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Cyclooxygenase-2 (COX-2) and tyrosine kinase, which are involved in the biosynthesis of prostaglandin E2 (PGE2) in mouse calvarial osteoblasts, are stimulated by cytokine interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and/or interleukin-6 (IL-6). IL-1β and IL-6 and, to a lesser extent, TNF-α, enhances COX-2 mRNA levels in calvarial osteoblasts. Simultaneous treatment with IL-6 and IL-1β and TNF-α resulted in enhanced COX-2 mRNA levels accompanied by the cooperative stimulation of PGE2 biosynthesis compared to cells treated with IL-1β or TNF-α or IL-6 alone. In contrast, the presence of TGF-β reduced COX-2 mRNA level, PGE2 biosynthesis and bone resorption induced by IL-1β, TNF-α, IL-6 or a combination thereof. However, neither IL-1β, TNF-α, IL-6 nor a combination of IL-1β, TNF-α, IL-6 enhanced COX-1 mRNA levels in calvarial osteoblasts. A novel Src tyrosine kinase inhibitor, Herbimycin A (HERB), reduced COX-2 mRNA levels as well as PGE2 production induced by IL-1β, TNF-α and IL-6 or a combination of IL-1β, TNF-α, IL-6, whereas COX-1 mRNA levels remained unaffected. Finally, HERB was found to inhibit in vitro bone resorption. These results indicate that the cooperative effects of IL-β, TNF-α, IL-6 on PGE2 production are due to the enhanced expression of the COX-2 gene and that tyrosine kinase(s) are involved in COX-2 signal transduction in mouse calvarial osteoblasts. Thus, the Src family of kinase inhibitors may be useful in treating diseases associated with elevated bone loss. [Copyright &y& Elsevier]

We hypothesize that homocysteinemia causes oxidative stress, decreases the aortic ability to generate prostacyclin and that antioxidants have a protective role. Four groups of eight rats each were fed for 8 weeks the control diet (group A), control diet with folic acid omitted and excess methionine (Me) added to drinking water (group B), diet B + 500 mg/kg of Vitamin C (group C) or diet B + 60 mg/kg Vitamin B6 (group D). The three groups of rats fed folic acid deficient (FD) diets (groups B, C and D) were homocysteinemic as indicated by the significant increase in their serum homocysteine (HC) concentration. Rats fed diet B had oxidative stress as indicated by an increase in serum thiobarbituric acid reactive substances (TBARS) and advanced oxidation protein products (AOPP) and urinary isoprostanes and had a decreased ability of their aortas to generate prostacyclin.Homocysteinemic rats fed a FD diet + Vitamin C (group C) or Vitamin B6 (group D) also had high levels of serum homocysteine but the oxidative stress markers and the ability of their aortas to generate prostacyclin returned to normal. This indicates that the homocysteinemic effect is through an oxidative mechanism and that Vitamin C as a free radical scavenger prevents these effects. Serum Vitamin C and liver glutathione concentrations significantly increased in rats fed excess Vitamin B6 compared to the control or FD rats. This may explain why Vitamin B6 has an antioxidative effect. [Copyright &y& Elsevier]

Reactive oxygen species (ROS) are produced in the course of normal metabolism and they serve important physiological functions. However, because of their high reactivity, accumulation of ROS beyond the immediate needs of the cell may affect cellular structure and functional integrity, by bringing about oxidative degradation of critical molecules, such as the DNA, proteins, and lipids. Although cells possess an intricate network of defense mechanisms to neutralize excess ROS and reduce oxidative stress, some tissues, especially the brain, are much more vulnerable to oxidative stress because of their elevated consumption of oxygen and the consequent generation of large amounts of ROS. For the same reason, the mitochondrial DNA (mtDNA) of brain cells is highly susceptible to structural alterations resulting in mitochondrial dysfunction. Several lines of evidence strongly suggest that these effects of ROS may be etiologically related to a number of neurodegenerative disorders.Nutraceutical antioxidants are dietary supplements that can exert positive pharmacological effects on specific human diseases by neutralizing the negative effects of ROS. The present communication concentrates on a review of recent concepts and methodological developments, some of them based on the results of work from our own laboratory, on the following aspects: (1) the complex interactions and complementary interrelationships between oxidative stress, mitochondrial dysfunction, and various forms of neural degeneration; (2) fractionation and isolation of substances with antioxidant properties from plant materials, which are extensively used in the human diet and, therefore, can be expected to be less toxic in any pharmacological intervention; (3) recent developments in methodologies that can be used for the assay of oxidative stress and determination of biological activities of exogenous and endogenous antioxidants; and (4) presentation of simple procedures based on polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) of the resulting amplicon for investigations of structural alterations in mtDNA. [Copyright &y& Elsevier]

We present evidence that the ATP binding-cassette transporter-2 (ABCA2) is a sterol-responsive gene that has a role in the trafficking of low-density lipoprotein-derived free cholesterol (LDL-FC). In HepG2 cells ABCA2 was coordinately expressed with other sterol-responsive genes. Stable constitutive expression of ABCA2 in Chinese hamster ovary cells (CHOA2) was accompanied by an increase the expression of the low-density lipoprotein receptor (LDLR) and other genes involved in the regulation of cholesterol homeostasis. LDLR mRNA was elevated greater than ninefold and 3-hydroxy-3-methylglutaryl CoA synthase (HMGCoA S) expression was elevated sevenfold in CHOA2 cells. The increase in LDLR expression was regulated at the level of transcription; however, culture of CHO and CHOA2 cells in medium containing lipoprotein-deficient serum (LPDS) results in similar levels of LDLR promoter expression. No differences were measured in the dose-dependent uptake of fluorescently labeled 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchorate-LDL (DiI-LDL) between CHO and CHOA2 cells cultured in medium containing LPDS. Ultraviolet microscopy revealed a similar distribution of the DiI-LDL label in cytoplasmic vesicles. We measured an LDL dose-dependent reduction in esterification of LDL-FC in intact CHOA2 cells cultured in medium containing LPDS, however, no significant difference was measured in acylcoenzyme A:cholesterol acyltransferase (ACAT) activity in cell-free extracts of CHO and CHOA2 cells. CHO cells or CHOA2 cells treated with the hydrophobic amine, U18666A, showed similar filipin staining of unesterified cholesterol in cytoplasmic vesicles. Addition of progesterone or U18666A to CHO cells elevated ABCA2 expression. Finally, we found that ABCA2 expression was elevated in Niemann–Pick type C1 (NPC1) fibroblasts and in Familial Hypercholesterolemia (FHC) fibroblasts. [Copyright &y& Elsevier]

Fatty acids of the n-3 and n-6 families containing a photoactive conjugated tetraene group near the carboxylate were prepared from several naturally occurring fatty acids by sequential iodolactonization and treatment with excess 1,8-diazabicyclo[5.4.0]undec-7-ene. The new conjugated fatty acids include 5E,7E,9E,11Z,14Z- and 5E,7E,9E,11E,14Z-eicosapentaenoic acids derived from arachidonic acid; 5E,7E,9E,11Z,14Z,17Z- and 5E,7E,9E,11E,14Z,17Z-eicosahexaenoic acids from eicosapentaenoic acid; and 4E,6E,8E,10Z,13Z,16Z,19Z- and 4E,6E,8E,10E,13Z,16Z,19Z-docosaheptaenoic acids from docosahexaenoic acid. All of the newly synthesized fatty acids were characterized by UV, 1H NMR and mass spectroscopy. These new products represent the first examples of directed conjugation of methylene interrupted double bond systems. The products can be synthesized in gram quantities and in high yields (>50%). Interestingly, it did not prove possible to synthesize fatty acids having a triene group near the carboxyl group even using mild conditions and different synthetic approaches. Once initiated, the isomerization always continued until a tetraene group was formed. Because of the sensitivity of the tetraene group to light, these fatty acids have the potential for being used in tracking fatty acid movements in cells employing fluorescence techniques and in UV light-induced cross linking to membrane proteins. [Copyright &y& Elsevier]

Several studies have demonstrated that C57 and DBA mice exhibit behavioural differences in diverse learning tasks as well as variations in the expression of long-term potentiation (LTP) in the hippocampus. In the present investigation, we tested the possibility that these differences between the two strains might be attributable to differential regulation of hippocampal α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors by calcium-dependent mechanisms. Using in vitro receptor autoradiography, we found that calcium treatment of C57 mice sections resulted in a marked increase of 3H-AMPA binding in areas CA3 and CA1 of the hippocampus and in the dentate gyrus. However, we discovered that the ability of calcium to upregulate 3H-AMPA binding in the DBA strain was much lower than in corresponding regions from the C57 strain. Western blot and immunohistochemical experiments indicated that truncation of AMPA receptor subunits by calcium-dependent mechanisms was possibly not responsible for the binding differences, as no significant variations in glutamate receptor subunit 1 (GluR1) and GluR2/3 immunoreactivity were observed between the two strains after calcium treatment. Interestingly, we found that strain-related variations in the regulation of 3H-AMPA binding by calcium were totally eliminated when brain sections were preincubated with preferential inhibitors of lipoxygenase (LO) pathways of arachidonic acid (AA) metabolism. Taken together, these results suggest that calcium-induced regulation of AMPA receptors varies between the two strains and that this variation might be linked to the production of specific AA metabolites. [Copyright &y& Elsevier]

