Methyl Arachidonyl Fluorophosphonate Research Articles

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.

Lysophospholipase I identified as a ghrelin deacylation enzyme in rat stomach

Ghrelin, discovered in rat stomach as an endogenous growth hormone secretagogue, is octanoylated at the Ser3 residue. Since this octanoylation is essential for the functions of ghrelin, the enzymes that catalyze acylation for ghrelin biosynthesis and deacylation (deactivation step) must be considered as important regulators. We found that rat stomach homogenate contained ghrelin deacylation activity, and we isolated the active fractions by column chromatography. After sequencing and expressing candidate proteins, the ghrelin deacylation enzyme in the stomach was identified as lysophospholipase I (LysoPLA I). The enzyme properties were examined using recombinant rat LysoPLA I expressed in Escherichia coli. K m and V max values were determined as 6.5 μM and 2.3 μmol/min/mg for ghrelin and 2.2 × 10 2 μM and 0.5 μmol/min/mg for lysophosphatidylcholine (LysoPC), respectively. The deacylation of both substrates was inhibited by methyl arachidonyl fluorophosphonate (MAFP), which is known as an irreversible inhibitor of LysoPLA I. These results reveal that LysoPLA I catalyzes the removal of n-octanoic acid from ghrelin to form des-acyl ghrelin. Identification of the ghrelin deacylation enzyme in the stomach and a deacylation inhibitor will be helpful in investigating ghrelin biosynthesis.

Up-regulation of the enzymes involved in prostacyclin synthesis via Ras induces vascular endothelial growth factor

The constitutive activation of Ras is an important step in the development and progression of several different cancers and is known to increase the level of cyclooxygenase 2 (COX-2). Prostaglandins are the downstream bioactive lipid mediators produced by the COX-2 enzyme. We sought to determine the role of Ras-induced up-regulation of the enzymes involved in prostacyclin biosynthesis in nontransformed rat intestinal epithelial cells (IECs). Messenger RNA (mRNA) and protein expression were analyzed by Northern and Western analysis, respectively, to determine the level of enzymes induced by Ras. In vitro assays were used to determine the production of vascular endothelial growth factor (VEGF) and prostaglandins as well as the promoter and enzymatic activation of the rate-limiting enzyme in prostaglandin production (phospholipase A(2) [cPLA(2)]). The inducible expression of Ha-Ras(V12) increased the production of prostaglandin (PG)F(2alpha) and prostacyclin by 2- and 13-fold, respectively. The induction of Ha-Ras(V12) also up-regulated the mRNA and protein levels of cPLA(2), COX-2, and prostacyclin synthase, as well as the promoter and enzyme activity of cPLA(2). Furthermore, oncogenic Ras increased the production of the pro-angiogenic factor VEGF. The increase of VEGF was abolished after treatment with celecoxib, a selective COX-2 inhibitor. The addition of PGI 2 alone also induced the expression of VEGF. Inducible Ha-Ras(V12) increases the production of PGI(2) through the coordinate up-regulation of cPLA(2), COX-2, and prostacyclin synthase (PGIS). The production of PGI(2) leads to an increase in the level of the pro-angiogenic factor VEGF, which is known to play a crucial role in the regulation of tumor-associated angiogenesis.

Translocation of phospholipase A 2 to membranes by oxidized LDL and hydroxyoctadecadienoic acid to contribute to cholesteryl ester formation

We examined the mechanisms underlying the activation of group IVA cytosolic phospholipase A 2 (cPLA 2α) contributing to the supply of fatty acids required for the formation of cholesteryl ester in oxidized low-density lipoprotein (oxLDL)-stimulated macrophages. The possible involvement of oxidized lipids was also examined. In [ 3H]arachidonic acid-labeled mouse peritoneal macrophages, oxLDL stimulated the release of arachidonic acid, which was suppressed by methyl arachidonyl fluorophosphonate (MAFP), a cPLA 2α inhibitor. oxLDL induced an increase in PLA 2α levels in the membrane fraction without affecting those in whole cells or the activity in the lysate. Among 13-hydroxyoctadecadienoic acid (13-HODE), 7-ketocholesterol, and 25-hydroxycholesterol, oxidized lipids present in oxLDL particles, only 13-HODE induced the release of arachidonic acid, which was also sensitive to MAFP. Under conditions where addition of Ca 2+ to the cell lysate induced an increase in cPLA 2α protein in the membrane fraction, preincubation with 13-HODE facilitated the Ca 2+-dependent translocation of cPLA 2α. Furthermore, 13-HODE increased cholesteryl ester formation in the presence of [ 3H]cholesterol. These results suggest that 13-HODE mediates the oxLDL-induced activation of cPLA 2α through an increase in cPLA 2α protein in the membranes, thus contributing, in part, to the supply of fatty acids required for the esterification of cholesterol in macrophages.

