6-(bromomethylene)tetrahydro-3-(1-naphthaleneyl)-2h-pyran-2-one and arachidonyltrifluoromethane

We previously reported that lysophosphatidic acid (LPA) initiates nerve injury-induced neuropathic pain and its underlying mechanisms. In addition, we recently demonstrated that intrathecal injection of LPA induces de novo LPA production through the action of autotaxin (ATX), which converts lysophosphatidylcholine to LPA. Here, we examined nerve injury-induced de novo LPA production by using a highly sensitive biological titration assay with B103 cells expressing LPA1 receptors. Nerve injury caused high levels of LPA production in the ipsilateral sides of the spinal dorsal horn and dorsal roots, but not in the dorsal root ganglion, spinal nerve, or sciatic nerve. Nerve injury-induced LPA production reached its maximum at 3 h after injury, followed by a rapid decline by 6 h. The LPA production was significantly attenuated in ATX heterozygous mutant mice, whereas the concentration and activity of ATX in cerebrospinal fluid were not affected by nerve injury. On the other hand, the activities of cytosolic phospholipase A2 (cPLA2) and calcium-independent phospholipase A2 (iPLA2) were enhanced, with peaks at 1 h after injury. Both de novo LPA production and neuropathic pain-like behaviors were substantially abolished by intrathecal injection of arachidonyl trifluoromethyl ketone, a mixed inhibitor of cPLA2 and iPLA2, or bromoenol lactone, an iPLA2 inhibitor, at 1 h after injury. However, administration of these inhibitors at 6 h after injury had no significant effect on neuropathic pain. These findings provide evidence that PLA2- and ATX-mediated de novo LPA production in the early phase is involved in nerve injury-induced neuropathic pain.

Topics: Animals; Arachidonic Acids; Blotting, Western; Cell Line; Injections, Spinal; Lysophosphatidylcholines; Lysophospholipids; Male; Mice; Mice, Inbred C57BL; Multienzyme Complexes; Naphthalenes; Neuralgia; Pain Measurement; Phosphodiesterase I; Phospholipase A2 Inhibitors; Phospholipases A2; Phosphoric Diester Hydrolases; Posterior Horn Cells; Pyrones; Pyrophosphatases; Sciatic Nerve; Spinal Nerve Roots

Monocyte chemoattractant protein-1 (MCP-1) directs migration of blood monocytes to inflamed tissues. Despite the central role of chemotaxis in immune responses, the regulation of chemotaxis by signal transduction pathways and their in vivo significance remain to be thoroughly deciphered. In this study, we examined the intracellular location and functions of two recently identified regulators of chemotaxis, Ca(2+)-independent phospholipase (iPLA(2)beta) and cytosolic phospholipase (cPLA(2)alpha), and substantiate their in vivo importance. These enzymes are cytoplasmic in unstimulated monocytes. Upon MCP-1 stimulation, iPLA(2)beta is recruited to the membrane-enriched pseudopod. In contrast, cPLA(2)alpha is recruited to the endoplasmic reticulum. Although iPLA(2)beta or cPLA(2)alpha antisense oligodeoxyribonucleotide (ODN)-treated monocytes display reduced speed, iPLA(2)beta also regulates directionality and actin polymerization. iPLA(2)beta or cPLA(2)alpha antisense ODN-treated adoptively transferred mouse monocytes display a profound defect in migration to the peritoneum in vivo. These converging observations reveal that iPLA(2)beta and cPLA(2)alpha regulate monocyte migration from different intracellular locations, with iPLA(2)beta acting as a critical regulator of the cellular compass, and identify them as potential targets for antiinflammatory strategies.