Treatment of bovine pulmonary artery endothelial cells with the calcium ionophore, A23187, stimulates the cell membrane associated protease activity, phospholipase A2 (PLA2) activity, and arachidonic acid (AA) release from the cells. Pretreatment of the cells with arachidonyl-trifluomethylketone (AACOCF3), a cPLA2 inhibitor, but not bromoenollactone (BEL), a iPLA2 inhibitor, prevents A23187 stimulated PLA2 activity and AA release without producing an appreciable alteration of the protease activity. Pretreatment of the cells with aprotinin, an ambient protease inhibitor, prevents the increase in the protease activity and cPLA2 activity in the membrane and AA release from the cells caused by both low and high doses of A23187, and also inhibits protein kinase C (PKC) activity caused by high doses of A23187. Immunoblot study of the endothelial cell membrane isolated from A23187 (10 μM)-treated cells with polyclonal PKCα antibody elicited an increase in the 80 kDa immunoreactive protein band along with an additional 47 kDa immunoreactive fragment. Pretreatment of the cells with aprotinin abolished the 47 kDa immunoreactive fragment in the immunoblot. Immunoblot study of the endothelial membrane with polyclonal cPLA2 antibody revealed that treatment of the cells with A23187 dose-dependently increases cPLA2 immunoreactive protein profile in the membrane. It therefore appears from the present study that treatment of the cells with a low dose of A23187 (1 μM) causes a small increase in an aprotinin-sensitive protease activity and that stimulates cPLA2 activity in the cell membrane without an involvement of PKC. By contrast, treatment of the cells with a high dose of 10 μM of A23187 causes optimum increase in the protease activity and that plays an important role in activating PKCα, which subsequently stimulates cPLA2 activity in the cell membrane. Although pretreatment of the cells with pertussis toxin caused ADP ribosylation of a 41 kDa protein in the cell membrane, it did not inhibit the cPLA2 activity and AA release caused by both low and high doses of A23187. [Copyright &y& Elsevier]

The integral membrane protein flavocytochrome b (Cyt b) comprises the catalytic core of the human phagocyte NADPH oxidase complex and serves to initiate a cascade of reactive oxygen species that participate in the elimination of infectious agents. Superoxide production by the NADPH oxidase complex has been shown to be specifically regulated by the enzymatic generation of lipid second messengers following phagocyte activation. In the present study, a Cyt b-specific monoclonal antibody (mAb 44.1) was labeled with Cascade Blue (CCB) and used in resonance energy transfer (RET) studies probing the effects of a panel of lipid species on the structure of Cyt b. The binding of CCB-mAb 44.1 to immunoaffinity-purified Cyt b was both highly specific and resulted in significant quenching of the steady state donor fluorescence. Titration of the CCB-mAb 44.1:Cyt b complex with the anionic amphiphile lithium dodecyl sulfate (LDS) resulted in a saturable relaxation of fluorescence quenching due to conformational changes in Cyt b at concentrations of the amphiphile required for maximum rates of superoxide production by Cyt b in cell-free assays. Similar results were observed for the anionic amphiphile arachidonic acid (AA), although no relaxation of fluorescence quenching was observed for arachidonate methyl ester (AA-ME). Saturable relaxation of fluorescence quenching was also observed with the anionic, 18:1 phospholipids phosphatidic acid (DOPA) and phosphatidylserine (DOPS), while no relaxation was observed upon addition of the neutral 18:1 lipids phosphatidylcholine (DOPC), phosphatidylethanolamine (DOPE) or diacylglycerol (DAG) at similar levels. Further examination of a variety of phosphatidic acid (PA) species demonstrated DOPA to both potently induce conformational changes in Cyt b and to cause more dramatic conformational changes than PA species with shorter, saturated acyl chains. The data presented in this study support the hypothesis that second messenger lipids, such as AA and PA, directly bind to flavocytochrome b and modulate conformational states relevant to the activation of superoxide production. [Copyright &y& Elsevier]

Histamine has been proposed to be an important modulator of developing neurons, but its mechanism of action remains unclear. In embryonic chick dorsal root ganglion neurons we found that histamine activates, through the pyrilamine-sensitive H1 receptor, a K-selective, background channel. The K channel activated by histamine was also activated by arachidonic acid in a dose-dependent way, with a KD of 4 μM and a slope of 2.5, had a unitary conductance of about 150 pS (symmetrical 140 KCl) and a moderate voltage dependence. The channel was insensitive to the classical K channel blockers tetraethylammonium, charybdotoxin, 4-aminopyridine, but inhibited by millimolar Ba2+. Channel activity could also be increased by lowering the intracellular pH from 7.2 to 5.5, or by applying negative pressure pulses through the patch pipette. Experiments aimed at delineating the metabotropic pathway leading to K channel activation by histamine indicated the involvement of a pertussis toxin-insensitive G protein, and a quinacrine-sensitive cytosolic phospholipase A2. The histamine-induced K channel activation was observed only with elevated internal Ca2+ (achieved using 0.5 μM ionomycin or elevated external KCl). An increase in the histamine-induced phosphoinositide hydrolysis was also observed upon internal Ca2+ elevation, showing the presence of a Ca2+ dependent step upstream to inositol 1,4,5-triphosphate production. In view of the functional importance of K conductances during cell differentiation, we propose that histamine activation of this K channel may have a significant role during normal development of embryonic chick neurons. [Copyright &y& Elsevier]

Isoketals are highly reactive γ-ketoaldehydes formed by the oxidation of arachidonic acid that rapidly adduct to proteins. To investigate the formation of isoketal adducts in vivo, we isolated and characterized a single-chain antibody from a phage displayed recombinant ScFv library that bound a model peptide adducted with synthetic 15-E2-isoketal. Recognition of isoketal adduct by this anti-isoketal adduct single-chain antibody was essentially independent of the amino acid sequence of adducted peptides or proteins. The antibody did not cross-react with 4-hydroxynonenal or 4-oxononanal adducts or with 15-F2t-isoprostane (8-iso-prostaglandin F2α). We investigated the formation of isoketal adducts in a well-established model of oxidative injury, hyperoxia. Exposure to >98% oxygen for 7 h dramatically increased both the number of immunoreactive airway epithelial cells and the intensity of immunoreactivity compared with animals exposed to normal room air (21% oxygen). We conclude that isoketal adducts form in epithelial cells as a result of high oxygen exposure and that this single-chain antibody provides a valuable tool to localize the formation of isoketal adducts in tissues in vivo. [Copyright &y& Elsevier]

Hydroxy fatty acids from Euglena gracilis were identified by reverse-phase high performance liquid chromatography coupled to a mass spectrometer run in atmospheric pressure chemical ionization positive ion mode. These metabolites were converted to methyl esters to improve stability and chromatographic properties. A detection limit of 20 pg/μl per injection was determined for 5-HETE methyl ester based on the signal to noise ratio of the m/z 317 ion which corresponds to the loss of a hydroxyl group (M-17) and the major fragment in all HETE methyl esters studied. This is the first report for these metabolites in E. gracilis. [Copyright &y& Elsevier]

We have recently identified hyperforin, a lipophilic constituent of the herb Hypericum perforatum (St. John’s wort), as a dual inhibitor of the proinflammatory enzymes cyclooxygenase-1 and 5-lipoxygenase. The aim of the present study was to further elucidate antiinflammatory properties and respective targets of hyperforin. We found that hyperforin inhibited the generation of reactive oxygen species (ROS) as well as the release of leukocyte elastase (degranulation) in human isolated polymorphonuclear leukocytes (PMNL), challenged by the G protein-coupled receptor (GPCR) ligand N-formyl-methionyl-leucyl-phenylalanine (fMLP) with an ic50≈0.3 μM. When PMNL were stimulated with phorbol-12-myristate-13-acetate (PMA) or ionomycin, hyperforin (up to 10 μM) failed to inhibit ROS production and elastase release, respectively. Moreover, hyperforin blocked receptor-mediated Ca2+ mobilization (ic50≈0.4 and 4 μM, respectively) in PMNL and monocytic cells, and caused a rapid decline of the intracellular Ca2+ concentration in resting cells. In contrast, the Ca2+ influx induced by ionomycin or thapsigargin was not suppressed. Comparative studies with the specific phospholipase C inhibitor U-73122 and hyperforin revealed similarities between both compounds. Thus, U-73122 and hyperforin blocked fMLP- and PAF-induced Ca2+ mobilization, ROS formation, and elastase release, but failed to suppress these responses when cells were stimulated by PMA or ionomycin. Also, both compounds rapidly decreased basal Ca2+ levels in resting cells and led to a rapid decline of the Ca2+ elevations evoked by fMLP or PAF. Our data suggest that hyperforin targets component(s) within G protein signaling cascades that regulate Ca2+ homeostasis, coupled to proinflammatory leukocyte functions. [Copyright &y& Elsevier]

