Release Of Free Arachidonic Acid Research Articles

A simplified method for preventing postmortem alterations of brain prostanoids for true in situ level quantification

Dramatic postmortem prostanoid (PG) enzymatic synthesis in the brain causes a significant artifact during PG analysis. Thus, enzyme deactivation is required for an accurate in situ endogenous PG quantification. To date, the only method for preventing postmortem brain PG increase with tissue structure preservation is fixation by head-focused microwave irradiation (MW), which is considered the gold standard method, allowing for rapid in situ heat-denaturation of enzymes. However, MW requires costly equipment that suffers in reproducibility, causing tissue loss and metabolite degradation if overheated. Our recent study indicates that PGs are not synthesized in the ischemic brain unless metabolically active tissue is exposed to atmospheric O2. Based on this finding, we proposed a simple and reproducible alternative method to prevent postmortem PG increase by slow enzyme denaturation before craniotomy. To test this approach, mice were decapitated directly into boiling saline. Brain temperature reached 100°C after ∼140 s during boiling, though 3 min boiling was required to completely prevent postmortem PG synthesis, but not free arachidonic acid release. To validate this fixation method, brain basal and lipopolysaccharide (LPS)-induced PG were analyzed in unfixed, MW, and boiled tissues. Basal and LPS-induced PG levels were not different between MW and boiled brains. However, unfixed tissue showed a significant postmortem increase in PG at basal conditions, with lesser differences upon LPS treatment compared to fixed tissue. These data indicate for the first time that boiling effectively prevents postmortem PG alterations, allowing for a reproducible, inexpensive, and conventionally accessible tissue fixation method for PG analysis.

Molecular Mechanisms for the Inflammation-Resolving Actions of Lenabasum

A first-in-class cannabinoid analog called lenabasum that is a CB2 agonist is being developed as an inflammation-resolving drug candidate. Thus far, specific therapeutic targets include scleroderma, cystic fibrosis, dermatomyositis, and lupus, all of which represent unmet medical needs. Two somewhat-independent molecular mechanisms for this type of action are here proposed. Both pathways initially involve the release of free arachidonic acid after activation of the CB2 receptor and phospholipase A2 by lenabasum. The pathways then diverge into a cyclooxygenase 2-mediated and a lipoxygenase-mediated route. The former leads to increased levels of the cyclopentenone prostaglandin 15-deoxy-Δ12,14-prostaglandin-J2 that can activate the NLPR3 inflammasome, which in turn releases caspase-3, leading to apoptosis and the resolution of chronic inflammation. The lipoxygenase-mediated pathway stimulates the production of lipoxin A4 as well as other signaling molecules called specialized proresolving mediators. These also have inflammation-resolving actions. It is not well understood under which conditions each of these mechanisms operates and whether there is crosstalk between them. Thus, much remains to be learned about the mechanisms describing the actions of lenabasum. SIGNIFICANCE STATEMENT: The resolution of chronic inflammation is a major unmet medical need. The synthetic nonpsychoactive cannabinoid lenabasum could provide a safe and effective drug for this purpose. Two putative molecular mechanisms are suggested to better understand how lenabasum produces this action. In both, different metabolites of arachidonic acid act as mediators.

Cytosolic and Calcium-Independent Phospholipases A2 Activation and Prostaglandins E2 Are Associated with Escherichia coli-Induced Reduction of Insulin Secretion in INS-1E Cells.

It is suspected that microbial infections take part in the pathogenesis of diabetes mellitus type 1 (T1DM). Glucose-induced insulin secretion is accompanied by the release of free arachidonic acid (AA) mainly by cytosolic- and calcium independent phospholipases A2 (cPLA2 and iPLA2). Insulinoma cell line (INS-1E) was infected with E. coli isolated from the blood culture of a patient with sepsis. Invasion assay, Scanning Electron Microscopy and Transmission Electron Microscopy demonstrated the capacity of E. coli to enter cells, which was reduced by PLA2 inhibitors. Glucose-induced insulin secretion was significantly increased after acute infection (8h) but significantly decreased after chronic infection (72h). PLA2 activities, cPLA2, iPLA2, phospho-cPLA2, and COX-2 expressions were increased after acute and, even more, after chronic E. coli infection. The silencing of the two isoforms of PLA2s, with specific cPLA2- or iPLA2-siRNAs, reduced insulin secretion after acute infection and determined a rise in insulin release after chronic infection. Prostaglandins E2 (PGE2) production was significantly elevated in INS-1E after long-term E. coli infection and the restored insulin secretion in presence of L798106, a specific EP3 antagonist, and NS-398, a COX-2 inhibitor, and the reduction of insulin secretion in presence of sulprostone, a specific EP3 agonist, revealed their involvement in the effects triggered by bacterial infection. The results obtained demonstrated that cPLA2 and iPLA2 play a key role in insulin secretion process after E. coli infection. The high concentration of AA released is transformed into PGE2, which could be responsible for the reduced insulin secretion.