Cyclohexylcarbamic acid 3'- or 4'-substituted biphenyl-3-yl esters as fatty acid amide hydrolase inhibitors: synthesis, quantitative structure-activity relationships, and molecular modeling studies.

Fatty acid amide hydrolase (FAAH) is a promising target for modulating endocannabinoid and fatty acid ethanolamide signaling, which may have important therapeutic potential. We recently described a new class of O-arylcarbamate inhibitors of FAAH, including the cyclohexylcarbamic acid biphenyl-3-yl ester URB524 (half-maximal inhibitory concentration, IC(50) = 63 nM), which have significant anxiolytic-like properties in rats. In the present study, by introducing a selected group of substituents at the meta and para positions of the distal phenyl ring of URB524, we have characterized structure-activity profiles for this series of compounds and shown that introduction of small polar groups in the meta position greatly improves inhibitory potency. Most potent in the series was the m-carbamoyl derivative URB597 (4i, IC(50) = 4.6 nM). Furthermore, quantitative structure-activity relationship (QSAR) analysis of an extended set of meta-substituted derivatives revealed a negative correlation between potency and lipophilicity and suggested that small-sized substituents may undertake polar interactions with the binding pocket of the enzyme. Docking studies and molecular dynamics simulations, using the crystal structure of FAAH, indicated that the O-biphenyl scaffold of the carbamate inhibitors can be accommodated within a lipophilic region of the substrate-binding site, where their folded shape mimics the initial 10-12 carbon atoms of the arachidonyl moiety of anandamide (a natural FAAH substrate) and methyl arachidonyl fluorophosphonate (a nonselective FAAH inhibitor). Moreover, substituents at the meta position of the distal phenyl ring can form hydrogen bonds with atoms located on the polar section of a narrow channel pointing toward the membrane-associated side of the enzyme. The structure-activity characterization reported here should help optimize the pharmacodynamic and pharmacokinetic properties of this class of compounds.

Role of Group VIA Calcium-independent Phospholipase A2 in Arachidonic Acid Release, Phospholipid Fatty Acid Incorporation, and Apoptosis in U937 Cells Responding to Hydrogen Peroxide

Group VIA calcium-independent phospholipase A2 (iPLA2) has been shown to play a major role in regulating basal phospholipid deacylation reactions in certain cell types. More recently, roles for this enzyme have also been suggested in the destruction of membrane phospholipid during apoptosis and after oxidant injury. Proposed iPLA2 roles have rested heavily on the use of bromoenol lactone as an iPLA2-specific inhibitor, but this compound actually inhibits other enzymes and lipid pathways unrelated to PLA2, which makes it difficult to define the contribution of iPLA2 to specific functions. In previous work, we pioneered the use of antisense technology to decrease cellular iPLA2 activity as an alternative approach to study iPLA2 functions. In the present study, we followed the opposite strategy and prepared U937 cells that exhibited enhanced iPLA activity by stably expressing a plasmid containing iPLA2 cDNA. Compared with control cells, the iPLA2 -overexpressing U937 cells showed elevated responses to hydrogen peroxide with regard to both arachidonic acid mobilization and incorporation of the fatty acid into phospholipids, thus providing additional evidence for the key role that iPLA2 plays in these events. Long-term exposure of the cells to hydrogen peroxide resulted in cell death by apoptosis, and this process was accelerated in the iPLA2-overexpressing cells. Increased phospholipid hydrolysis and fatty acid release also occurred in these cells. Unexpectedly, however, abrogation of U937 cell iPLA2 activity by either methyl arachidonyl fluorophosphonate or an antisense oligonucleotide did not delay or decrease the extent of apoptosis induced by hydrogen peroxide. These results indicate that, although iPLA2-mediated phospholipid hydrolysis occurs during apoptosis, iPLA2 may actually be dispensable for the apoptotic process to occur. Thus, beyond a mere destructive role, iPLA2 may play other roles during apoptosis.