The current study examined the metabolism of phospholipid (PL) in the whole cell homogenate and in the nuclear fraction in proliferative and non-proliferative uterine stromal cells (UIII cells). Growth arrested cells were obtained either from contact-inhibited confluent cells or from proliferative cells treated with aristolochic acid (AR) for 2 days. Fatty acid composition and fatty acid amount of both total and nuclear PL were not significantly different between proliferative, confluent and AR-treated cells. In contrast, marked differences were observed in the incorporation of [3H]AA, with greater incorporation in proliferative cells than in confluent or AR-treated cells, particularly in nuclear PL. Considering endogenous level of arachidonic acid (AA) in total and nuclear PL, we found that AA turnover in nuclear PL was especially high compared to that in total PL and that this difference was accentuated in proliferative cells compared to non-proliferative cells. Interestingly, [3H]AA incorporation and AA turnover in proliferative, confluent and AR-treated cells vary accordingly to the expression, activity and/or content of pancreatic phospholipase A2 (PLA2-I) in the nuclear compartment of these cells that we reported in previous studies. The changes in metabolism of nuclear PL during cell proliferation are consistent with an enhanced PL hydrolysis that could involve PLA2-I. [Copyright &y& Elsevier]

In this study, the role of exogenous fatty acids in the regulation of proteolipid protein (PLP) gene expression was investigated using the following model culture system: C6 glioma cells expressing the green-fluorescent protein (eGFP) driven by different segments of PLP promoter. Eicosapentanoic acid (EPA; 20:5 n-3), but not arachidonic acid (AA; 20:4 n-6), induced a significant increase in medium fluorescence intensity (MFI) determined by fluorescence-activated cell sorting (FACS). The induction of PLP promoter was time-dependent showing maximal activity between 24 and 48 h after EPA exposure. PLP promoter activation was dependent on fatty acid concentration, with maximum activation at 200 μM. Northern blot analysis confirmed the fluorescence data in C6 cells incubated with EPA. Furthermore, this treatment increased the adenylyl cyclase-cyclic AMP (cAMP) levels and the mitogen-activated protein kinase (MAPK) activation in C6 cells. PLP promoter activity was inhibited by pre-treatment with H89 (protein kinase A (PKA) inhibitor), but not with PD98059 (MAPK inhibitor), suggesting that EPA stimulates the expression of PLP via cAMP-mediated pathways. [Copyright &y& Elsevier]

Amyloid β protein (Aβ)- and human group IIA secretory phospholipase A2 (sPLA2-IIA)-induced neuronal cell death have been established as in vitro models for Alzheimer’s disease (AD) and stroke. Both sPLA2-IIA and Aβ causes neuronal apoptosis by increasing the influx of Ca2+ through L-type voltage-sensitive Ca2+ channel (L-VSCC). In the present study, we evaluated effects of a selective L-VSCC blocker, S-(+)-methyl 4,7-dihydro-3-isobutyl-6-methyl-4-(3-nitro-phenyl)thieno[2,3-b]pyridine-5-carboxylate (S-312-d), on Aβ- and sPLA2-IIA-induced neuronal apoptosis in primary cultures of rat cortical neurons. S-312-d significantly rescued cortical neurons from Aβ- and sPLA2-IIA-induced cell death. Both cell death stimuli caused the appearance of apoptotic features such as plasma membrane blebs, chromatin condensation, and DNA fragmentation. S-312-d completely suppressed these apoptotic features. Before apoptosis, the two death ligands markedly enhanced an influx of Ca2+ into neurons. S-312-d significantly prevented neurons from sPLA2-IIA- and Aβ-induced Ca2+ influx. Furthermore, the neuroprotective effect of S-312-d was more potent than that of another L-VSCC blocker, nimodipine. On the other hand, blockers of other VSCCs such as the N-type and P/Q-type calcium channels had no effect on the neuronal cell death, apoptotic features and Ca2+ influx. In conclusion, we demonstrated that S-312-d rescues cortical neurons from Aβ- and sPLA2-IIA-induced apoptosis. [Copyright &y& Elsevier]

The present study was aimed at examining the mechanism whereby an otherwise non-toxic concentration of peroxynitrite promotes a rapid necrotic response in U937 cells in which cytosolic phospholipase A2 is pharmacologically inhibited or genetically depleted. We found that loss of viable cells is appreciable 15 min after addition of peroxynitrite, does not further increase at 30 min and is mediated by mitochondrial permeability transition (MPT). Both MPT and toxicity were prevented by exogenous arachidonic acid (AA). Various experimental approaches produced results consistent with the notion that the AA-dependent protective mechanism takes place 10–15 min after addition of peroxynitrite. The observation that the extent of DNA strand scission induced by peroxynitrite did not vary under conditions of different AA availability suggests that this event is either upstream to mitochondrial dysfunction or irrelevant for cytotoxicity. Collectively, these data indicate that a non-toxic concentration of peroxynitrite commits U937 cells to MTP-dependent necrosis that is however prevented by endogenous AA. Thus, mitochondria are a likely target of the cytoprotective signalling triggered by AA. [Copyright &y& Elsevier]

Surgery at any location in the body leads to surgical stress response and alterations in normal body homeostasis. The intestine is extremely sensitive to surgical stress even at remote locations and the gastrointestinal tract plays an important role in the development of postsurgical complications such as sepsis, the systemic immune response syndrome (SIRS), and multiple organ failure syndrome (MOFS). The generation of free radicals and subsequent biochemical alterations at the cellular and subcellular level in the intestine has been suggested to play an important role in this process. These oxidative stress-induced events in the mucosa might act as an initiator of distant organ damage and also facilitate bacterial adherence onto the epithelium and translocation into the systemic circulation. This review attempts to highlight the important role of intestine and oxygen free radicals in initiating post-surgical complications. [Copyright &y& Elsevier]

We have demonstrated that magnolol suppressed thromboxane B2 (TXB2) and leukotriene B4 (LTB4) formation in A23187-stimulated rat neutrophils. Maximum inhibition was obtained with about 10 μM magnolol. Magnolol was more effective in the inhibition of cyclooxygenase (COX) activity than in the inhibition of 5-lipoxygenase (5-LO) activity as assessed by means of enzyme activity determination in vitro and COX and 5-LO metabolic capacity analyses in vivo. Magnolol alone stimulated cytosolic phospholipase A2 (cPLA2) phosphorylation and the translocation of 5-LO and cPLA2 to the membrane, and evoked arachidonic acid (AA) release. Recruitment of both 5-LO and cPLA2 to the membranes was suppressed by EGTA. Arachidonyl trifluoromethyl ketone (AACOCF3), a PLA2 inhibitor, bromoenol lactone (BEL), a Ca2+-independent PLA2 (iPLA2) inhibitor, and EGTA suppressed the magnolol-induced AA release. However, none of the follows affected magnolol-induced AA-release: 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole (SB203580), a p38 mitogen-activated protein kinase (MAPK) inhibitor, 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene (U0126), a MAPK kinase (MEK) inhibitor, or 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide (GF109203X), a protein kinase C (PKC) inhibitor. In addition, magnolol at 30 μM did not stimulate the p38 MAPK and extracellular signal-regulated kinase 2 (ERK2) enzyme activities. These results indicated that magnolol inhibits the formation of prostaglandins and leukotrienes in A23187-stimulated rat neutrophils, probably through a direct blockade of COX and 5-LO activities. The stimulatory effects of magnolol at high concentration on the membrane association of 5-LO and cPLA2 are attributable to the elevation of [Ca2+]i, and on the AA release is likely via activation of cPLA2 and iPLA2. [Copyright &y& Elsevier]