Topics: Actins; Adoptive Transfer; Animals; Arachidonic Acids; Cells, Cultured; Chemokine CCL2; Chemotaxis; Female; Group IV Phospholipases A2; Group VI Phospholipases A2; Humans; Leukocytes, Mononuclear; Mice; Mice, Inbred BALB C; Monocytes; Naphthalenes; Oligodeoxyribonucleotides, Antisense; Peritonitis; Pyrones

Group IIA secretory phospholipase A(2) (sPLA(2)-IIA) is a prototypic sPLA(2) enzyme that may play roles in modification of eicosanoid biosynthesis as well as antibacterial defense. In several cell types, inducible expression of sPLA(2) by pro-inflammatory stimuli is attenuated by group IVA cytosolic PLA(2) (cPLA(2)alpha) inhibitors such as arachidonyl trifluoromethyl ketone, leading to the proposal that prior activation of cPLA(2)alpha is required for de novo induction of sPLA(2). However, because of the broad specificity of several cPLA(2)alpha inhibitors used so far, a more comprehensive approach is needed to evaluate the relevance of this ambiguous pathway. Here, we provide evidence that the induction of sPLA(2)-IIA by pro-inflammatory stimuli requires group VIB calcium-independent PLA(2) (iPLA(2)gamma), rather than cPLA(2)alpha, in rat fibroblastic 3Y1 cells. Results with small interfering RNA unexpectedly showed that the cytokine induction of sPLA(2)-IIA in cPLA(2)alpha knockdown cells, in which cPLA(2)alpha protein was undetectable, was similar to that in replicate control cells. By contrast, knockdown of iPLA(2)gamma, another arachidonyl trifluoromethyl ketone-sensitive intracellular PLA(2), markedly reduced the cytokine-induced expression of sPLA(2)-IIA. Supporting this finding, the R-enantiomer of bromoenol lactone, an iPLA(2)gamma inhibitor, suppressed the cytokine-induced sPLA(2)-IIA expression, whereas (S)-bromoenol lactone, an iPLA(2)beta inhibitor, failed to do so. Moreover, lipopolysaccharide-stimulated sPLA(2)-IIA expression was also abolished by knockdown of iPLA(2)gamma. These findings open new insight into a novel regulatory role of iPLA(2)gamma in stimulus-coupled sPLA(2)-IIA expression.

Topics: Animals; Arachidonic Acids; Calcium; Cell Line; Cytokines; Eicosanoids; Enzyme Induction; Fibroblasts; Group II Phospholipases A2; Group VI Phospholipases A2; Inflammation; Lipopolysaccharides; Naphthalenes; Phosphodiesterase Inhibitors; Phospholipases A; Phospholipases A2; Pyrones; Rats

A considerable body of evidence indicates that phospholipase A(2) (PLA(2)) enzymes participate in long-term potentiation (LTP) of excitatory synaptic transmission. In the present study, we have undertaken experiments to identify which calcium-independent isoform of PLA(2) is involved in synaptic plasticity and to determine whether calcium-independent PLA(2) (iPLA(2)) contributes to post-synaptic processes of LTP. Using field recordings from rat CA1 hippocampal slices, we found that theta-burst stimulation (TBS)-induced LTP of field excitatory post-synaptic potentials (fEPSPs) was abolished by the iPLA(2) inhibitor bromoenol lactone (BEL) but not by the Ca(2+)-dependent PLA(2) inhibitor arachidonyl trifluoromethyl ketone (AACOCF(3)). The ionic currents generated during TBS were not affected during iPLA(2) inhibition as BEL by itself had no effect on the magnitude of facilitation during burst responses. In addition, (R)-BEL, an enantioselective inhibitor of iPLA(2)gamma, precluded TBS-induced LTP, an action that was not replicated by the iPLA(2)beta inhibitors (S)-BEL and methyl arachidonyl fluorophosphonate. (R)-BEL was, however, ineffective on pre-established LTP. Finally, BEL also prevented the potentiation of fEPSPs elicited by brief exposure to 50 microM N-methyl-d-aspartate, as well as the associated up-regulation of alpha-amino-3-hydroxy-5-methylisoxazole-propionate (AMPA) receptor GluR1 subunit levels and the increase of (3)H-AMPA binding in crude synaptic fractions. Collectively, these results unravel a new role for iPLA(2)gamma in LTP, which appears to favor the insertion of AMPA receptors at post-synaptic membranes.