RNA interference suggests a primary role for monoacylglycerol lipase in the degradation of the endocannabinoid 2-arachidonoylglycerol.

The endogenous cannabinoid 2-arachidonoylglycerol (2-AG) is produced by neurons and other cells in a stimulus-dependent manner and undergoes rapid biological inactivation through transport into cells and catalytic hydrolysis. The enzymatic pathways responsible for 2-AG degradation are only partially understood. We have shown previously that overexpression of monoacylglycerol lipase (MGL), a cytosolic serine hydrolase that cleaves 1- and 2-monoacylglycerols to fatty acid and glycerol, reduces stimulus-dependent 2-AG accumulation in primary cultures of rat brain neurons. We report here that RNA interference-mediated silencing of MGL expression greatly enhances 2-AG accumulation in HeLa cells. After stimulation with the calcium ionophore ionomycin, 2-AG levels in MGL-silenced cells were comparable with those found in cells in which 2-AG degradation had been blocked using methyl arachidonyl fluorophosphonate, a nonselective inhibitor of 2-AG hydrolysis. The results indicate that MGL plays an important role in the degradation of endogenous 2-AG in intact HeLa cells. Furthermore, immunodepletion experiments show that MGL accounts for at least 50% of the total 2-AG-hydrolyzing activity in soluble fractions of rat brain, suggesting that this enzyme also contributes to 2-AG deactivation in the central nervous system.

Catalytic features, regulation and function of myocardial phospholipase A2.

Phospholipase A2 (PLA2) catalyzes the hydrolysis of sn-2 fatty acids from membrane phospholipids resulting in the production of several biologically active phospholipid metabolites such as lysophospholipids, arachidonic acid, eicosanoids and platelet-activating factor. The majority of myocardial PLA2 activity is membrane-associated and does not require Ca2+ for activity (iPLA2). Myocardial iPLA2 demonstrates unique characteristics when compared to other PLA2 isoforms described previously, including a selectivity for plasmalogen phospholipids and resistance to inhibition by methyl arachidonyl fluorophosphonate. Activation of myocardial iPLA2 results in the production of lysoplasmenylcholine and arachidonic acid, both of which can change the electrophysiologic properties of the myocardium. Arachidonic acid can modulate ion channel activity via protein kinase C activation and has been demonstrated to decrease gap junctional conductance. Lysoplasmenylcholine directly produces action potential derangements and alters calcium cycling in cardiac myocytes. Thus, inhibition of iPLA2 activity to block production of phospholipid metabolites that mediate pathologic changes in the myocardium would be of considerable benefit. However, there are situations where inhibition of PLA2 activity would be detrimental to the myocardium, in particular if iPLA2 acts as a phospholipid repair enzyme following oxidative damage. Although little is known regarding the function of cPLA2 or sPLA2 in the myocardium, it is possible that they may be important for signal transduction or may modulate the activity of iPLA2.

Lactosylceramide recruits PKCalpha/epsilon and phospholipase A2 to stimulate PECAM-1 expression in human monocytes and adhesion to endothelial cells.

Despite the importance of platelet/endothelial cell adhesion molecule-1 (PECAM-1, CD31) in the adhesion and diapedesis of monocytes/lymphocytes, little is known about the mechanisms by which it is regulated. We explored the role of a glycosphingolipid, lactosylceramide (LacCer), in modulating PECAM-1 expression and cell adhesion in human monocytes. We observed that LacCer specifically exerted a time-dependent increase in PECAM-1 expression in U-937 cells. Maximal increase in PECAM-1 protein occurred after incubation with LacCer for 60 min. LacCer activated PKCalpha and -epsilon by translocating them from cytosol to membrane. This was accompanied by the activation of phospholipase A(2) (PLA(2)) and the increase of cell adhesion, which were abrogated by chelerythrine chloride, 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide and 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole (GO 6976) (PKC inhibitors). Similarly, bromoenol lactone (a Ca(2+)-independent PLA(2) inhibitor) and methyl arachidonyl fluorophosphonate (an inhibitor of cytosolic PLA(2) and Ca(2+)-independent PLA(2)) inhibited LacCer-induced PLA(2) activity. Bromophenacyl bromide (a PLA(2) inhibitor) abrogated LacCer-induced PECAM-1 expression, and this was bypassed by arachidonic acid. Furthermore, the arachidonate-induced up-regulation of PECAM-1 was abrogated by indomethacin [a cyclooxygenase (COX)-1 and -2 inhibitor] or N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide (a COX-2 inhibitor) but not nordihydroguaiaretic acid (a lipoxygenase inhibitor). In sum, PKCalpha/epsilon are the primary targets for the activation of LacCer. Downstream activation of intracellular Ca(2+)-independent PLA(2) and/or cytosolic PLA(2) results in the production of arachidonic acid, which in turn serves as a precursor for prostaglandins that subsequently stimulate PECAM-1 expression and cell adhesion. These findings may be relevant in explaining the role of LacCer in the regulation of PECAM-1 and related pathophysiology.

