cell as well as human microglial neurotoxicity

Activated microglia have been observed in various neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis, and multiple sclerosis. They may exacerbate neuronal damage by secreting various toxic molecules. The list of candidate toxins includes proteases. Since it is currently not known which, if any, proteases are involved in human microglia neurotoxicity, we studied the effects of a panel of protease inhibitors on the toxicity of cell-free supernatants of stimulated human microglia and THP-1 monocytic cells to human SH-SY5Y cells. Five structurally distinct inhibitors that are known to inhibit chymotrypsin-like proteases were partially protective. They included chymostatin, AEBSF (Pefabloc SC), alpha1-antichymotrypsin, bromoenol lactone, and 3,4-dichloroisocoumarin. The data suggest that certain protease inhibitors could inhibit microglial-mediated toxicity. They might represent a novel class of drugs with benefit in diseases where overactivity of microglia contributes to the pathogenesis.

Ca 2+-independent phospholipase A2 participates in the vesicular transport of milk proteins

Changes in the lipid composition of intracellular membranes are believed to take part in the molecular processes that sustain traffic between organelles of the endocytic and exocytic transport pathways. Here, we investigated the participation of the calcium-independent phospholipase A2 in the secretory pathway of mammary epithelial cells. Treatment with bromoenol lactone, a suicide substrate which interferes with the production of lysophospholipids by the calcium-independent phospholipase A2, resulted in the reduction of milk proteins secretion. The inhibitor slowed down transport of the caseins from the endoplasmic reticulum to the Golgi apparatus and affected the distribution of p58 and p23, indicating that the optimal process of transport of these proteins between the endoplasmic reticulum, the endoplasmic reticulum/Golgi intermediate compartment and/or the cis-side of the Golgi was dependent upon the production of lysolipids. Moreover, bromoenol lactone was found to delay the rate of protein transport from the trans-Golgi network to the plasma membrane. Concomitantly, membrane-bound structures containing casein accumulated in the juxtanuclear Golgi region. We concluded from these results that efficient formation of post-Golgi carriers also requires the phospholipase activity. These data further support the participation of calcium-independent phospholipase A2 in membrane trafficking and shed a new light on the tubulo/vesicular transport of milk protein through the secretory pathway.

Calcium-independent phospholipase A2 is regulated by a novel protein kinase C in human coronary artery endothelial cells

We demonstrated previously that thrombin stimulation of endothelial cells activates a membrane-associated, Ca(2+)-independent phospholipase A2 (iPLA2) that selectively hydrolyzes arachidonylated plasmalogen phospholipids. We report that incubation of human coronary artery endothelial cells (HCAEC) with phorbol 12-myristate 13-acetate (PMA) to activate protein kinase C (PKC) resulted in hydrolysis of cellular phospholipids similar to that observed with thrombin stimulation (0.05 IU/ml; 10 min). Thrombin stimulation resulted in a decrease in arachidonylated plasmenylcholine (2.7 +/- 0.1 vs. 5.3 +/- 0.4 nmol PO4/mg of protein) and plasmenylethanolamine (7.5 +/- 1.0 vs. 12.0 +/- 0.9 nmol PO4/mg of protein). Incubation with PMA resulted in decreases in arachidonylated plasmenylcholine (3.2 +/- 0.3 nmol PO4/mg of protein) and plasmenylethanolamine (6.0 +/- 1.0 nmol PO4/mg of protein). Incubation of HCAEC with the selective iPLA2 inhibitor bromoenol lactone (5 mM; 10 min) inhibited accelerated plasmalogen phospholipid hydrolysis in response to both PMA and thrombin stimulation. Incubation of HCAEC with PMA (100 nM; 5 min) resulted in increased arachidonic acid release (7.1 +/- 0.3 vs. 1.1 +/- 0.1%) and increased production of lysoplasmenylcholine (1.4 +/- 0.2 vs. 0.6 +/- 0.1 nmol PO4/mg of protein), similar to the responses observed with thrombin stimulation. Downregulation of PKC by prolonged exposure to PMA (100 nM; 24 h) completely inhibited thrombin-stimulated increases in arachidonic acid release (7.1 +/- 0.6 to 0.5 +/- 0.1%) and lysoplasmenylcholine production (2.0 +/- 0.1 to 0.2 +/- 0.1 nmol PO4/mg of protein). These data suggest that PKC activates iPLA2 in HCAEC, leading to accelerated plasmalogen phospholipid hydrolysis and increased phospholipid metabolite production.