Arachidonic acid (AA; 20:4, n-6) has been implicated in cell damage in the brain under ischemia–reperfusion and other pathological conditions. In our experiments, PC12 cells exposed to >10 μM AA died within 1–2 hr, as assessed by the LDH release assay. Since AA is known to induce Ca2+/cation-permeable conductance in the plasma membrane, we investigated whether Ca2+ influx plays a role in this acute cell death. We found that extracellular Ca2+ was not required for the toxic effect of AA. In fact, the removal of extracellular Ca2+ dramatically accelerated its development: the half-time of the toxic effect of 40 μM AA decreased from 70.1±0.3 min in the presence of 5 mM Ca2+ to 7.4±0.3 min in the Ca-free solution. The extent of cell killing depended only weakly on AA concentration and ion composition, remaining within the 70–95% range. The AA-induced acute death was not affected by inhibitors of AA metabolism (nordihydroguaiaretic acid, indomethacin, proadifen), whereas some antioxidants tested (deferoxamine and ellagic acid), but not all (melatonin), partially suppressed it. Also, it was not affected by changes in the extracellular ionic strength or mimicked by an acetylenic analog of AA 5,8,11,14-eicosatetraynoic acid. We conclude that lethal injuries sustained by cells during short exposures to AA were caused by the fatty acid itself and were not mediated by the AA-induced influx of Ca2+/cations. Moreover, direct physical effects of AA on the plasma membrane (changes in membrane fluidity or detergent-like action) were also excluded. [Copyright &y& Elsevier]

Chronic ethanol consumption causes oxidative damage in the liver, and induction of cytochrome P450 2E1 (CYP2E1) is one pathway involved in oxidative stress produced by ethanol. The hepatic accumulation of iron and polyunsaturated fatty acids significantly contributes to ethanol hepatotoxicity in the intragastric infusion model of ethanol treatment. The objective of this study was to analyze the effect of the green tea flavanol epigallocatechin-3-gallate (EGCG), which has been shown to prevent alcohol-induced liver damage, on CYP2E1-mediated toxicity in HepG2 cells overexpressing CYP2E1 (E47 cells). Treatment of E47 cells with arachidonic acid plus iron (AA + Fe) was previously reported to produce synergistic toxicity in E47 cells by a mechanism dependent on CYP2E1 activity and involving oxidative stress and lipid peroxidation. EGCG protected E47 cells against toxicity and loss of viability induced by AA+Fe; EGCG had no effect on CYP2E1 activity. Prevention of this toxicity was associated with a reduction in oxidative damage as reflected by decreased generation of reactive oxygen species, a decrease in lipid peroxidation, and maintenance of intracellular glutathione in cells challenged by AA+Fe in the presence of EGCG. AA+Fe treatment caused a decline in the mitochondrial membrane potential, which was also blocked by EGCG. In conclusion, EGCG exerts a protective action on CYP2E1-dependent oxidative stress and toxicity that may contribute to preventing alcohol-induced liver injury, and may be useful in preventing toxicity by various hepatotoxins activated by CYP2E1 to reactive intermediates. [Copyright &y& Elsevier]

Exposure of PC12 cells to A23187 or thapsigargin caused a concentration-dependent release of arachidonic acid (AA) mediated by cytosolic phospholipase A2 (PLA2). Under the same conditions, however, analysis of nitric oxide (NO) formation revealed that activation of NO synthase (NOS) is best described by a bell-shaped curve. Reduced detection of NO observed at increasing A23187 or thapsigargin concentrations was not due to formation of peroxynitrite or to activation of NO-consuming processes, but rather to AA-dependent inhibition of NOS activity. Furthermore, NO formation observed under optimal conditions for NOS activity was suppressed by AA as well as by the PLA2 activator melittin. Finally, the effects of AA were not the consequence of direct enzyme inhibition, because this lipid messenger failed to inhibit formation of NO by purified neuronal NOS, but were mediated by an AA-dependent signaling and not by downstream products of the cyclooxygenase and lipoxygenase pathways. In conclusion, the present study underscores a novel mechanism whereby endogenous, or exogenous, AA promotes inhibition of NOS activity. Because AA is generated in response to various agonists acting on membrane receptors and extensively released in inflammatory conditions, these findings have important physiopathological implications. [Copyright &y& Elsevier]

Several regulated Ca2+ entry pathways have been identified, with capacitative Ca2+ entry (CCE) being the most characterized. In the present study, we examined Ca2+ entry pathways regulated by arachidonic acid (AA) in mouse parotid acini. AA induced Ca2+ release from intracellular stores, and increased Ca2+ entry. AA inhibited thapsigargin (Tg)-induced CCE, whereas AA activated Ca2+ entry when CCE was blocked by gadolinium (Gd3+). AA-induced Ca2+ entry was associated with depletion of calcium from ryanodine-sensitive stores; both AA-induced Ca2+ release and Ca2+ entry were inhibited by tetracaine and the nitric oxide synthase (NOS) inhibitor, 7-nitroindazole (7-NI). The nitric oxide (NO) donor, 1,2,3,4-ox-triazolium,5-amino-3-(3,4-dichlorophenyl)-chloride (GEA 3162), but not 8-bromo-cGMP, mimicked the effects of AA in inhibiting CCE. Results suggest that AA acts via nitric acid to inhibit the CCE pathway that is selective for Ca2+, and to activate a second Ca2+ entry pathway that is dependent on depletion of Ca2+ from ryanodine-sensitive stores. [Copyright &y& Elsevier]

Renal ischaemia is associated with accumulation of fatty acids (FA) and mobilisation of arachidonic acid (AA). Given the capacity of UDP-glucuronosyltransferase (UGT) isoforms to metabolise both drugs and FA, we hypothesised that FA would inhibit renal drug glucuronidation. The effect of FA (C2:0–C20:5) on 4-methylumbelliferone (4-MU) glucuronidation was investigated using human kidney cortical microsomes (HKCM) and recombinant UGT1A9 and UGT2B7 as the enzyme sources. 4-MU glucuronidation exhibited Michaelis–Menten kinetics with HKCM (apparent Km (Kmapp) 20.3 μM), weak substrate inhibition with UGT1A9 (Kmapp 10.2 μM, Ksi 289.6 μM), and sigmoid kinetics with UGT2B7 (S50app440.6 μM) Similarly, biphasic UDP-glucuronic acid (UDPGA) kinetics were observed with HKCM (S50 354.3 μM) and UGT1A9 (S50 88.2 μM). In contrast, the Michaelis–Menten kinetics for UDPGA observed with UGT2B7 (Kmapp 493.2 μM) suggested that kinetic interactions with UGTs were specific to the xenobiotic substrate and the co-substrate (UDPGA). FA (C16:1–C20:5) significantly inhibited (25–93%) HKCM, UGT1A9 or UGT2B7 catalysed 4-MU glucuronidation. Although linoleic acid (LA) and AA were both competitive inhibitors of 4-MU glucuronidation by HKCM (Kiapp 6.34 and 0.15 μM, respectively), only LA was a competitive inhibitor of UGT1A9 (Kiapp 4.06 μM). In contrast, inhibition of UGT1A9 by AA exhibited atypical kinetics. These data indicate that LA and AA are potent inhibitors of 4-MU glucuronidation catalysed by human kidney UGTs and recombinant UGT1A9 and UGT2B7. It is conceivable therefore that during periods of renal ischaemia FA may impair renal drug glucuronidation thus compromising the protective capacity of the kidney against drug-induced nephrotoxicity. [Copyright &y& Elsevier]

The effects of four conjugated linoleic acid (CLA) isomers on in vitro collagen-induced human platelet aggregation and thromboxane (TXB2, the inactive metabolite of the proaggregatory TXA2) production were examined. As the free fatty acid (FFA), 9t, 11t-CLA was the most effective inhibitor of these two processes (I50s of 2.2 and 4 μM, respectively) and the 9c, 11c-CLA was the least effective (I50s of 8.3 and 37 μM) of the isomers tested. When platelets were preesterified with either 25 μM 9t, 11t-CLA or 9c, 11c-CLA, CLA incorporation in total platelet lipids increased from 0.24% to 0.31% and 0.38%, and most of this increase was found to be in the phosphatidyl choline and phosphatidyl ethanolamine subclasses. The decrease in arachidonic acid (AA) content in total fatty acids or phospholipids was an order of magnitude greater. Furthermore, no significant differences between platelets prelabeled with either 9t, 11t- or 9c, 11c-CLA in the inhibition of collagen-induced aggregation and TXB2 formation were observed. However, platelets prelabeled with 9c, 11c-CLA stimulated basal TXB2 production (4-fold) which was not observed with platelets pretreated with either 9t, 11t-CLA, linoleic acid or stearic acid. This enhancement was associated with a 2.4–5-fold increase in the release of endogenous AA. Our results suggest that the presence of a conjugated cis, cis double bond appears to change the lipid environment sufficiently to stimulate the basal platelet phospholipase activity, which in turn increases the formation of TXB2. [Copyright &y& Elsevier]