Cyclic stretch-induced cPLA2 mediates ERK 1/2 signaling in rabbit proximal tubule cells

Recent evidence from this laboratory have demonstrated a critical role of phospholipase A2 (PLA2) and arachidonic acid in angiotensin II type 2 (AT2) receptor-mediated kinase activation in renal epithelium independent of phosphoinositide- specific phospholipase C (PLC) and without the necessity of eicosanoid biosynthesis. In the present study, we investigated whether cyclic stress phosphorylates and activates the mitogen-activated protein kinase (MAPK) pathway and whether PLA2 activation mediates mechanotransduction in renal epithelial cells. The rational for studying kidney epithelial cells relates to their similarity to podocytes, which undergo mechanical stretch related to changes in intraglomerular pressure. To produce strain or stretch, primary cultures of rabbit proximal tubular cell cells are grown in tissue culture wells having a collagen-coated Silastic deformable membrane bottoms and applying vacuum to the well to generate alternating cycles of stretch and relaxation (30 cycles/min). We found that cyclic stretching of rabbit proximal tubular cells caused a time- and intensity-dependent activation of extracellular signal-regulated kinases 1 and 2 (ERK 1/2) in proximal tubular cells as detected by its phosphorylation. In addition, mechanical stretch induced PLA2 activation and a subsequent rapid release of arachidonic acid. Inhibition of PLA2 by mepacrine and methyl arachidonyl fluorophosphonate ketone (AACOCF3) attenuated both arachidonic acid release and ERK 1/2 activation by cyclic stretch, supporting the importance of PLA2 as a mediator of mechanotransduction in renal proximal tubular cells. A requirement for extracellular Ca2+ and stretch-activated Ca2+ channels was also documented. Complete inhibition of ERK 1/2 by PD98059, a MAPK kinase (MEK) inhibitor, did not suppress stretch- induced PLA2 activation and arachidonic acid release, suggesting the later events were upstream of ERK 1/2. Cyclic stretch also caused rapid phosphorylation of the EGF receptor kinase and c-Src. Furthermore, arachidonic acid itself induced time- and dose-dependent phosphorylation of c-Src. In addition, the c-Src inhibitor PP2 and selective EGF receptor kinase inhibitor AG1478 attenuated both ERK 1/2 and EGF receptor phosphorylation by cyclic stretch. PLA2 dependence for ERK 1/2 activation in response to cyclic stretch in proximal tubular epithelial cells was established in this report. In addition, these findings indicate cyclic stretch increased the tyrosine phosphorylation of the EGF receptor and c-Src and that c-Src acts upstream of the EGF receptor to mediate its phosphorylation, whereby both are critical for stretch- induced ERK 1/2 activation in rabbit proximal tubular cells. These observations documents for the first time a mechanism of mechanical stretch-induced kinase activation mediated by stretch activated Ca2+ channels and PLA2-dependent release of arachidonic acid.