Topics: alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Arachidonic Acids; Blotting, Western; Calcium; Drug Interactions; Electric Stimulation; Gene Expression Regulation; Hippocampus; Immunoprecipitation; In Vitro Techniques; Long-Term Potentiation; Male; N-Methylaspartate; Naphthalenes; Neurons; Organophosphonates; Patch-Clamp Techniques; Phosphodiesterase Inhibitors; Phospholipases A; Protein Binding; Protein Isoforms; Pyrones; Rats; Receptors, AMPA; Synaptic Transmission; Time Factors

Lysophosphatidic acid (LPA) is both a potential marker and a therapeutic target for ovarian cancer. It is critical to identify the sources of elevated LPA levels in ascites and blood of patients with ovarian cancer. We show here that human peritoneal mesothelial cells constitutively produce LPA, which accounts for a significant portion of the chemotactic activity of the conditioned medium from peritoneal mesothelial cells to ovarian cancer cells. Both production of LPA by peritoneal mesothelial cells and the chemotactic activity in the conditioned medium can be blocked by HELSS [an inhibitor of the calcium-independent phospholipase A(2) (iPLA(2))] and AACOCF(3) [an inhibitor of both cytosolic PLA(2) (cPLA(2)) and iPLA(2)]. Moreover, cell-based enzymatic activity assays for PLA(2) indicate that peritoneal mesothelial cells have strong constitutive PLA(2) activity. Receptors for LPA, LPA(2), and LPA(3) are involved in the conditioned medium-induced chemotactic activity. Invasion of ovarian cancer cells into peritoneal mesothelial cells has also been analyzed and shown to require PLA(2), LPA receptors, and the mitogen-activated protein/extracellular signal-regulated kinase kinase/extracellular signal-regulated kinase signaling pathway. Thus, we show here, for the first time, that human peritoneal mesothelial cells constitutively produce bioactive lipid signaling molecules, such as LPA, via iPLA(2) and/or cPLA(2) activities. Conditioned medium from peritoneal mesothelial cells stimulate migration, adhesion, and invasion of ovarian cancer cells, and may play similar roles in vivo.

Topics: Arachidonic Acids; Cell Adhesion; Cell Line, Tumor; Cell Movement; Collagen Type I; Culture Media, Conditioned; Cytosol; Epithelium; Extracellular Signal-Regulated MAP Kinases; Female; Group VI Phospholipases A2; Humans; Isoenzymes; Lysophospholipids; Naphthalenes; Ovarian Neoplasms; Peritoneal Cavity; Peritoneum; Phosphodiesterase Inhibitors; Phospholipases A; Proto-Oncogene Proteins c-akt; Pyrones

Heavy metals are environmental pollutants able to produce different cellular effects, such as an alteration of Ca2+ homeostasis and lysosomal membrane destabilisation. The latter is one of the most used stress indices in biomonitoring programs. Recently, it has been demonstrated that cytosolic calcium increase can modulate lysosomal membrane destabilisation via activation of Ca(2+)-dependent phospholipase A2 (cPLA2). The aim of this work was to investigate the possible involvement of Ca(2+)-activated PLA2 in lysosomal membrane destabilisation induced by heavy metals in mussel haemolymph cells. We have studied the effects of Hg2+ and Cu2+ on free cytosolic calcium using Fura2/AM-loaded cells and lysosomal membrane destabilisation using neutral red (NR) staining. Hg2+ induced a [Ca2+]i rise from 100 to 780 nM in 30 min, and a lysosome destaining of 70% after 60 min that indicates destabilisation of lysosomal membranes. Both effects were reduced in a Ca(2+)-free medium, suggesting a cause-effect relationship. Exposure to Cu2+ produced the same effects, but with an intensity of about 50% respect to Hg2+. Metal-induced lysosomal destabilisation was also reduced in cells pre-exposed to a specific Ca(2+)-dependent cPLA2 inhibitor (AACOCF3). Conversely, haemocyte pretreatment with a Ca(2+)-independent PLA2 inhibitor (bromoenol-lactone (BEL)) did not prevent the destabilizing effect of heavy metals on lysosomes. Exposure to heavy metals also produced an increase in lysosomal volume of 1.8-2-folds, that was prevented by pre-incubation with AACOCF3 but not with BEL. These data indicate an involvement of cPLA2 in lysosomal membrane destabilisation induced by heavy metals.