Transcription Factor RUNX1 Regulates Pctp (Phosphatidylcholine Transfer Protein) in Platelets: A Potential Role in Regulating Platelet Function

Transcription factor (TF) mutations are increasingly recognized to play a major role in inherited platelet abnormalities. RUNX1, a critical hematopoietic TF, acts in a combinatorial manner with other TFs to regulate numerous megakaryocyte (MK)/platelet genes. Human RUNX1 haplodeficiency is associated with thrombocytopenia, numerous platelet function defects, and increased leukemia risk. We have previously described a patient with a heterozygous RUNX1 nonsense mutation in the conserved runt domain necessary for DNA binding (Sun et al Blood 2004;103;948-54). The patient's platelet abnormalities included impaired aggregation and secretion in response to multiple agonists, granule deficiency, and decreased phosphorylation of myosin light chain and pleckstrin, activation of GPIIb-IIIa, 12-HETE production, and platelet protein kinase C-θ. Transcript expression profiling of patient platelets (Sun et al, J Thromb Haemost 2007;5:146-54) showed numerous genes were significantly downregulated, including myosin light chain (MYL9), platelet factor 4 (PF4), protein kinase C-θ (PRCKQ), and 12-lipoxygenase (ALOX12); these have been shown by us to be regulated by RUNX1. The profiling data also showed 10-fold downregulation of phosphatidylcholine transfer protein (PCTP) gene (fold change ratio 0.09, p=0.02) in the patient compared with normal controls. PCTP is a member of the START (Steroidogenic Acute Regulatory Protein-Related Transfer) domain superfamily and is responsible for the intermembrane transfer of phosphatidylcholine (PC), a major plasma membrane phospholipid. Several findings indicate that PCTP is important for platelet function. PC is the main fraction of platelet phospholipids and source of arachidonic acid (AA) upon activation. Release of free AA from PC is the rate-limiting step in thromboxane production. PC is also a substrate for phospholipase D, which yields phosphatidic acid that can generate the second messenger diacylglycerol. Importantly, platelet PCTP has recently been demonstrated to have a major role in the racial differences in platelet responses - increased PCTP expression has been linked to the higher platelet aggregation and calcium mobilization induced by thrombin receptor protease-activated receptor 4 (PAR4) in blacks as compared to whites (Edelstein et al Nat Med 2013;19:1609-16). Little is known regarding the regulation of PCTP in MKs/platelets.Based on the decreased platelet PCTP expression in our patient, we pursued the hypothesis that PCTP is regulated by RUNX1. Corrected total cellular immunofluorescence with anti-PCTP antibody showed significantly reduced platelet PCTP expression by 58% in our patient compared to a normal control. In silico analysis revealed 5 RUNX1 consensus binding sites up to 995 bp of the PCTP 5' upstream region from ATG. To assess for interaction of RUNX1 with the PCTP promoter, chromatin immunoprecipitation (ChIP) assay with anti-RUNX1 antibody was performed using human erythroid leukemia (HEL) cells treated with phorbol 12-myristate 13-acetate (PMA) for 24 hours to induce megakaryocytic transformation. The ChIP studies showed RUNX1 binding to PCTP chromatin in the regions encompassing RUNX1 binding sites 1 (-232/-227) and 2 (-345/-340), at site 3 (-519/-514), and encompassing sites 4 and 5 (-861/-856, -884/-879). Electrophoretic mobility shift assay (EMSA) using PMA-treated HEL cell nuclear extracts showed RUNX1 binding to DNA probes (28-37 bp) containing site 1 (-232/-227) and both sites 4 and 5 (-861/-856, -884/-879). To assess the effect of modulating RUNX1 expression on PCTP expression, PMA-treated HEL cells were transfected with RUNX1 overexpression plasmid or siRNA. PCTP mRNA and protein expression were increased with RUNX1 overexpression and reduced with RUNX1 knockdown, suggesting that PCTP is regulated by RUNX1.Conclusions: Our results provide evidence that PCTP is a direct transcriptional target of and regulated by RUNX1, and a cogent molecular mechanism for downregulation of platelet PCTP in our patient with RUNX1 haplodeficiency. Regulation of PCTP by RUNX1 may be relevant to the platelet dysfunction in RUNX1 haplodeficiency as well as to racial differences in platelet responses linked to the differential platelet expression of PCTP. DisclosuresNo relevant conflicts of interest to declare.