Monoglyceride lipase-like enzymatic activity is responsible for hydrolysis of 2-arachidonoylglycerol in rat cerebellar membranes

2-Arachidonoylglycerol (2-AG) is an endogenous cannabinoid that binds to CB1 and CB2 cannabinoid receptors, inducing cannabimimetic effects. However, the cannabimimetic effects of 2-AG are weak in vivo due to its rapid enzymatic hydrolysis. The enzymatic hydrolysis of 2-AG has been proposed to mainly occur by monoglyceride lipase (monoacylglycerol lipase). Fatty acid amide hydrolase (FAAH), the enzyme responsible for the hydrolysis of N-arachidonoylethanolamide (AEA), is also able to hydrolyse 2-AG. In the present study, we investigated the hydrolysis of endocannabinoids in rat cerebellar membranes and observed that enzymatic activity towards 2-AG was 50-fold higher than that towards AEA. Furthermore, various inhibitors for 2-AG hydrolase activity were studied in rat cerebellar membranes. 2-AG hydrolysis was inhibited by methyl arachidonylfluorophosphonate, hexadecylsulphonyl fluoride and phenylmethylsulphonyl fluoride with ic 50 values of 2.2 nM, 241 nM and 155 μM, respectively. Potent FAAH inhibitors, such as OL-53 and URB597, did not inhibit the hydrolysis of 2-AG, suggesting that 2-AG is inactivated in rat cerebellar membranes by an enzyme distinct of FAAH. The observation that the hydrolysis of 1(3)-AG and 2-AG occurred at equal rates supports the role of MGL in 2-AG inactivation. This enzyme assay provides a useful method for future inhibition studies of 2-AG degrading enzyme(s) in brain membrane preparation having considerably higher MGL-like activity when compared to FAAH activity.

Activation of Na+-Independent Mg2+ Efflux by 20-Hydroxyeicosatetraenoic Acid in Rat Renal Epithelial Cells

Renal epithelial cells may have Mg(2+) transport pathways that regulate intracellular free Mg(2+) concentration ([Mg(2+)](i)) and reabsorption into the body. In mag-fura 2 fluorescent measurement, extracellular Mg(2+) removal induced a Na(+)-independent [Mg(2+)](i) decrease. The [Mg(2+)](i) decrease was suppressed by methyl arachidonyl fluorophosphonate, a cytosolic and Ca(2+)-independent phospholipase A(2) (iPLA(2)) inhibitor, and bromoenol lactone, an iPLA(2) inhibitor, but it was not suppressed by a secretory phospholipase A(2) inhibitor. On the contrary, the [Mg(2+)](i) decrease was enhanced by the addition of exogenous arachidonic acid (AA). Next, we examined the effect of AA metabolite inhibitors on the [Mg(2+)](i) decrease. 17-octadecynoic acid inhibited the [Mg(2+)](i) decrease, but indomethacin and nordihydroguaiaretic acid did not. In the 17-octadecynoic acid-treated cells, 20-hydroxy-(5Z,8Z,11Z,14Z)-eicosatetraenoic acid (20-HETE) recovered the [Mg(2+)](i) decrease. Nicardipine inhibited both the basal and the 20-HETE-enhanced [Mg(2+)](i) decrease. These results suggest that 20-HETE is a key mediator in the activation of Na(+)-independent Mg(2+) efflux.

Bromoenol Lactone Promotes Cell Death by a Mechanism Involving Phosphatidate Phosphohydrolase-1 Rather than Calcium-independent Phospholipase A2

Originally described as a serine protease inhibitor, bromoenol lactone (BEL) has recently been found to potently inhibit Group VI calcium-independent phospholipase A2 (iPLA2). Thus, BEL is widely used to define biological roles of iPLA2 in cells. However, BEL is also known to inhibit another key enzyme of phospholipid metabolism, namely the magnesium-dependent phosphatidate phosphohydrolase-1 (PAP-1). In this work we report that BEL is able to promote apoptosis in a variety of cell lines, including U937, THP-1, and MonoMac (human phagocyte), RAW264.7 (murine macrophage), Jurkat (human T lymphocyte), and GH3 (human pituitary). In these cells, long term treatment with BEL (up to 24 h) results in increased annexin-V binding to the cell surface and nuclear DNA damage, as detected by staining with both DAPI and propidium iodide. At earlier times (2 h), BEL induces the proteolysis of procaspase-9 and procaspase-3 and increases cleavage of poly(ADP-ribose) polymerase. These changes are preceded by variations in the mitochondrial membrane potential. All these effects of BEL are not mimicked by the iPLA2 inhibitor methylarachidonyl fluorophosphonate or by treating the cells with a specific iPLA2 antisense oligonucleotide. However, propranolol, a PAP-1 inhibitor, is able to reproduce these effects, suggesting that it is the inhibition of PAP-1 and not of iPLA2 that is involved in BEL-induced cell death. In support of this view, BEL-induced apoptosis is accompanied by a very strong inhibition of PAP-1-regulated events, such as incorporation of [3H]choline into phospholipids and de novo incorporation of [3H]arachidonic acid into triacylglycerol. Collectively, these results stress the role of PAP-1 as a key enzyme for cell integrity and survival and in turn caution against the use of BEL in studies involving long incubation times, due to the capacity of this drug to induce apoptosis in a variety of cells.