Although the expression of the prototypic secretory phospholipase A2 (sPLA2), group IIA (sPLA2-IIA), is known to be up-regulated during inflammation, it remains uncertain if other sPLA2 enzymes display similar or distinct profiles of induction under pathological conditions. In this study, we investigated the expression of several sPLA2s in rodent inflammation models. In lipopolysaccharide (LPS)-treated mice, the expression of sPLA2-V, and to a lesser extent that of sPLA2-IID, -IIE, and -IIF, were increased, whereas that of sPLA2-X was rather constant, in distinct tissues. 12-O-Tetradecanoylphorbol-13-acetate (TPA)-induced mouse ear edema, in which the expression of sPLA2-IID, -IIF and -V was increased, was significantly reduced by YM-26734, a competitive sPLA2-IIA inhibitor that turned out to inhibit sPLA2-IID, -IIE, -V and -X as well. In contrast, sPLA2-IIA was dominant in carageenin-induced pleurisy in rats, where the accumulation of exudate fluids and leukocytes was significantly ameliorated by YM-26734. These results indicate that distinct sPLA2s can participate in inflammatory diseases according to tissues, animal species, and types of inflammation. [Copyright &y& Elsevier]

Phospholipase A2 plays a key role in phagocytic cell functions. By screening a synthetic hexapeptide combinatorial library, we identified 24 novel peptides based on their ability to stimulate arachidonic acid release associated with cytosolic phospholipase A2 activity in differentiated HL60 cells. The identified peptides, that contain the consensus sequence (K/R/M)KYY(P/V/Y)M, also induce intracellular calcium release in a pertussis toxin-sensitive manner showing specific action on phagocytic leukocytes, but not on other cells. Functionally, the peptides stimulate superoxide generation and chemotactic migration in human neutrophils and monocytes. Four of the tested active peptides were ligands for formyl peptide receptor like 1. Among these, two peptides with the consensus sequence (R/M)KYYYM can induce intracellular calcium release in undifferentiated HL60 cells that do not express formyl peptide receptor like 1, indicating usage of other receptor(s). A study of intracellular signaling in differentiated HL60 cells induced by the peptides has revealed that four of the novel peptides can induce extracellular signal-regulated protein kinase activation via shared and distinct signaling pathways, based on their dependence of phospatidylinositol-3-kinase, protein kinase C, and MEK. These peptides provide previously unavailable tools for study of differential signaling in leukocytes. [Copyright &y& Elsevier]

The cyclic 1,N2-propanodeoxyguanosine adducts, derived from α,β-unsaturated aldehydes or enals, including acrolein (Acr), crotonaldehyde (Cro), and trans-4-hydroxy-2-nonenal (HNE), have been detected as endogenous DNA lesions in rodent and human tissues. Collective evidence has indicated that the oxidative metabolism of polyunsaturated fatty acids (PUFAs) is an important pathway for endogenous formation of these adducts. In a recent study, we examined the specific role of different types of fatty acids, ω-3 and ω-6 PUFAs, in the formation of cyclic adducts of Acr, Cro, and HNE. Our studies showed that the incubation of deoxyguanosine 5′-monophosphate with ω-3 or ω-6 fatty acids under oxidative conditions in the presence of ferrous sulfate yielded different amounts of Acr, Cro, and HNE adducts, depending on the types of fatty acids. We observed that Acr- and Cro-dG adducts are primarily formed from ω-3, and the adducts derived from longer chain enals, such as HNE, were detected exclusively from ω-6 fatty acids. Acr adducts are also formed from ω-6 fatty acids, but to a lesser extent; the yields of Acr adducts are proportional to the number of double bonds present in the PUFAs. Two previously unknown cyclic adducts, one from pentenal and the other from heptenal, were detected as products from ω-3 and ω-6 fatty acids, respectively. Because ω-6 PUFAs are known to be involved in the promotion of tumorigenesis, we investigated the role of HNE adducts in p53 gene mutation by mapping the HNE binding to the human p53 gene with UvrABC nuclease and determined the formation of HNE-dG adducts in the gene. The results showed that HNE-dG adducts are preferentially formed in a sequence-specific manner at the third base of codon 249 in the p53 gene, a mutational hotspot in human cancers. The DNA repair study using plasmid DNA containing HNE-dG adducts as a substrate in HeLa cell extracts showed that HNE adducts are readily repaired, and that nucleotide excision repair appears to be a major pathway involved. Together, results of these studies provide a better understanding of the involvement of different PUFAs in DNA damage and their possible roles in tumorigenesis. [Copyright &y& Elsevier]

Dexamethasone-treated WEHI7.2 mouse thymoma cells readily undergo apoptosis. WEHI7.2 variants that overexpress catalase (CAT38) or Bcl-2 (Hb12) show a delay or lack of apoptosis, respectively, when treated with dexamethasone. This is accompanied by a delay or lack of cytochrome c release from the mitochondria suggesting that alterations in the signaling phase of apoptosis are responsible for the observed resistance. Because membranes are a rich source of signaling molecules, we have used 31P NMR spectroscopy to compare phospholipids and their metabolites in WEHI7.2, CAT38 and Hb12 cells after dexamethasone treatment. Increased lysophosphatidylcholine (lysoPtdC) content accompanied phosphatidylserine (PtdS) externalization in the WEHI7.2 cells. Both changes were delayed in CAT38 cells suggesting phosphatidylcholine (PtdC) metabolites may play a role in steroid-induced apoptotic signaling. The steroid-resistant Hb12 cells showed a dramatic increase in glycerophosphocholine (GPC) content, suggesting increased phospholipid turnover may contribute to the anti-apoptotic mechanism of Bcl-2. [Copyright &y& Elsevier]

Flavonoids from plant origin show anti-inflammatory activity in vitro and in vivo. In addition to inhibition of inflammation-associated enzymes, such as cyclooxygenases (COX) and lipoxygenases, they have been found to regulate the expression of inflammation-associated proteins from in vitro experiments. In order to prove in vivo behavior and the potential for beneficial use against inflammatory skin disorders, the effect of wogonin (5,7-dihydroxy-8-methoxyflavone) on in vivo expression of several inflammation-associated genes was examined in the intact as well as in the inflamed mouse skin by reverse transcriptase–polymerase chain reaction analysis. When applied topically on the intact skin, only a high dose treatment of wogonin (1000 μg/ear/3 days) slightly increased COX-1 and fibronectin mRNA. On the other hand, wogonin at the doses of 250–1000 μg/ear/3 days potently lowered mRNA levels of COX-2 and tumor necrosis factor-α with less effect on intercellular adhesion molecule-1 and interleukin-1β in a sub-chronic skin inflammation model of tetradecanoylphorbol-13-acetate-induced ear edema (multiple treatment). The decrease of prostaglandin E2 concentration (27.3–34.3%) was concomitantly observed in the wogonin-treated groups. A similar effect was also observed in an acute inflammation model of arachidonic acid-induced ear edema. From the present study, wogonin was proved to differentially regulate the expression of inflammation-associated genes in vivo and to become a useful therapeutic agent for skin inflammatory diseases mainly due to its modulation of the expression of proinflammatory molecules. [Copyright &y& Elsevier]

In the last decade, the knowledge of the pathogenesis of portal hypertension and cirrhosis has increased dramatically. In portal hypertension, almost all the known vasoactive systems/substances are activated or increased and the most recent studies have stressed the importance of the endothelial factors, in particular, prostaglandins. Prostaglandins are formed following the oxygenation of arachidonic acid by the cyclooxygenase (Cox) pathway. An important consideration in portal hypertension and cirrhosis in the periphery is the altered hemodynamic profile and its contributory role in controlling endothelial release of these vasoactive substances. Prostaglandins are released from the endothelium in response to both humoral and mechanical stimuli and can profoundly affect both intrahepatic and peripheral vascular resistance. Within the liver, intrahepatic resistance is altered due to a diminution in sinusoidal responsiveness to vasodilators and an increase in prostanoid vasoconstrictor responsiveness. This review will examine the contributory role of both hormonal and/or hemodynamic force-induced changes in prostaglandin production and signaling in cirrhosis and portal hypertension and the consequence of these changes on the structural and functional response of both the vasculature and the liver. [Copyright &y& Elsevier]

The anticarcinogenic properties of conjugated linoleic acid (CLA) are, at least partially, attributed to its ability to interrupt the n-6 polyunsaturated fatty acid (PUFA) metabolic pathway for the biosynthesis of eicosanoids, including prostaglandins (PG). Both PGE2 and PGF2α play key roles in parturition. In the present study, we compared the effects of CLA (a mixture of cis- and trans-9, 11- and -10, 12-octadecadienoic acid) and linoleic acid (LA) on PG production by cells isolated from maternal intercotyledonary endometrium, fetal allantochorion and amnion from late pregnant ewes. The results demonstrated that supplementation of LA and CLA significantly affected both the proportions and the amounts of PGs produced by all three tissue types. The ability of the uterus and placenta to respond to oxytocin (OT, endometrium only) and lipopolysaccharide (LPS) was also affected. LA inhibited PGE2 and PGF2α production in the absence or presence of either oxytocin or LPS. In endometrial cells with or without oxytocin or LPS, CLA dose-dependently suppressed PGF2α generation, whereas low doses of CLA (20 μM) increased PGE2 generation. Supplementation with CLA therefore increased the PGE2/PGF2α ratio in the endometrial cells. These results suggest that dietary supplementation of LA or CLA may affect both the initiation and progression of parturition. [Copyright &y& Elsevier]