Characterization of the pattern of the nongenomic signaling pathway through which TCDD-induces early inflammatory responses in U937 human macrophages

2,3,7,8-Tetrachlorodibenzo( p)dioxin (TCDD) has been known to induce inflammatory signaling in a number of cell types and tissues. We found that in U937 macrophages TCDD causes rapid activation of cytosolic phospholipase A2 (cPLA2) within 30 min as judged by the increase in the serine 505 phosphorylated form of cPLA2 protein and the increased cellular release of free arachidonic acid. This initial action of TCDD is accompanied with the up-regulation of an important inflammation marker, COX-2 mRNA expression within 1 h, and by 3 h, several other markers become up-regulated. These effects appear to be dependent on the initial increase in the intracellular concentration of Ca 2+, and activation of cPLA2 and COX-2. A comparative study among three different human cell lines showed that activation of COX-2 within 1 h of action of TCDD is a common feature exhibited by all cell lines. On the other hand, the U937 macrophage line appears to be unique among them with respect to its ability to activate TNF-α and IL-8 mRNA expressions, and not requiring Src kinase in propagating the initial signaling of cPLA2. Based on the rapidity of activation of cPLA2 and COX-2, which occurs within 1 h of cell exposure to TCDD, when no change in mRNA expression of CYP1A1 has been observed, it is apparent that this unique action of TCDD is carried out through a distinct “nongenomic” pathway which, is clearly discernable from the classical, “genomic” action pathway of the AhR by not requiring the participation of ARNT.

Formation of eicosanoids, E2/D2 isoprostanes, and docosanoids following decapitation-induced ischemia, measured in high-energy-microwaved rat brain

Inflammatory lipid mediators derived from arachidonic acid (AA) and docosahexaenoic acid (DHA) modify the pathophysiology of brain ischemia. The goal of this work was to investigate the formation of eicosanoids and docosanoids generated from AA and DHA, respectively, during no-flow cerebral ischemia. Rats were subjected to head-focused microwave irradiation 5 min following decapitation (complete ischemia) or prior to decapitation (controls). Brain lipids were extracted and analyzed by reverse-phase liquid chromatography-tandem mass spectrometry. After complete ischemia, brain AA, DHA, and docosapentaenoic acid concentrations increased 18-, 5- and 4-fold compared with controls, respectively. Prostaglandin E(2) (PGE(2)) and PGD(2) could not be detected in control microwaved rat brain, suggesting little endogenous PGE(2)/D(2) production in the brain in the absence of experimental manipulation. Concentrations of thromboxane B(2), E(2)/D(2)-isoprostanes, 5-hydroxyeicosatetraenoic acid (5-HETE), 5-oxo-eicosatetraenoic acid, and 12-HETE were significantly elevated in ischemic brains. In addition, DHA products such as mono-, di- and trihydroxy-DHA were detected in control and ischemic brains. Monohydroxy-DHA, identified as 17-hydroxy-DHA and thought to be the immediate precursor of neuroprotectin D(1), was 6.5-fold higher in ischemic than in control brain. The present study demonstrated increased formation of eicosanoids, E(2)/D(2)-IsoPs, and docosanoids following cerebral ischemia. A balance of these lipid mediators may mediate immediate events of ischemic injury and recovery.

An improved LC-MS/MS procedure for brain prostanoid analysis using brain fixation with head-focused microwave irradiation and liquid-liquid extraction

High-performance liquid chromatography with tandem mass spectrometry detection (LC-MS/MS) allows a highly selective, sensitive, simultaneous analysis for prostanoids (PG) without derivatization. However, high chemical background noise reduces LC-MS/MS selectivity and sensitivity for brain PG analysis. Four common methods using different solvent systems for PG extraction were tested. Although these methods had the same recovery of PG, the modified acetone extraction followed by liquid/liquid purification had the greatest sensitivity. This method combined with hexane/2-propanol extraction permits the simultaneous analysis of other lipid molecules and PG in the same extract. We also determined that PG mass in brain powder stored at -80 degrees C was reduced 2- to 4- fold in 4 weeks; however, PG were stable for long periods (>3 months) in hexane/2-propanol extracts. PG mass was increased significantly when mice were euthanized by decapitation and the brains rapidly flash-frozen rather than euthanized using head-focused microwave irradiation. This reduction is not the result of PG trapping or destruction in microwave-irradiated brains, demonstrating its importance in limiting mass artifacts during brain PG analysis. Our improved procedure for brain PG analysis provides a reliable, rapid means to detect changes in brain PG mass under both basal and pathological conditions and demonstrates the importance of sample preparation in this process.