Localization of Group V Phospholipase A2 in Caveolin-enriched Granules in Activated P388D1 Macrophage-like Cells

In murine P388D1 macrophages, the generation of prostaglandin E2 in response to long term stimulation by lipopolysaccharide involves the action of Group V secreted phospholipase A2 (PLA2), Group IV cytosolic PLA2 (cPLA2), and cyclooxygenase-2 (COX-2). There is an initial activation of cPLA2 that induces expression of Group V PLA2, which in turn induces both the expression of COX-2 and most of the arachidonic acid substrate for COX-2-dependent prostaglandin E2 generation. Because Group V PLA2 is a secreted enzyme, it has been assumed that after cellular stimulation, it must be released to the extracellular medium and re-associates with the outer membrane to release arachidonic acid from phospholipids. In the present study, confocal laser scanning microscopy experiments utilizing both immunofluorescence and green fluorescent protein-labeled Group V PLA2 shows that chronic exposure of the macrophages to lipopolysaccharide results in Group V PLA2 being associated with caveolin-2-containing granules close to the perinuclear region. Heparin, a cell-impermeable complex carbohydrate with high affinity for Group V PLA2, blocks that association, suggesting that the granules are formed by internalization of the Group V sPLA2 previously associated with the outer cellular surface. Localization of Group V PLA2 in perinuclear granules is not observed if the cells are treated with the Group IV PLA2 inhibitor methyl arachidonyl fluorophosphonate, confirming the important role for Group IV PLA2 in the activation process. Cellular staining with antibodies against COX-2 reveals the presence of COX-2-rich granules in close proximity to those containing Group V PLA2. Collectively, these results suggest that encapsulation of Group V PLA2 into granules brings the enzyme to the perinuclear envelope during cell activation where it may be closer to Group IV PLA2 and COX-2 for efficient prostaglandin synthesis.

Role of EETs in regulation of endothelial permeability in rat lung.

This study tested the hypothesis that epoxyeicosatrienoic acids (EETs) derived from arachidonic acid via P-450 epoxygenases are soluble factors linking depletion of endoplasmic reticulum Ca(2+) stores and store-dependent regulation of endothelial cell (EC) permeability in rat lung. EC permeability was measured via the capillary filtration coefficient (K(f,c)) in isolated, perfused rat lungs. 14,15-EET and 5,6-EET increased EC permeability, a response that was significantly different from that of 8,9-EET, 11,12-EET, and vehicle control. The permeability response to 14,15-EET was not significantly attenuated by the nonspecific Ca(2+) channel blocker Gd(3+) (P = 0.068). In lungs perfused with low [Ca(2+)], 14,15-EET tended to increase EC permeability, although a significant increase in K(f,c) was observed only following Ca(2+) add-back. As positive control, we showed that the 3.7-fold increase in K(f,c) evoked by thapsigargin (TG), a known activator of store depletion-induced Ca(2+) entry, was blocked by both Gd(3+) and low [Ca(2+)] buffer. Nonetheless, the permeability response to TG could not be blocked by the phospholipase A(2) inhibitors mepacrine or methyl arachidonyl fluorophosphonate or the P-450 epoxygenase inhibitors 17-octadecynoic acid or propargyloxyphenyl hexanoic acid. Similarly, combined pretreatment with ibuprofen and dicyclohexylurea to block EET metabolism had no effect on the permeability response to TG. We conclude that EETs have a heterogeneous impact on EC permeability. Despite a requirement for Ca(2+) entry with both TG and 14,15-EET, our data suggest that distinct signaling pathways or heterogeneity in EC responsiveness is responsible for the observed EC injury evoked by EETs and store depletion in the isolated rat lung.