Topics: Animals; Arachidonic Acids; Bivalvia; Calcium; Copper; Environmental Monitoring; Enzyme Activation; Hemocytes; Italy; Lysosomes; Membranes; Mercury; Naphthalenes; Phospholipases A; Phospholipases A2; Pyrones; Water Pollutants, Chemical

We investigated changes in cytosolic phospholipase A(2) (cPLA(2)) and calcium-independent PLA(2) (iPLA(2)) activities in bovine retina capillary pericytes after stimulation with 50 microM amyloid-beta (Abeta) (1-42) and its (25-35) fragment, over 24 h (mild, sublethal model of cell damage). In the presence of Abeta peptides, we found that cPLA(2) activity was increased and translocated from the cytosolic fraction to the membrane system, particularly in the nuclear region. Reversed-sequence Abeta(35-25) peptide did not stimulate or induce cPLA(2) translocation. Exposure to both Abeta peptides had no significant effect on cPLA(2) protein content as tested by Western immunoblot analysis. The addition of Abetas to quiescent pericytes was followed by phosphorylation of cPLA(2) and arachidonic acid release. Treatment with inhibitors (AACOCF(3), staurosporine and cycloheximide) resulted in a sharp decrease in basal and stimulated cPLA(2) activity. Inactivating effects of bromoenol lactone (BEL), inhibitor of iPLA(2), demonstrated that the stimulation of total PLA(2) activity by Abetas was mediated by both PLA(2) enzymes. Taken together with our previous observations that both Abeta peptides may induce hydrolysis of phosphatidylcholine, the present results provide evidence that this process is cooperatively mediated by cPLA(2) activation/translocation and iPLA(2) activation. The effect is very likely triggered by a mild prooxidant mechanism which was not able to divert the cell to degeneration. The data confirm the hypothesis that pericytes could be a target of potential vascular damage and reactivity during processes involving amyloid accumulation.

Topics: Amyloid beta-Peptides; Animals; Arachidonic Acid; Arachidonic Acids; bcl-2-Associated X Protein; Caspase Inhibitors; Caspases; Cattle; Cells, Cultured; Cycloheximide; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Gene Expression; Group VI Phospholipases A2; Naphthalenes; Oligopeptides; Peptide Fragments; Pericytes; Phospholipases A; Phospholipases A2; Phosphorylation; Poly(ADP-ribose) Polymerases; Protein Transport; Proto-Oncogene Proteins c-bcl-2; Pyrones; Retinal Artery; Staurosporine; Subcellular Fractions

We have earlier demonstrated that dopamine stimulates the liberation of the prostaglandin E(2) (PGE(2)) precursor, arachidonic acid, in Chinese hamster ovary cells transfected with the rat dopamine D(2) receptor (long isoform), also without concomitant administration of a Ca(2+)-releasing agent [Nilsson et al., Br J Pharmacol 1998;124:1651-8]. In the present report, we show that dopamine, under the same conditions, also induces a concentration-dependent increase in the production of PGE(2), with a maximal effect of 235% at approximately 100 microM, and with an EC(50) of 794 nM. The effect was counteracted by the D(2) antagonist eticlopride, pertussis toxin, the inhibitor of intracellular Ca(2+) release TMB-8, incubation in Ca(2+)-free experimental medium, and PKC desensitization obtained by chronic pretreatment with the phorbol ester TPA. It was also antagonized by the non-specific cyclooxygenase (COX) inhibitor, indomethacin, and by the selective COX-2 inhibitor, NS-398, but not by the specific COX-1 inhibitor, valeryl salicylate. Both the non-specific phospholipase A(2) inhibitor, quinacrine, and an inhibitor of cPLA(2) and iPLA(2), AACOF3, counteracted the effect; in contrast, a selective iPLA(2) inhibitor, BEL, and a selective sPLA(2) inhibitor, TAPC, were ineffective. No effects of dopamine were obtained in control cells mock-transfected with the p3C vector only. The results reinforce previous assumptions that dopamine may interact with eicosanoid metabolism by means of D(2) receptor activation, and implicate an involvement of cPLA(2) and COX-2 in this effect. It is suggested that measurement of dopamine-induced PGE(2) production may serve as a convenient way to study D(2) receptor function in vitro.