Phospholipase A2 Activation Regulates Cytotoxicity of Methylmercury in Vascular Endothelial Cells

Mercury has been identified as a risk factor for cardiovascular disease among humans. Through diet, mainly fish consumption, humans are exposed to methylmercury, the biomethylated organic form of environmental mercury. As the endothelium is an important player in homeostasis of the cardiovascular system, here, the authors tested their hypothesis that methylmercury activates the lipid signaling enzyme phospholipase A(2) (PLA(2)) in vascular endothelial cells (ECs), causing upstream regulation of cytotoxicity. To test this hypothesis, the authors used bovine pulmonary artery ECs (BPAECs) cultured in monolayers, following labeling of their membrane phospholipids with [(3)H]arachidonic acid (AA). The cells were exposed to methylmercury chloride (MMC) and then the release of free AA (index of PLA(2) activity) and lactate dehydrogenase (LDH; index of cytotoxicity) were determined by liquid scintillation counting and spectrophotometry, respectively. MMC significantly activated PLA(2) in a dose-dependent (5 to 15 microM) and time-dependent (0 to 60 min) fashion. Sulfhydryl (thiol-protective) agents, calcium chelators, antioxidants, and PLA(2)-specific inhibitors attenuated the MMC-induced PLA(2) activation, suggesting the role of thiols, reactive oxygen species (ROS), and calcium in the activation of PLA(2) in BPAECs. MMC also induced the loss of thiols and increase of lipid peroxidation in BPAECs. MMC induced cytotoxicity in BPAECs as observed by the altered cell morphology and LDH leak, which was significantly attenuated by PLA(2) inhibitors. This study established that PLA(2) activation through thiols, calcium, and oxidative stress was associated with the cytotoxicity of MMC in BPAECs, drawing attention to the involvement of PLA(2) signaling in the methylmercury-induced vascular endothelial dysfunctions.

TLR3-Dependent Induction of Nitric Oxide Synthase in RAW 264.7 Macrophage-Like Cells via a Cytosolic Phospholipase A2/Cyclooxygenase-2 Pathway

dsRNA is a by-product of viral replication capable of inducing an inflammatory response when recognized by phagocyte cells. In this study, we identify group IVA cytosolic phospholipase A2 (cPLA2alpha) as an effector of the antiviral response. Treatment of RAW 264.7 murine macrophage-like cells with the dsRNA analog polyinosinic:polycytidylic acid (poly-IC) promotes the release of free arachidonic acid that is subsequently converted into PGE2 by the de novo-synthesized cyclooxygenase-2 (COX-2) enzyme. These processes are blocked by the selective cPLA2alpha inhibitor pyrrophenone, pointing out to cPLA2alpha as the effector involved. In keeping with this observation, the cPLA2alpha phosphorylation state increases after cellular treatment with poly-IC. Inhibition of cPLA2alpha expression and activity by either small interfering RNA (siRNA) or pyrrophenone leads to inhibition of the expression of the inducible NO synthase (iNOS) gene. Moreover, COX-2-derived PGE2 production appears to participate in iNOS expression, because siRNA inhibition of COX-2 also leads to inhibition of iNOS, the latter of which is restored by exogenous addition of PGE2. Finally, cellular depletion of TLR3 by siRNA inhibits COX-2 expression, PGE2 generation, and iNOS induction by poly-IC. Collectively, these findings suggest a model for macrophage activation in response to dsRNA, whereby engagement of TLR3 leads to cPLA2alpha-mediated arachidonic acid mobilization and COX-2-mediated PGE2 production, which cooperate to induce the expression of iNOS.

Mercury Activates Phospholipase A2 and Induces Formation of Arachidonic Acid Metabolites in Vascular Endothelial Cells