Group VIA Phospholipase A2 Forms a Signaling Complex with the Calcium/Calmodulin-dependent Protein Kinase IIβ Expressed in Pancreatic Islet β-Cells

Insulin-secreting pancreatic islet beta-cells express a Group VIA Ca(2+)-independent phospholipase A(2) (iPLA(2)beta) that contains a calmodulin binding site and protein interaction domains. We identified Ca(2+)/calmodulin-dependent protein kinase IIbeta (CaMKIIbeta) as a potential iPLA(2)beta-interacting protein by yeast two-hybrid screening of a cDNA library using iPLA(2)beta cDNA as bait. Cloning CaMKIIbeta cDNA from a rat islet library revealed that one dominant CaMKIIbeta isoform mRNA is expressed by adult islets and is not observed in brain or neonatal islets and that there is high conservation of the isoform expressed by rat and human beta-cells. Binary two-hybrid assays using DNA encoding this isoform as bait and iPLA(2)beta DNA as prey confirmed interaction of the enzymes, as did assays with CaMKIIbeta as prey and iPLA(2)beta bait. His-tagged CaMKIIbeta immobilized on metal affinity matrices bound iPLA(2)beta, and this did not require exogenous calmodulin and was not prevented by a calmodulin antagonist or the Ca(2+) chelator EGTA. Activities of both enzymes increased upon their association, and iPLA(2)beta reaction products reduced CaMKIIbeta activity. Both the iPLA(2)beta inhibitor bromoenol lactone and the CaMKIIbeta inhibitor KN93 reduced arachidonate release from INS-1 insulinoma cells, and both inhibit insulin secretion. CaMKIIbeta and iPLA(2)beta can be coimmunoprecipitated from INS-1 cells, and forskolin, which amplifies glucose-induced insulin secretion, increases the abundance of the immunoprecipitatable complex. These findings suggest that iPLA(2)beta and CaMKIIbeta form a signaling complex in beta-cells, consistent with reports that both enzymes participate in insulin secretion and that their expression is coinduced upon differentiation of pancreatic progenitor to endocrine progenitor cells.

Modulation of phospholipase A2 activity in primary cultures of rat cortical neurons

In neurons, phospholipase A2 (PLA2) plays a central role in the regulation of membrane phospholipid metabolism. We have addressed the pharmacological modulation of PLA2 in primary cultures of rat cortical neurons. Inhibition curves were obtained in 4 day-in-culture neurons treated for 30 minutes with either the dual PLA2 inhibitor methyl arachidonyl fluorophosphonate (MAFP), or the iPLA2 inhibitor bromoenol lactone (BEL). Full inhibition was achieved with 100 and 250 microM of MAFP, or 10 and 20 microM of BEL. Conversely, a dose-dependent activation of PLA2 was obtained with 10-20 microg/ml of melitin. PLA2 inhibition with MAFP or BEL was not acutely toxic for cultured neurons. However, sustained inhibition of the enzyme precluded the development of neurites, and resulted in long-term loss of neuronal viability. We present a model of pharmacological challenge of PLA2 in vitro, which can be further used to address the involvement of the enzyme in neurodevelopment and neurodegeneration models.

AMPA receptor phosphorylation is selectively regulated by constitutive phospholipase A2and 5-lipoxygenase activities