Topics: Animals; Arachidonic Acids; Calcium; Calcium Channel Blockers; CHO Cells; Cricetinae; Cyclooxygenase 2; Dinoprostone; Dopamine; Dopamine Antagonists; Drug Interactions; Enzyme Inhibitors; Gallic Acid; Indomethacin; Isoenzymes; Naphthalenes; Nitrobenzenes; Phosphatidylcholines; Prostaglandin-Endoperoxide Synthases; Pyrones; Quinacrine; Receptors, Dopamine D2; Salicylamides; Salicylates; Sulfonamides; Tetradecanoylphorbol Acetate; Transfection

The mechanism of lysosome activation by 17beta-estradiol has been studied in mussel blood cells. Cell treatment with estradiol induced a sustained increase of cytosolic free Ca2+ that was completely prevented by preincubating the cells with the Ca2+ chelator BAPTA-AM. Estradiol treatment was also followed by destabilization of the lysosomal membranes, as detected in terms of the lysosomes' increased permeability to neutral red. The effect of estradiol on lysosomes was almost completely prevented by preincubation with the inhibitor of cytosolic Ca2+ -dependent PLA2 (cPLA2), arachidonyl trifluoromethyl ketone (AACOCF3), and was significantly reduced by preincubation with BAPTA-AM. In contrast, it was virtually unaffected by preincubation with the inhibitor of Ca2+ -independent PLA2, (E)-6-(bromomethylene)tetrahydro-3-(1-naphtalenyl)-2H-pyran-2-one (BEL). The Ca2+ ionophore A-23187 yielded similar effects on [Ca2+](i) and lysosomes. Exposure to estradiol also resulted in cPLA2 translocation from cytosol to membranes, lysosome enlargement, and increased protein degradation. These results suggest that the destabilization of lysosomal membranes following cell exposure to estradiol occurs mainly through a Ca2+ -dependent mechanism involving activation of Ca2+ -dependent PLA2. This mechanism promotes lysosome fusion and catabolic activities and may mediate short-term estradiol effects.

Topics: Actins; Animals; Arachidonic Acids; Bivalvia; Calcium; Calcium Signaling; Carbon Radioisotopes; Cytosol; Enzyme Activation; Enzyme Inhibitors; Estradiol; Lysosomes; Microscopy, Confocal; Naphthalenes; Phalloidine; Phosphodiesterase Inhibitors; Phospholipases A; Phospholipases A2; Pyrones; Valine

Changes in endothelium functions during ischemia are thought to be of importance in numerous pathological conditions, with, for instance, an increase in the release of inflammatory mediators like prostaglandins. Here, we showed that hypoxia increases phospholipase A(2) (PLA(2)) activity in human umbilical vein endothelial cells. Both basal PLA(2) activity and PG synthesis are sensitive to BEL and AACOCF3, respectively, inhibitors of calcium-independent PLA(2) (iPLA(2)) and cytosolic PLA(2) (cPLA(2)), while OPC, an inhibitor of soluble PLA(2) (sPLA(2)) only inhibited the hypoxia-induced AA release and PGF(2alpha) synthesis. Hypoxia does not alter expression of iPLA(2), sPLA(2) and cPLA(2) and cycloheximide did not inhibit PLA(2) activation, indicating that hypoxia-induced increase in PLA(2) activity is due to activation rather than induction. However, mRNA levels for sPLA(2) displayed a 2-fold increase after 2 hr incubation under hypoxia. BAPTA, an intracellular calcium chelator, partially inhibited the AA release in normoxia and in hypoxia. Direct assays of specific PLA(2) activity showed an increase in sPLA(2) activity but not in cPLA(2) activity after 2hr hypoxia. Taken together, these results indicate that the hypoxia-induced increase in PLA(2) activity is mostly due to the activation of sPLA(2).