ABSTRACTCurrently, mercury has been identified as a risk factor in cardiovascular diseases among humans. Here, we tested our hypothesis that mercury modulates the activity of the vascular endothelial cell (EC) lipid signaling enzyme phospholipase A2 (PLA2), which is an important player in the EC barrier functions. Monolayers of bovine pulmonary artery ECs (BPAECs) in culture, following labeling of membrane phospholipids with [3H]arachidonic acid (AA), were exposed to the inorganic form of mercury, mercury chloride, and the release of free AA (index of PLA2 activity) and formation of AA metabolites were determined by liquid scintillation counting and enzyme immunoassay, respectively. Mercury chloride significantly activated PLA2 in BPAECs in a dose-dependent (0 to 50 μM) and time-dependent (0 to 120 min) fashion. Metal chelators significantly attenuated mercury-induced PLA2 activation, suggesting that cellular mercury–ligand interaction is required for the enzyme activation and that chelators are suitable blockers for mercury-induced PLA2 activation in ECs. Sulfhydryl (thiol-protective) agents, calcium chelating agents, and cPLA2-specific inhibitor also significantly attenuated the mercury-induced PLA2, suggesting the role of thiol and calcium in the activation of cPLA2 in BPAECs. Significant formation of AA metabolites, including the release of total prostaglandins, thromboxane B2, and 8-isoprostane, were observed in BPAECs following their exposure to mercury chloride. Mercury chloride induced cytotoxicity as observed by the altered cell morphology and enhanced trypan blue uptake, which was attenuated by the cPLA2 inhibitor AACOCF3. The results of this study revealed that inorganic mercury-induced PLA2 activation through the thiol and calcium signaling and the formation of bioactive AA metabolites further demonstrated the association of PLA2 with the cytotoxicity of mercury in ECs. Overall, the results of the current study underscore the importance of PLA2 signaling in mercury-induced endothelial dysfunctions.

Phospholipases and Galactolipases Trigger Oxylipin‐Mediated Wound‐Activated Defence in the Red Alga Gracilaria chilensis against Epiphytes

We investigated the wound response of the commercially important red alga, Gracilaria chilensis, in order to obtain insight into its interaction with epiphytic pests. After wounding, the host releases free fatty acids as well as the hydroxylated eicosanoids, 8R-hydroxy eicosatetraenoic acid (8-HETE) and 7S,8R-dihydroxy eicosatetraenoic acid (7,8-di-HETE). While the release of free arachidonic acid and subsequent formation of 8-HETE is controlled by phospholipase A, 7,8-di-HETE production is independent of this lipase. This dihydroxylated fatty acid might be directly released from galactolipids. Physiologically relevant concentrations of oxylipins reduced spore settlement of Acrochaetium sp. (Rhodophyta, Acrochaetiaceae) and suppressed the development of hapteria in Ceramium rubrum (Rhodophyta, Ceramiaceae) when these model epiphytes were exposed to artificial surfaces that contained 8-HETE or 7,8-di-HETE. Thus, the immediate release of oxylipins can be seen as G. chilensis defence against epiphytes.

Leptin augments alveolar macrophage leukotriene synthesis by increasing phospholipase activity and enhancing group IVC iPLA2 (cPLA2gamma) protein expression.

Leptin is a hormone secreted by adipocytes in correlation with total body fat mass. In addition to regulating energy homeostasis, leptin modulates immune functions such as macrophage phagocytosis and cytokine synthesis. Previously, we reported defective leukotriene synthesis in macrophages from leptin-deficient mice that could be restored with exogenous leptin. In the present study, we utilized macrophages from normal rodents to explore the mechanism by which leptin could enhance cellular leukotriene synthesis. Leptin pretreatment of either rat alveolar or murine peritoneal macrophages for 16 h dose dependently increased the synthesis of leukotriene B4 and cysteinyl leukotrienes in response to calcium ionophore or the particulate zymosan. Leptin also enhanced calcium ionophore-stimulated release of free arachidonic acid. Calcium-dependent and -independent arachidonoyl-selective phospholipase activities in macrophage lysates were likewise increased following leptin treatment. Immunoblot analysis of leptin-treated cells revealed that group IVC iPLA2 (cPLA2gamma) protein expression increased approximately 80%. These data demonstrate for the first time that phospholipase A2 activity and cPLA2gamma protein levels in alveolar macrophages represent targets for upregulation by leptin and provide previously unrecognized mechanisms by which this hormone can promote inflammatory responses.

Congenital disorders of platelet signal transduction.