Background: Based on recent studies, pungent constituents of ginger (Zingiber officinale) and related substances represent a potential new class of anti-platelet agents. The ability of 20 pungent constituents of ginger and related substances to inhibit arachidonic acid (AA) induced platelet activation in human whole blood was studied. Methods: Anti-platelet activity of the compounds was measured in vitro by the Chrono Log whole blood platelet aggregometer. Molecular hydrophobicity (log P) was measured by reversed-phase high-performance liquid chromatography. COX-1 (ovine) inhibitory effect of [8]-paradol and analogues 1 and 5 was carried out using a COX-1 inhibitor assay kit. Results: [8]-Gingerol, [8]-shogaol, [8]-paradol and gingerol analogues (1 and 5) exhibited anti-platelet activities with IC50 values ranging from 3 to 7 μM, whilst under similar conditions the IC50 value for aspirin was 20±11 μM. The COX-1 inhibitory activity of [8]-paradol (IC50=4±1 μM) was more potent than the gingerol analogues (1 and 5) (IC50 approximately 20 μM). Conclusion: The above findings show that gingerol compounds and their derivatives are more potent anti-platelet agents than aspirin under the conditions described in this study. [8]-Paradol, a natural constituent of ginger, was found to be the most potent COX-1 inhibitor and anti platelet aggregation agent. The mechanism underlying AA-induced platelet aggregation inhibition may be related to attenuation of COX-1/Tx synthase enzymatic activity. Lastly, important features of phenolic compounds for inhibition of AA-induced platelet aggregation and COX-1 activity were revealed in this study. [Copyright &y& Elsevier]

Cytotoxic drug-induced nausea and vomiting are the side effects most feared by cancer patients. Emesis is an instinctive defense reaction caused by the somatoautonomic nerve reflex, which is integrated in the medulla oblongata. Emesis caused by cytotoxic drugs such as cisplatin is associated with an increase in the concentration of 5-hydroxytryptamine (5-HT) in the intestine and the brainstem. It is proposed that cytotoxic drugs evoke 5-HT release from the enterochromaffin (EC) cells in the intestinal mucosa and that the released 5-HT stimulates the 5-HT receptors on the adjacent vagal afferent nerves. The depolarization of the vagal afferent nerves stimulates the vomiting center in the brainstem and eventually induces a vomiting reflex. 5-HT released from EC cells seems to mediate the cisplatin-induced emesis sensitive to 5-HT3 receptor antagonists. The release of 5-HT from the EC cells, however, is regulated by polymodal mechanisms on autoreceptors or heteroreceptors. The precise role of 5-HT on the occurrence of vomiting has not been fully elucidated. The present review aims to describe the role of 5-HT in anticancer drug-induced emesis from the viewpoint of 5-HT release and afferent vagus nerve activity. Various methods for predicting emesis are also evaluated. [Copyright &y& Elsevier]

Distinct functional coupling between cyclooxygenases (COXs) and specific terminal prostanoid synthases leads to phase-specific production of particular prostaglandins (PGs). In this study, we examined the coupling between COX isozymes and PGF synthase (PGFS). Co-transfection of COXs with PGFS-I belonging to the aldo-keto reductase family into HEK293 cells resulted in increased production of PGF2α only when a high concentration of exogenous arachidonic acid (AA) was supplied. However, this enzyme failed to produce PGF2α from endogenous AA, even though significant increase in PGF2α production occurred in cells transfected with COX-2 alone. This poor COX/PGFS-I coupling was likely to arise from their distinct subcellular localization. Measurement of PGF2α-synthetic enzyme activity in homogenates of several cells revealed another type of PGFS activity that was membrane-bound, glutathione (GSH)-activated, and stimulus-inducible. In vivo, membrane-bound PGFS activity was elevated in the lung of lipopolysaccharide-treated mice. Taken together, our results suggest the presence of a novel, membrane-associated form of PGFS that is stimulus-inducible and is likely to be preferentially coupled with COX-2. [Copyright &y& Elsevier]

Long-chain polyunsaturated fatty acid (LC-PUFA) composition of neural membranes is a key factor for brain development, in chemical communication of neurons and probably also their survival in response to injury. Viability of cholinergic neurons was tested during brain development following dietary supplementation of fish oil LC-PUFAs (docosahexaenoic acid [DHA], eicosapentaenoic acid, arachidonic acid) in the food of mother rats. Excitotoxic injury was introduced by N-methyl-d,l-aspartate (NMDA) injection into the cholinergic nucleus basalis magnocellularis of 14-day-old rats. The degree of loss of cholinergic cell bodies, and the extend of axonal and dendritic disintegration were measured following immunocytochemical staining of cell bodies and dendrites for choline acetyltransferase and p75 low-affinity neurotrophin receptor and by histochemical staining of acetylcholinesterase-positive fibres in the parietal neocortex. The impact of different feeding regimens on fatty acid composition of neural membrane phospholipids was also assayed at 12 days of age. Supplementation of LC-PUFAs resulted in a resistance against NMDA-induced excitotoxic degeneration of cholinergic neurones in the infant rats. More cholinergic cells survived, the dendritic involution of surviving neurons in the penumbra region decreased, and the degeneration of axons at the superficial layers of parietal neocortex also attenuated after supplementing LC-PUFAs. A marked increment in DHA content in all types of phospholipids was obtained in the forebrain neuronal membrane fraction of supplemented rats. It is concluded that fish oil LC-PUFAs, first of all DHA, is responsible for the neuroprotective action on developing cholinergic neurons against glutamate cytotoxicity. [Copyright &y& Elsevier]

The salivary glands and saliva from the lone star tick Amblyomma americanum (L.) were analyzed for the presence of the two endogenous agonists of cannabinoid receptors, N-arachidonoylethanolamine (anandamide) and 2-arachidonoylglycerol (2-AG), as well as of the anandamide congener, N-palmitoylethanolamine (PEA), an anti-inflammatory and analgesic mediator that is inactive at cannabinoid receptors. Two very sensitive mass-spectrometric techniques were used for this purpose. Both 2-AG and PEA, as well as other N-acylethanolamines (NAEs), were identified in salivary glands, but anandamide was below detection. The levels of 2-AG were considerably higher in the salivary glands of partially fed than replete females. Ex vivo gland stimulation with arachidonic acid increased the levels of 2-AG, but not of PEA or other NAEs, and caused the formation of anandamide and of the potent analgesic compound N-arachidonoylglycine. Instead, the amounts of anandamide, 2-AG and PEA were not influenced by treatment of salivary glands with dopamine, which stimulates saliva secretion. The possible biosynthetic precursors of anandamide, PEA and other NAEs were also detected in salivary glands, whereas only PEA was detected in tick saliva. These data demonstrate for the first time that the salivary glands of an obligate ectoparasite species can make endocannabinoids and/or related congeners with analgesic and anti-inflammatory activity, which possibly participate in the inhibition of the host defense reactions. [Copyright &y& Elsevier]

Cyclooxygenase (COX) is the rate-limiting enzyme for the biosynthesis of prostaglandins in monocytes/macrophages. The COX-1 is constitutively expressed in most tissues and may be involved in cellular homeostasis, whereas the COX-2 is an inducible enzyme that may play an important role in inflammation and mitogenesis. When U937 monocytic cells were incubated with retinoic acid (RA) for 48 h, cell differentiation took place with concomitant increases in prostaglandin E2 (PGE2) production and COX activity. In this study, the mechanism of RA (all-trans- or 9-cis-RA)-induced enhancement of PGE2 biosynthesis in U937 cells was examined. Treatment of cells with all-trans- or 9-cis-RA up to 48 h caused an increase in PGE2 production in a time- and dose-dependent manner. Both RA isomers caused the enhancement of PGE2 production and the up-regulation of COX-1 expression at the protein and mRNA levels. The increase in COX-1 mRNA was found to precede the increase in COX-1 protein expression. Interestingly, the COX-2 protein and COX-2 mRNA were not detected in U937 cells, and their levels remained undetectable during the entire course of RA treatment. We conclude that treatment of U937 cells by RA for 48 h caused the initiation of cell differentiation, which was found to be concomitant with a significant increase in PGE2 production mediated via the up-regulation of COX-1 mRNA and protein expression. [Copyright &y& Elsevier]