The present investigation provides the first indication that constitutive, calcium-independent phospholipase A2 activity (iPLA2) modulates phosphorylation of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) subtype of glutamate receptors. Preincubation of frozen-thawed brain sections with two iPLA2 inhibitors, bromoenol lactone (BEL) or palmitoyl trifluoromethyl ketone (PACO), produced a dose-dependent enhancement in phosphorylation at both Ser831 and Ser845 sites on the GluR1 subunit of AMPA receptors. This effect was not associated with changes in phosphorylation at the Ser sites of either the GluR2/3 subunits of AMPA receptors or the NR1 subunits of N-methyl-D-aspartate (NMDA) receptors, nor was it reproduced by inhibition of the calcium-dependent form of PLA2 activity. These results suggest that the effects of these inhibitors are selective to GluR1 subunits and that they are dependent on iPLA2 activity. The ability of iPLA2 inhibitors to increase GluR1 phosphorylation was mimicked by the 5-lipoxygenase (5-LO) inhibitor MK-886, but not by blockers of 12-lipoxygenase (12-LO) or cyclooxygenase. Additional experiments indicated that calcium-mediated truncation of GluR1 subunits was reduced by iPLA2 inhibitors, an effect that was not correlated with overall changes in the distribution of AMPA receptors between intracellular and membrane compartments prepared from whole brain sections. However, quantitative autoradiographic analysis indicated enhanced 3H-AMPA binding to the CA1 stratum radiatum of the hippocampus in BEL-treated sections. Saturation kinetics experiments demonstrated that this binding augmentation was due to an increase in the maximal number of AMPA binding sites. Altogether, our results point to the conclusion that basal iPLA2 activity, through the generation of 5-LO metabolites, regulates AMPA receptor phosphorylation of GluR1 subunits, an effect that might selectively influence the number of membrane receptors in area CA1 of the hippocampus.

Involvement of phospholipase A2 and lipoxygenase in lipopolysaccharide-induced inducible nitric oxide synthase expression in glial cells

The present study underlines the importance of phospholipase A2 (PLA2)- and lipoxygenase (LO)-mediated signaling processes in the regulation of inducible nitric oxide synthase (iNOS) gene expression. In glial cells, lipopolysaccharide (LPS) induced the activities of PLA2 (calcium-independent PLA2; iPLA2 and cytosolic PLA2; cPLA2) as well as gene expression of iNOS. The inhibition of cPLA2 by methyl arachidonyl fluorophosphates (MAFP) or antisense oligomer against cPLA2 and inhibition of iPLA2 by bromoenol lactone reduced the LPS-induced iNOS gene expression and NFkappaB activation. In addition, the inhibition of LO by nordihydroguaiaretic acid (NDGA; general LO inhibitor) or MK886 (5-LO inhibitor), but not baicalein (12-LO inhibitor), completely abrogated the LPS-induced iNOS expression. Because NDGA could abrogate the LPS-induced activation of NFkappaB, while MK886 had no effect on it, LO-mediated inhibition of iNOS gene induction by LPS may involve an NFkappaB-dependent or -independent (by 5-LO) pathway. In contrast to LO, however, the cyclooxygenase (COX) may not be involved in the regulation of LPS-mediated induction of iNOS gene because COX inhibition by indomethacin (general COX inhibitor), SC560 (COX-1 inhibitor), and NS398 (COX-2 inhibitor) affected neither the LPS-induced iNOS expression nor activation of NFkappaB. These results indicate a role for cPLA2 and iPLA2 in LPS-mediated iNOS gene induction in glial cells and the involvement of LO in these reactions.

Synthesis of Iodoenol Lactone Derivatives from the Baylis-Hillman Adducts Using Iodolactonization Protocol