After injury to the blood vessel, platelets adhere to the exposed subendothelium by a process (adhesion) that involves the interaction of a plasma protein, von Willebrand factor (vWF), and a specific protein on the platelet surface, glycoprotein Ib (GPIb; the Figure⇓). Adhesion is followed by recruitment of additional platelets that form clumps, a process called aggregation (cohesion). This involves binding of fibrinogen to specific platelet surface receptors—a complex comprising glycoproteins IIb-IIIa (GPIIb-IIIa). Activated platelets release the contents of their granules (secretion or release reaction), such as ADP and serotonin from dense granules, which subsequently cause recruitment of additional platelets. In addition, platelets play a major role in coagulation mechanisms; several key enzymatic reactions occur on the platelet membrane–lipoprotein surface. A number of physiological agonists interact with specific receptors on the platelet surface to induce responses, including a change in platelet shape from discoid to spherical, aggregation, secretion, and thromboxane A2 (TxA2) production. Other agonists such as prostacyclin inhibit these responses. Ligation of the platelet receptors initiates the production or release of several intracellular messenger molecules, including Ca2+ ions, products of phosphoinositide (PI) hydrolysis by phospholipase C (PLC; diacylglycerol [DG] and inositol 1,4,5-triphosphate [InsP3]), TxA2, and cyclic nucleotides (cAMP; the Figure⇓). These subsequently induce or modulate the various platelet responses of Ca2+ mobilization, protein phosphorylation, aggregation, secretion, and liberation of arachidonic acid. The interaction between the agonist receptors and the key intracellular effector enzymes (eg, PLA2, PLC, adenylyl cyclase) is mediated by a group of GTP-binding proteins that are modulated by GTP. As in most secretory cells, platelet activation results in …

PLA 2 activity in Tetrahymena pyriformis. Effects of inhibitors and stimulators

Phospholipase A 2 (PLA 2) is an enzyme which participates in signalling mechanisms cleaving arachidonate from sn-2 position of glycerophospholipids. In this study we have verified the existence of a PLA 2-like activity in the free living protozoan, Tetrahymena pyriformis GL. This activity is Ca 2+-independent, EDTA (10 mM) has no effect on its activity. Quinacrine (0.1 mM) and 4-bromophenacyl bromide (BPB; 0.1 mM) inhibited, melittin (20 μg/ml significantly stimulated the PLA 2 activity and the release of free arachidonic acid (AA) from 1-acyl 2- 14C-arachidonyl-3-phosphatidylethanolamine substrate. Melittin stimulated PLA 2 hyperactivity is Ca 2+-dependent. There was no considerable alteration in the PLA 2 activity by stimulation of the activity by tyrosine kinase (with vanadate, H 20 2), phospholipase C (PLC) (with phorbol 12, 13-dibutyrate) or G-proteins (with NaF, AlF 4 thus in Tetrahymena PLA 2 activity seems to be independent of these—in Tetrahymena (also functioning)—signalling pathways. Treatment with quinacrine and BPB leads to decreased synthesis and disturbed breakdown of phospholipids and phosphoinositides. These findings suggest that PLA 2 activity is in connection with the phospholipid metabolism of Tetrahymena.

Arachidonic acid as an endogenous signal for the glutathione-induced feeding response in Hydra

Phospholipase A2 -derived arachidonic acid (AA) and related metabolic products represent an important pathway involved in the regulation of growth and morphogenesis as well as in oxidative processes in cnidarian tissues. Here we present data on the participation of AA in the glutathione (GSH)-induced feeding response in Hydra vulgaris. Under conditions in which it produces the feeding response (which consists mainly of mouth opening followed by mouth closure), GSH dose-dependently induced the release of free arachidonic acid from live polyps. Phospholipase A2 inhibitors blocked this release and enhanced the duration of GSH-induced Hydra mouth opening. Accordingly, AA and, to a smaller extent, -linolenic acid, were found to reduce the duration of the feeding response in a 10–100 M concentration range, and this effect resulted mainly from earlier times of mouth closure. 11-(R)-hydroxy-eicosa-5Z,8Z,12E,14Z-tetraenoic acid, a lipoxygenase metabolite of AA in H. vulgaris, reproduced the effects of its precursor fatty acid at lower concentrations. The kinetics of GSH-induced arachi donate release and GSH-induced feeding response were correlated, suggesting that AA might modulate the mechanisms controlling mouth closure. Finally, the role of AA in the facilitation of the mouth-closing phase of the feeding response was further supported by the finding that: (1) exogenous AA reversed the effect on the feeding response of -aminobutyric acid, which normally delays the times of mouth closure, and (2) endogenous AA was also released in the absence of GSH from live polyps stimulated with antho-RFamide, a neuropeptide abundant in hydrozoans. We suggest that in Hydra AA may act as a second messenger following chemical stimulation.

Priming of human peripheral blood mononuclear cells with lipopolysaccharides for enhanced arachidonic acid release and leukotriene synthesis.