Increased production of prostaglandin E2 (PGE2) by the combined activities of cyclooxygenase-2 (COX-2) and microsomal glutathione S-transferase 1-like 1 (MGST1-L1) enhances the progression of colorectal cancer. To assess how chemopreventive agents influence colon tumorigenesis, the modulation of PGE2 production by indolo[3,2-b]carbazole (ICZ), β-naphthoflavone (β-NF), and tert-butylhydroquinone (tBHQ), as well as the nonsteroidal anti-inflammatory drug Piroxicam, has been studied in the human HCA-7 colon carcinoma cell line. We have found that these xenobiotics both down-regulate and up-regulate the expression of COX-2 and MGST1-L1. They can also either inhibit or stimulate PGE2 synthesis. COX-2 mRNA levels were increased significantly by those compounds that activate transcription through the xenobiotic responsive element (XRE) and/or the antioxidant responsive element (ARE). A possible ARE enhancer was identified in the COX-2 promoter, and reporter gene experiments demonstrated that tBHQ induction of a transgene driven by the 5′-flanking region of COX-2 was increased by co-transfection with an expression vector for the Nrf2 transcription factor. By contrast, only compounds such as ICZ and β-NF which activate the XRE increased the mRNA levels of MGST1-L1. While the ARE-specific inducer tBHQ did not modulate the basal expression of MGST1-L1, it was found to act as an antagonist of interleukin-1β-stimulated MGST1-L1 overexpression. Changes in COX-2 and MGST1-L1 expression were not always coincident with a corresponding change in PGE2 production by human colon carcinoma cells. Importantly, dietary compounds can modulate PGE2 biosynthesis, and this is likely to influence colon tumorigenesis. [Copyright &y& Elsevier]

Geiji-Bokryung-Hwan (GBH), consisting of herbes of Cinnamomi ramulus (Geiji), Poria cocos (Bokryun), Mountan cortex radicis (Mokdanpi), Paeoniae radix (Jakyak), and Persicae semen (Doin), on antiplatelet activity in human platelet suspensions was studied. The mechanism involved in the antiplatelet activity of GBH in human platelet suspensions was investigated. GBH did not significantly affect the thromboxane synthetase activity of aspirin-treated platelet microsomes and GBH (15 and 30 μg/ml) significantly inhibited [3H]arachidonic acid released in collagen-activated platelets but not in unactivated-platelets. Nitric oxide (NO) production in human platelets was measured by a chemiluminesence detection method in this study. GBH did not significantly affect nitrate production in collagen (10 μg/ml)-induced human platelet aggregation. Various concentrations of GBH (0, 5, 10, 15, and 30 μg/ml) dose-dependently inhibited [3H]inositol monophosphate formation stimulated by collagen (10 μg/ml) in [3H]myoinositol-loaded platelets at different incubation times (1, 2, 3, and 5 min). These results indicated that the antiplatelet activity of GBH may possibly be due to the inhibition of phospholipase C (PLC) activity, leading to reduce phosphoinositide breakdown, followed by the inhibition of thromboxane A2 formation, and then inhibition of [Ca2+]i mobilization of platelet aggregation stimulated by agonists. In conclusion, GBH suppressed PLC in a dose-dependent manner, and may have pharmaceutical applications. These data suggest that GBH extracts merit investigation as a potential anti-atherosclerogenic agent in humans. [Copyright &y& Elsevier]

Resident rat peritoneal macrophages synthesize a variety of prostanoids and leukotrienes from arachidonic acid. Overnight treatment with lipopolysaccharide (LPS) induces the synthesis of cyclooxygenase-2 (COX-2) and an altered prostanoid profile that emphasizes the preferential conversion of arachidonic acid to prostacyclin and prostaglandin E2. In these studies, we report that exposure to LPS also caused a strong suppression of 5-lipoxygenase but not 12-lipoxygenase activity, indicated by the inhibition of synthesis of both leukotriene B4 and 5-hydroxyeicosatetraenoic acid (5-HETE), but not of 12-HETE. Inhibition of 5-lipoxygenase activity by LPS was both time- and dose-dependent. Treatment of macrophages with prostaglandin E2 partially inhibited leukotriene synthesis, and cyclooxygenase inhibitors partially blocked the inhibition of leukotriene generation in LPS-treated cells. In addition to COX-2, nitric oxide synthase (NOS) was also induced by LPS. Treatment of macrophages with an NO donor mimicked the ability of LPS to significantly reduce leukotriene B4 synthesis. Inhibition of NOS activity in LPS-treated cells blunted the suppression of leukotriene synthesis. Inhibition of both inducible NOS and COX completely eliminated leukotriene suppression. Finally, macrophages exposed to prolonged LPS demonstrated impaired killing of Klebsiella pneumoniae and the combination of NOS and COX inhibitors restored killing to the control level. These results indicate that prolonged exposure to LPS severely inhibits leukotriene production via the combined action of COX and NOS products. The shift in mediator profile, to one that minimizes leukotrienes and emphasizes prostacyclin, prostaglandin E2 and NO, provides a signal that reduces leukocyte function, as indicated by impaired killing of Gram-negative bacteria. [Copyright &y& Elsevier]

Several developments during the past 15 years have profoundly affected our understanding of the vomeronasal system (VNS) of vertebrates. In the mid 1990s, the vomeronasal epithelium of mammals was found to contain two populations of receptor cells, based on their expression of G-proteins. These two populations of neurons were subsequently found to project their axons to different parts of the accessory olfactory bulb (AOB), forming the basis of segregated pathways with possibly heterogeneous functions. A related discovery was the cloning of members of at least two gene families of putative vomeronasal G-protein-coupled receptors (GPRs) in the vomeronasal epithelium. Ligand binding to these receptors was found to activate a phospholipase C (PLC)-dependent signal transduction pathway that primarily involves an increase in intracellular inositol-tris-phosphate and intracellular calcium. In contrast to what was previously believed, neuron replacement in the vomeronasal epithelium appears to occur through a process of vertical migration in most mammals. New anatomical studies of the central pathways of the olfactory and vomeronasal systems indicated that these two systems converge on neurons in the telencephalon, providing an anatomical substrate for functional interactions. Combined anatomical, physiological and behavioral studies in mice provided new information that furthered our understanding of one of the most striking pheromonal phenomena, the Bruce effect. Finally, contrary to prior observations, new anatomical studies indicated that a vomeronasal organ (VNO) was present in human adults and reports were published indicating that this system might be functional. These latter observations are still controversial and require confirmation from independent laboratories. [Copyright &y& Elsevier]

Inhibition of platelet cyclooxygenase (COX)-1 is involved in aspirin cardioprotection observed in clinical trials, but, in some patients, aspirin is unable to protect from thrombotic complications. An incomplete suppression of platelet thromboxane (TX) A2 biosynthesis has been assumed to participate in the phenomenon of aspirin resistance, as a consequence of the following possible mechanisms: (i) COX-2 expression in newly formed platelets; (ii) pharmacodynamic interactions between aspirin and coadministered nonsteroidal antiinflammatory drugs (e.g. ibuprofen); (iii) expression of variant isoforms of COX-1 with reduced sensitivity to irreversible inactivation at Ser529. Furthermore, aspirin failure may be due to enhanced formation of vasoactive and/or proaggregatory eicosanoids despite an almost complete suppression of platelet TXA2 biosynthesis by aspirin. Thus, in a subset of patients with unstable angina treated with low-dose aspirin, to almost completely block platelet COX-1 activity, enhanced TXA2 biosynthesis in vivo has been demonstrated, presumably through an increased generation of COX-2-dependent PGH2 in plaque monocytes/macrophages or activated vascular cells. The concomitant increased formation of 8-iso-PGF2α, one of the most abundant F2-isoprostanes in humans, generated by free-radical catalyzed arachidonate peroxidation, may be involved in aspirin resistance because of its capacity to induce platelet and vascular smooth muscle cell activation through the interaction with thromboxane receptors (TPs). Finally, enhanced production of vasoactive cysteinyl leukotrienes (cys-LTs) occurs in unstable angina despite conventional antithrombotic and antianginal treatment. The use of selective pharmacological tools (i.e. highly selective COX-2 inhibitors, TP antagonists, cys-LT inhibitors and antagonists) will allow to ascertain the contribution of aspirin insensitive eicosanoid biosynthesis in aspirin cardioprotective failure. [Copyright &y& Elsevier]

The present study was designed to assess the effect of supplementation with dietary cytidine (5′)-diphosphocholine (CDP-choline), a source of cytidine and choline, on memory in young and older rats. Although the hippocampal-dependent memory deficits in aged rats are well documented, cognitive functioning in early aging has not been as thoroughly evaluated. Female Sprague–Dawley rats (3 or 15 months of age) consumed either a control diet or a diet supplemented with CDP-choline (approximately 500 mg/kg/day) for 8 weeks, after which they were trained to perform spatial and cued versions of the Morris water maze. Compared with young rats, aged rats exhibited a selective deficit in spatial memory tasks that required rats to retain information for 24 h or longer. CDP-choline supplementation protected against the development of this deficit, but had no memory-enhancing effect in normal young rats. These findings suggest that early-aged rats display a selective impairment in hippocampal-dependent long-term memory, and that dietary CDP-choline supplementation can protect against this deficit. [Copyright &y& Elsevier]