Recently, we have reported the preparation of benzocycloheptene derivatives from the intramolecular Friedel-Crafts reaction of triple bond-tethered methyl cinnamates. In the reaction, we could not obtain any lactone derivatives, which could be formed via the intramolecular attack of the oxygen atom of ester moiety to the activated triple bond by the acid catalyst (Scheme 1). We think that there may be a certain competition between arene moiety and ester group as nucleophiles toward the activated triple bond. From the previous results we concluded that the arene moiety is more nucleophilic than the oxygen atom of ester toward the in-situ generated vinyl cation. In order to increase the nucleophilicity of the oxygen atom of ester group we decided to hydrolyze the ester into acid and to examine the feasibility of iodolactonization in the presence of NaHCO3 as shown in Scheme 1. From the reaction, we could expect the formation of two types of lactones, 5-membered iodoenol lactone or 6membered α-pyrone. Iodoenol lactone derivatives have been studied extensively due to their usefulness as synthetic intermediates and their biological activities. Low molecular weight αpyrones have been shown to be potent HIV-1 protease inhibitors. Recently, Larock and Rossi have reported an elegant synthesis of isocoumarins and α-pyrones via electrophilic cyclization. We were stimulated by their works and envisioned that we could synthesize suitably substituted α-pyrones or iodoenol lactone derivatives starting from the Baylis-Hillman adducts as mentioned in Scheme 1. The starting material 1 was prepared from Baylis-Hillman acetate according to our previous paper. Base hydrolysis of 1 was carried out using LiOH in aqueous THF at room temperature to give 2 in 53-69% yields. The use of KOH or NaOH was less efficient. With 2 in our hands, we initially examined the reaction with iodine in the presence of NaHCO3 in CH3CN, which is a typical condition for the iodolactonization. Unfortunately, complex mixtures were observed on TLC. Among the various conditions, we finally found that the use of THF as solvent afforded the desired iodoenol lactones 3 in moderate to good yields (56-90%). The formation of 3 can be explained as iodolactonization involving iodonium intermediate (I) via 5-exo-dig manner as shown in Scheme 2. We could not find nor isolate the corresponding six-membered α-pyrones, which can be formed via 6-endo-dig fashion. As the other electrophile source we examined H2SO4 and N-bromosuccinimide (NBS) in order to check the possibility for the synthesis of protonor bromine-attached enol lactones. Fortunately, desired compounds were synthesized in moderate yields. As shown in Scheme 3, bromolactonization of 2a (THF, NBS, NaHCO3) gave the bromoenol lactone 4 in 72% yield. Sulfuric acid-mediated cyclization of 2c in acetonitrile afforded enol lactone 5 in 53% yield at

Identification and distribution of endoplasmic reticulum iPLA 2

Our laboratory demonstrated that endoplasmic reticulum iPLA 2 (ER-iPLA 2) activity protects renal cells from oxidant-induced cell death and lipid peroxidation. The goals of this study were to determine the PLA 2 isoform(s) responsible for ER-iPLA 2 activity in different species and tissues. ER-iPLA 2 activity was observed in microsomes from rabbit and rat kidney, heart, and brain as well as in human kidney (Caki-1 and HEK293) and glioblastoma (A172) cell lines. Reverse transcriptase-polymerase chain reaction results demonstrated the presence of iPLA 2γ (group VIB PLA 2) message in all tissues tested. Immunoblot analysis and PLA 2 inhibitor studies with methyl arachidonyl fluorophosphonate and enantiomers of bromoenol lactone demonstrated that the ER-iPLA 2 in rabbit kidney and heart and rat kidney is iPLA 2γ. These results demonstrate the expression of ER-iPLA 2γ (group VIB) across species and tissues, and suggest that iPLA 2γ may play critical roles in oxidant-induced cell injury.

Protease-activated receptor stimulation activates a Ca2+-independent phospholipase A2in bladder microvascular endothelial cells

Increased mast cell numbers and mast cell activation represent one of the prevalent etiologic theories for interstitial cystitis, an inflammatory condition in the bladder. This study was designed primarily to determine whether increased mast cell tryptase in the bladder wall may play a role in activating bladder endothelial cell phospholipase A(2) (PLA(2)), leading to increased inflammatory phospholipid metabolite accumulation, which may propagate the inflammatory process. We stimulated human bladder microvascular endothelial cells with thrombin or tryptase and measured the activation of PLA(2) and the production of multiple membrane phospholipid-derived inflammatory mediators. Thrombin and tryptase stimulation resulted in activation of a Ca(2+)-independent PLA(2), leading to increased release of arachidonic acid and prostacyclin and increased production of platelet-activating factor. These responses were blocked completely by pretreatment of human bladder microvascular endothelial cells with the Ca(2+)-independent PLA(2)-selective inhibitor bromoenol lactone. The combination of increased prostacyclin and platelet-activating factor in the bladder circulation may result in vasodilation and increased polymorphonuclear leukocyte adherence to the endothelium and may facilitate recruitment of polymorphonuclear leukocytes to the bladder wall of patients with interstitial cystitis.