In a previous study, we have shown that the ability of lipopolysaccharides (LPS) to prime isolated neutrophils for enhanced leukotriene B4 (LTB4) synthesis was dependent on the presence of plasma and involved the CD 14 antigen. In the present study, we have investigated the priming of human peripheral blood mononuclear cells (PBMC) with LPS for the subsequent release and metabolism of arachidonic acid. When PBMC were incubated with LPS for up to 2 h or when freshly isolated PBMC were stimulated with N-formyl-methionyl-leucyl-phenylalanine (fMLP) or with LPS alone, little or no synthesis of 5-lipoxygenase products nor arachidonic acid liberation were detected. However, the preincubation of PBMC with LPS for as little as 5 min primed cells for the subsequent synthesis of LTB4 upon stimulation with fMLP. Maximal priming was observed following a 15-min preincubation period and the priming effect was transient as cells preincubated with LPS for 90 min or more were no longer primed for leukotriene synthesis. Monocytes were found to be responsible for the enhanced response to fMLP since purified lymphocytes did not produce LTB4 nor LTC4 in contrast to monocyte-enriched suspensions. The priming for leukotriene synthesis coincided with an increased capacity for the release of free arachidonic acid as measured by mass spectrometry; LPS-primed cells released 8-15 times more arachidonic acid than unprimed cells within 1 min of stimulation with fMLP. Priming was observed with as little as 0.001-0.01 microg LPS/mL when cells were incubated in the presence of 10% autologous plasma. Interestingly, in the absence of plasma, priming was only observed at LPS concentrations of 0.1 microg/mL or greater. Pretreatment of cells with anti-CD14 antibodies significantly decreased the priming effect observed with 0.01 microg/mL LPS but did not affect priming with 1 microg/mL LPS. These results indicate that the priming of human PBMC with LPS for the subsequent synthesis of arachidonic acid metabolites via the 5-lipoxygenase pathway is dependent on plasma and CD14 at lower concentrations of LPS (0.001-0.01 microg/mL) but not at LPS concentrations of 0.1 microg/mL or greater.

Interaction of Platelet-Activating Factor with Rat Hepatocytes: Uptake, Translocation, Metabolism, and Effects on PAF-Acetylhydrolase Secretion and Protein Tyrosine Phosphorylation

In the present study the interaction of the phospholipid mediator platelet-activating factor (PAF) with rat hepatocytes in primary culture was examined. Following exposure to hepatocytes, exogenous [3H]alkyl-PAF was metabolized rapidly to [3H]lyso-PAF, the content of which was raised in the outer leaflet of the plasma membrane within the initial 5 min of incubation. Thereafter [3H]lyso-PAF was translocated into cells with concomitant reacylation to [3H]alkyl-acyl-glycerophosphocholine. A portion of untransformed [3H]PAF accumulated in the outer leaflet, and only a small amount of the [3H]PAF was translocated into the inner leaflet of the plasma membrane. Detectable levels of [3H]lyso-PAF were found in the medium of hepatocyte cultures at all times of incubation. These findings suggest that at least a portion of the cellular PAF-acetylhydrolase (PAF-AH) activity is located in the outer leaflet of the plasma membrane and can be secreted into the medium. Indeed, rat hepatocytes in culture released PAF-AH into the medium in a time-dependent fashion. Incubation of hepatocytes with exogenous PAF increased secretion of PAF-AH, whereas lyso-PAF and the nonhydrolyzable analog methylcarbamyl-PAF significantly reduced secretion. The structurally related PAF receptor antagonist CV 3988 markedly inhibited the activity of PAF-AH and also diminished its release by hepatocytes. In contrast, BN 50739 and WEB 2170, thienotriazolodiazepine PAF receptor antagonists, did not affect the PAF-AH activity, but increased its secretion by the cells. A full-length 3.8-kb mRNA encoding the cell surface PAF receptor was absent in hepatocytes as indicated by Northern blot analysis using the rat PAF receptor cDNA, whereas PAF receptor mRNA was readily detected in Kupffer cells. Upon incubation with hepatocytes, PAF induced tyrosine phosphorylation of proteins with molecular masses of 120–130 and 160–180 kDa and dephosphorylation of 80- to 90-kDa proteins; these responses were not inhibited by WEB 2170 and BN 50739. The protein tyrosine kinase inhibitor genistein abolished the release of free arachidonic acid, suggesting a crucial role for tyrosine phosphorylation in PAF-induced phospholipase A2activation in rat hepatocytes. Taken together, our data indicate that the interaction of PAF with rat hepatocytes is dependent upon its metabolism, involves protein tyrosine phosphorylation/dephosphorylation and arachidonic acid release, and does not involve the heteromeric G-protein-coupled PAF receptor which has been characterized in Kupffer cells. This metabolically regulated mechanism for PAF action on hepatocytes may be of potential biological importance in the liver under normal and pathological conditions.