Arachidonic acid-activated Na +-dependent Mg 2+ efflux in rat renal epithelial cells

Arachidonic acid (AA), a metabolite of membrane phospholipids, and its metabolites are increased in Mg 2+ deficiency. We examined whether the extracellular Mg 2+ concentration affects AA production and whether AA regulates a putative Na +-dependent Mg 2+ efflux pathway in renal epithelial NRK-52E cells. We used the cells cultured in 5 mM Mg 2+-containing medium for 2 days because they enable us to detect Na +-stimulated Mg 2+ efflux that was not observed in normal culture medium. Removal of extracellular Mg 2+ increased AA release both in the absence and presence of extracellular Na +. This was inhibited by methyl arachidonyl fluorophosphonate (MAFP, 10 μM), an inhibitor of cytosolic phospholipase A 2 (cPLA 2) and Ca 2+-independent phospholipase A 2 (iPLA 2), and bromoenol lactone (BEL, 10 μM), an inhibitor of iPLA 2. However, LY-311727 (10 μM), a secretory phospholipase A 2 (sPLA 2) inhibitor, had no inhibitory effect. Reverse transcriptase-polymerase chain reaction (RT-PCR) showed that NRK-52E cells express cPLA 2 and iPLA 2 mRNAs, but not sPLA 2. In the mag-fura 2 fluorescence measurements, extracellular Mg 2+ removal caused slight decrease in the intracellular free Mg 2+ concentration ([Mg 2+] i) in the Na +-free condition. The addition of Na + caused a rapid decrease in [Mg 2+] i, indicating the presence of a Na +-dependent Mg 2+ efflux pathway. The Na +-dependent [Mg 2+] i decrease was suppressed by MAFP and BEL. On the other hand, AA metabolite inhibitors, nordihydroguaiaretic acid (NDGA) (50 μM), indomethacin (10 μM) and 17-octadecynoic acid (ODYA) (10 μM), enhanced the Na +-dependent [Mg 2+] i decrease. Furthermore, the addition of exogenous AA (30 μM) enhanced the Na +-dependent [Mg 2+] i decrease, which was significantly inhibited by imipramine (0.1 mM), a putative Na +/Mg 2+-exchanger inhibitor. These results suggest that extracellular Mg 2+ removal elevates AA release mediated mainly by iPLA 2 and that AA upregulates the Na +-dependent Mg 2+ efflux in NRK-52E cells.

Inhibition of platelet-activating factor (PAF) acetylhydrolase by methyl arachidonyl fluorophosphonate potentiates PAF synthesis in thrombin-stimulated human coronary artery endothelial cells.

We have previously demonstrated that thrombin stimulation of endothelial cells results in increased membrane-associated, Ca(2+)-independent phospholipase A2 (iPLA2) activity, accelerated hydrolysis of membrane plasmalogen phospholipids, and production of several biologically active phospholipid metabolites, including prostacyclin and platelet-activating factor (PAF) that is abolished by pretreatment with the iPLA2-selective inhibitor bromoenol lactone. This study was designed to further investigate the role of alternative PLA2 inhibitors, including methyl arachidonyl fluorophosphonate (MAFP, an inhibitor of cytosolic PLA2 isoforms), on phospholipid turnover and PAF production from thrombin-stimulated human coronary artery endothelial cells (HCAECs). Paradoxically, pretreatment of HCAEC with MAFP (5-25 microM) resulted in a significant increase in PAF production in both unstimulated and thrombin-stimulated cells that was found to be a direct result of inhibition of PAF acetylhydrolase (PAF-AH) activity. Pretreatment with MAFP did not significantly inhibit HCAEC PLA2 activity, possibly due to the localization of PLA2 activity in the membrane fraction rather than the cytosol. Bromoenol lactone did not inhibit PAF-AH activity, even at concentrations as high as 20 microM. We conclude that MAFP augments thrombin-stimulated PAF production by inhibition of PAF catabolism without affecting membrane-associated iPLA2 activity.

Advanced glycation end products stimulate an enhanced neutrophil respiratory burst mediated through the activation of cytosolic phospholipase A2 and generation of arachidonic Acid.