The effects of dietary flaxseed (FS), and defatted flaxseed meal (FLM) on serum and tissue fatty acid profiles were investigated. Pregnant Sprague–Dawley rats were fed AIN-93 based diets balanced in calories, fat, nitrogen, and fiber. Diets contained 0, 20%, 40% FS or 13% or 26% FLM by weight. The control, FS and FLM diets differed in linoleic acid to α-linolenic acid (ALA) fatty acid ratio. These diets were fed continuously during gestation, suckling period and 8 weeks post-weaning (F1). FS fatty acids were bioavailable and metabolized by pregnant and F1 rats. ALA and eicosapentaenoic acid increased; linoleic and arachidonic acid decreased; and docosahexaeonic acid was unchanged in serum, ‘gastric milk’ and liver of FS and FLM-fed pregnant and F1 rats. FS more than FLM, changed fatty acids profiles, but FLM and 40% FS significantly reduced serum cholesterol. Dietary 40% FS may have increased oxidative stress as evidenced by a reduction in liver vitamin E. [Copyright &y& Elsevier]

This is a review of our present understanding of the mechanism by which the n−3 polyunsaturated fatty acids (PUFA) in fish oils prevent fatal ventricular arrhythmias in animals and cultured heart cells. A brief review of three clinical trials that suggest that these PUFAs prevent sudden cardiac death is also included in order to emphasize the potential importance of these fatty acids in human nutrition. The PUFAs act by stabilizing electrically every cardiac myocyte by modulating conductance of ion channels in the sarcolemma, particularly the fast, voltage-dependent sodium current and the L-type calcium currents, though other ion currents are also affected. Work in progress suggests that the primary site of action of the PUFAs may be on the phospholipid bilayer of the heart cells in the microdomains through which the ion channels penetrate the membrane bilayer in juxtaposition with the ion channels rather than directly on the channel protein itself. These PUFAs then allosterically alter the conformation and conductance of the channels. Both potential benefits and possible adverse effects of the PUFAs in man will be discussed. Knowing that the ion channels have been structurally conserved among all excitable tissues, we tested their effects on the electrophysiology of rat hippocampal CA1 neurons and found that the sodium and calcium ion channels in these neurons were also affected by PUFAs. An attempt to show the place of the PUFAs in human nutrition during the 2–4 million years of our evolution will conclude the review. [Copyright &y& Elsevier]

Recent evidence suggests that receptors may exist in an “inactive” conformation (designated R) in equilibrium with one or more so-called “active” conformations (R*, R**, R***, etc.) that have the ability to activate cellular effector mechanisms in the absence of an agonist (constitutive activity). In addition, many current models of receptor function suggest that different active conformations may differentially interact (qualitatively and/or quantitatively) with cellular effector mechanisms. One of the predictions stemming from these models is that constitutive receptor activity should be effector pathway dependent. We demonstrate that inverse agonism at the serotonin2C (5-HT2C) receptor is effector pathway dependent and that the experimental observations can be described by a three-state model of receptor function. Further, we provide evidence for protean ligand activity of SB 242084. The results emphasize the importance of associating the pharmacological properties of ligands with the experimental conditions and responses measured. [Copyright &y& Elsevier]

We examined the influence of diets supplemented with fish and vegetable oils on fatty acid and prostaglandin E2 (PGE2) contents in livers of non-7,12-dimethylbenz[a]anthracene (DMBA)- and DMBA-treated rats, and in DMBA-induced tumours. Decreased concentrations of saturated fatty acids and increased unsaturated fatty acid levels were observed in liver phospholipids of rats fed these oils. There was a marked difference in the concentrations of fatty acids found in the tumours and those present in liver lipids. Oleic acid was the main unsaturated fatty acid found in the tumour tissue. Both liver and tumour PGE2 contents were clearly correlated to the diet. The PGE2 concentrations were decreased in livers and tumours of rats fed fish (FO) and linseed oils (LO). [Copyright &y& Elsevier]

In addition to their reported antitumorigenic properties, various conjugated linoleic acid (CLA) isomers have also been shown to decrease prostanoid synthesis as a result of inhibiting the cyclooxygenase (COX) enzyme. We have previously reported that several CLA isomers inhibited both platelet aggregation and formation of thromboxane A2 (TXA2), a proaggregatory and vasoconstrictive agent. Since the interaction between platelets and vascular endothelial cells is essential to maintaining vascular homeostasis, we decided to investigate the effects of various CLA isomers on the production of endothelial prostacyclin (PGI2), a potent vasodilator and inhibitor of platelet function. Using interleukin 1-β (IL1-β)-stimulated human umbilical vein endothelial cells (HUVECs), we initially established that HUVECs of passage #2 should be used since these cells were most responsive to thrombin-induced conversion of endogenous arachidonic acid to PGI2, as monitored by the formation of its stable, inactive metabolite, 6-ketoPGF1α. In the first part of the study, the effects of CLA isomers in the free fatty acid form were tested. The 10(E), 12(Z)- and 9(Z), 11(E)-CLA isomers inhibited thrombin-induced 6-ketoPGF1α formation with I50''s of 2.6 and 5.5 μM, whereas the 9(Z), 11(Z)- and 9(E), 11(E)-CLA were ineffective at concentrations up to 60 μM. The inhibitory effect of the 10(E), 12(Z)-CLA was irreversible. Next, the effects of CLA incorporation into HUVECs on PGI2 generation was determined. An average 8-fold stimulation of 6-ketoPGF1α formation was obtained with quiescent IL1-β-exposed HUVECs pretreated for 18 h with 25 μM 9(Z), 11(Z)-CLA, whereas cells preincubated with the 10(E), 12(Z) isomer enhanced this eicosanoid 3-fold. Such IL1-β-treated HUVECs prelabeled with 25 μM 9(Z), 11(Z)-CLA became refractory to thrombin stimulation, as measured by 6-ketoPGF1α production, whereas a small, statistically insignificant, inhibition was observed upon thrombin treatment of HUVECs prelabeled with the 10(E), 12(Z) isomer. Qualitative similar results were obtained with resting or thrombin-stimulated platelets containing these esterified CLA isomers indicating that these effects occur with cells that contain either the COX-1 or COX-2 isozymes. The results of this in vitro study indicate that the effects of CLA on cellular prostanoid formation in endothelial cells and platelets can be either inhibitory or stimulatory, and this seems to depend not only on the specific CLA isomer and whether or not the CLA is in the free fatty acid form or esterified into cellular lipids, but also whether cells are in the resting or stimulated state. These findings suggest that in vivo, CLA might have multiple, complex effects on vascular homeostasis. [Copyright &y& Elsevier]

Renal vascular effects of cyclooxygenase and cytochrome P450 metabolites of arachidonic acid have been extensively studied, with major advances having been made. More recently, studies indicate that arachidonic acid metabolites of the lipoxygenase and cytochrome P450 pathway, such as hydroxyeicosatetraenoic acids and epoxyeicosatrienoic acids, play novel roles in glomerular mesangial and epithelial cells that are relevant to the pathogenesis of kidney disease associated with diabetes and hypertension. These studies demonstrate that eicosanoids generated during the actions of growth factors and vasoconstrictors can modulate disease processes by affecting vascular homeostasis, inflammation, cellular growth, apoptosis and oxidant stress. In addition, they highlight the important roles played by these oxidized lipids in mediating multiple physiological and pathological functions in the kidney through activation of key signal transduction pathways and genes. [Copyright &y& Elsevier]

Lipid mediators can exert their effects by interactions with well-characterised cell surface G-protein-linked receptors. Recently, a group of intracellular receptors have been identified that are activated by a large variety of lipid-derived mediators. Amongst these novel targets, the peroxisome proliferator-activated receptors (PPARs), a family of three (PPARα, β/δ and γ) nuclear receptor/transcription factors have become a major area for investigation. PPARs are found throughout the body, where they have diverse roles regulating lipid homeostasis, cellular differentiation, proliferation and the immune response. There is a great interest, therefore, in the roles of PPARs in a variety of pathological conditions, including diabetes, atherosclerosis, cancer and chronic inflammation. Although, a number of naturally occurring compounds can activate PPARs, it has been difficult, as yet, to characterise any of these mediators as truly endogenous ligands. These findings have lead to the suggestion that PPARs may act just as general lipid sensors. Acting as lipid sensors, PPARs may take changes in lipid/fatty acid balance in the diet or local metabolism and translate them to tissue-specific ligands, exerting tissue-specific effects. Using classical pharmacological criteria for endogenous mediator classification we will critically discuss the variety of pathways for putative ligand generation. [Copyright &y& Elsevier]