Involvement of an arachidonic-acid-dependent pathway in the interferon-beta-mediated expression of C202 gene in Ehrlich-ascites-tumor cells.

We have investigated the signal transduction mechanism of the expression of the C202 gene mediated by interferon beta (IFN-beta) in the murine Ehrlich's ascites tumor cell line. We have shown that treatment of cells with IFN-beta transiently enhances within minutes the release of free arachidonic acid through membrane phospholipase activity. Furthermore, prior treatment with either p-bromophenacyl bromide, an antagonist of both cytosolic and secretory phospholipase A2, or neomycin, which blocks phospholipase C activity, significantly decreased the activation of the murine IFN-beta-inducible gene, C202. Moreover, an increase of the expression of the C202 gene was observed after blocking of both the cyclooxygenase and lipoxygenase pathways. This suggests that further metabolism of arachidonic acid to epoxides via epoxygenase-catalysed pathways may be a mechanism by which second messengers for IFN-beta-mediated effects on C202 gene expression are generated. Taken together, these results indicate that lipids as second messengers may be important mediators in the IFN-beta-based activation of C202 gene expression.

Stimulation of proximal convoluted tubule phosphate transport by epidermal growth factor: signal transduction.

The present study investigated the signal-transduction pathway responsible for the epidermal growth factor (EGF) stimulation of phosphate transport (JPhos) in the rabbit proximal convoluted tubule (PCT). Genistein, 10(-4) M, bath and lumen, an inhibitor of EGF receptor tyrosine kinase activity, blocked the EGF effect on JPhos, consistent with a role for tyrosine kinase in the signal-transduction pathway. Both staurosporine (5 x 10(-8) M) and calphostin C (10(-8) M), inhibitors of protein kinase C, blocked the EGF stimulation of JPhos, indicating that protein kinase C is involved in EGF signaling. Intracellular calcium (Ca2+i) concentrations were measured in perfused tubules using fura PE3 to determine whether changes in Ca2+i were also part of the signaling pathway. After addition of 3 nM EGF, there was no change in Ca2+i, suggesting that stimulation of protein kinase C is not from phosphatidylinositol hydrolysis by phospholipase C-gamma. To determine whether phospholipase A2 (PLA2) is involved, the inhibitor mepacrine was used. Mepacrine (5 x 10(-5) M) had no direct effect on PCT transport but blocked the stimulatory effect of EGF on JPhos. PLA2 activity, assessed as free arachidonic acid release from proximal tubules in suspension, increased by 18.8% with 3 nM EGF. Thus the stimulation of JPhos by EGF is mediated via a signal-transduction pathway involving tyrosine kinase, protein kinase C, and PLA2.

Phospholipase participation in cannabinoid-induced release of free arachidonic acid

The exposure of cells in culture to cannabinoids results in a rapid and significant mobilization of phospholipid bound arachidonic acid. In vivo, this effect has been observed as a rise in eicosanoid tissue levels that may account for some of the pharmacological actions of Δ 9-tetrahydrocannabinol (THC), the major psychoactive cannabinoid. Fluoroaluminate pretreatment of mouse peritoneal cells potently reduced the cannabinoid response, while promoting arachidonate release on its own, consistent with earlier observations that this effect may be a receptor/G-protein-mediated process. Further support for receptor mediation was the demonstration of saturable, high-affinity cannabinoid binding in these cells. THC potency was reduced in the presence of ethanol, and was accompanied by significant increases in phosphatidylethanol (PdEt) levels, a unique product of phospholipase D (PLD) activity. THC-dependent arachidonate release was reduced partially in similar amounts by either propranolol or wortmannin, further implicating PLD as a mediator of THC action. A central role for diacylglyceride (DAG), a secondary product of PLD metabolism, in this THC-induced process, both as a source of arachidonate and as a stimulator of protein kinase C (PKC), is supported by the data in this report. Cells exposed to phorbol ester for 18 hr prior to THC challenge became less responsive, indicating a possible role for PKC. The involvement of PKC further suggests participation by phospholipase A 2 (PLA 2) whose activity may be regulated by the former. Treatment of cells with interleukin-1α, an agent known to elevate PLA 2 levels, caused an increase in the THC response, supporting a role for this enzyme in the release reaction. Direct evidence, by immunoblotting, for the activation and phosphorylation of PLA 2 by THC was also obtained. In summary, the evidence presented in this report indicates that THC-induced arachidonic acid release occurs through a series of events that are consistent with a receptor-mediated process involving the stimulation of one or more phospholipases.