Advanced glycation end products (AGEs) enhance NADPH oxidase, and hence respiratory burst activity, of stimulated neutrophils. They are thus potentially vasculopathic, especially in diabetes, uremia, and aging, in which AGEs classically accumulate. We investigated the underlying mechanisms. Neutrophils prelabeled with [3H]arachidonic acid display increased [3H]arachidonate release on exposure to AGE-albumin over exposure to albumin alone (by 151+/-16%, P<0.01). Arachidonic acid (AA) itself seems to mediate the AGE-augmented neutrophil respiratory burst (ascertained by chemiluminescence). Inhibitors of the cyclooxygenase pathway (indomethacin) and lipoxygenase pathway (MK-886) do not impair this AGE effect, excluding a contribution from AA metabolites. Cytosolic phospholipase A2 (cPLA2) controls AA generation. Its inhibition by methyl arachidonyl fluorophosphonate abrogates the AGE-enhanced activated neutrophil respiratory burst, and it is demonstrably stimulated in AGE-exposed neutrophils, as evidenced by isoform gel-shift and an increasingly membrane-translocated state in Western blots of neutrophil subfractions. Inhibition of other PLA2 isoforms, secretory PLA2 and calcium-independent PLA2, by manoalide and haloenol-lactone suicide substrate, respectively, does not affect this effect of AGEs relative to inhibitor-treated controls. The thiol antioxidant NAC reduces activation of cPLA2 (assessed by isoform gel-shift and membrane translocation), production of AA in AGE-albumin-exposed neutrophils (H3 release reduced to 104+/-17%, P=0.94 compared with albumin-exposed neutrophils), and the AGE-augmented neutrophil respiratory burst. AGE augmentation of the activated neutrophil respiratory burst requires AA generation, through which neutrophil NADPH oxidase may be upregulated, enhancing reactive oxygen species output. AA is generated by cPLA2, which may be stimulated through an AGE-activated redox-sensitive pathway.

Oxidized LDL activates phospholipase A2 to supply fatty acids required for cholesterol esterification

We examined the roles of phospholipase A2 (PLA2) in oxidized LDL (oxLDL)-induced cholesteryl ester formation in macrophages. In [3H]oleic acid-labeled RAW264.7 cells and mouse peritoneal macrophages, oxLDL induced [3H]cholesteryl oleate formation with an increase in free [3H]oleic acid and a decrease in [3H]phosphatidylcholine. The changes in these lipids were suppressed by methyl arachidonyl fluorophosphonate (MAFP), a cytosolic PLA2 (cPLA2) inhibitor. However, MAFP had no effect on the ACAT activity or the binding and/or uptake of oxLDL. Stimulation with oxLDL in the presence of [3H]cholesterol increased [3H]cholesteryl ester bearing fatty acyl chains derived from cellular and/or exogenous (oxLDL) lipids. The formation of cholesteryl ester under this condition was also inhibited by MAFP, and the inhibitory effect was reversed by adding oleic acid. While oxLDL did not affect the activity or amounts of cPLA2, preincubation with oxLDL enhanced the release of oleic acid and arachidonic acid induced by ionomycin in RAW264.7 cells. 13(S)-hydroxyoctadecadienoic acid, but not 7-ketocholesterol, also enhanced ionomycin-induced oleic acid release. These results suggest that oxLDL induces cPLA2 activation, which contributes, at least in part, to the supply of fatty acids required for the cholesteryl esterification, probably through the acceleration by oxidized lipids of the catalytic action of cPLA2 in macrophages.

Arachidonylsulfonyl derivatives as cannabinoid CB1 receptor and fatty acid amide hydrolase inhibitors.

Arachidonylsulfonyl fluoride (3), reported here for the first time, is similar in potency to its known methyl arachidonylfluorophosphonate (2) analogue as an inhibitor of mouse brain fatty acid amide hydrolase activity (IC(50) 0.1 nM) and cannabinoid CB1 agonist [3H]CP 55,940 binding (IC(50) 304-530 nM). Interestingly, 3 is much more selective than 2 as an inhibitor for fatty acid amide hydrolase relative to acetylcholinesterase, butyrylcholinesterase and neuropathy target esterase. N-(2-Hydroxyethyl)arachidonylsulfonamide (4) is at least 2500-fold less potent than N-(2-hydroxyethyl)arachidonamide (anandamide) (1) at the CB1 agonist site.