Topics: Arachidonic Acid; Arachidonic Acids; Cell Hypoxia; Cells, Cultured; Dinoprost; Endothelium, Vascular; Enzyme Inhibitors; Humans; Isoenzymes; Naphthalenes; Phospholipases A; Phosphorylcholine; Pyrones

Arachidonyltrifluoromethy ketone (AACOCF(3)), a phospholipase A(2) antagonist, reversibly induced dispersal of Golgi stack- and trans Golgi network (TGN)-resident proteins throughout the cytoplasm in NRK cells as followed by immunocytochemical staining of ManII and TGN38, respectively. The action of AACOCF(3) was partly blocked by other PLA(2) antagonists, suggesting it be not caused by a general inhibition of phospholipase A(2). AACOCF(3) neither dissociated beta-COP from membranes nor prevented brefeldin A-induced beta-COP release. Action of AACOCF(3) on the Golgi stack and TGN is different from that of brefeldin A and nordihydroguaiaretic acid. The most prominent difference is that the Golgi stack and TGN showed a similar sensitivity to AACOCF(3), while the TGN was dispersed more slowly than the Golgi stack in brefeldin A- or nordihydroguaiaretic acid-treated NRK cells. This novel action of AACOCF(3) may be used as pharmacological tool and give new insights into vesicle-mediated traffic and Golgi membrane dynamics.

Topics: Aminobenzoates; Animals; Arachidonic Acids; Biological Transport; Brefeldin A; Cell Line; Chlorobenzoates; Cinnamates; Coatomer Protein; Cytoplasm; Dose-Response Relationship, Drug; Endoplasmic Reticulum; Enzyme Inhibitors; Golgi Apparatus; Masoprocol; Membrane Proteins; Microscopy, Fluorescence; Naphthalenes; ortho-Aminobenzoates; Phospholipases A; Pyrones; Time Factors

1. We investigated the possible involvement of phospholipase A(2) (PLA(2)) and its products in long-term potentiation (LTP) in the CA1 neurotransmission of rat hippocampal slices. 2. Inhibitors of Ca(2+)-independent PLA(2) (iPLA(2)) prevented the induction of LTP without affecting the maintenance phase of LTP whereas Ca(2+)-dependent PLA(2) inhibitors were virtually ineffective, which suggests a pivotal role of iPLA(2) in the initiation of LTP. 3. We then investigated the effect of docosahexaenoic acid (DHA) and arachidonic acid (AA) on BEL (bromoenol lactone, an iPLA(2)-inhibitor) -impaired LTP, and found that either DHA or AA abolished the effect of BEL. However, DHA did not restore BEL-attenuated LTP when applied after the tetanus. DHA per se affected neither the induction nor maintenance of LTP. Linoleic acid had no effects, either. 4. These results suggest that DHA is crucial for the induction of LTP and that endogenously released DHA during tetanus is sufficient to trigger the formation of LTP.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Docosahexaenoic Acids; Dose-Response Relationship, Drug; Excitatory Postsynaptic Potentials; Hippocampus; In Vitro Techniques; Long-Term Potentiation; Male; Naphthalenes; Organophosphonates; Phosphodiesterase Inhibitors; Phospholipases A; Pyrones; Rats; Rats, Wistar; Time Factors

The Ca(2+)-sensing receptor (CaR) stimulates a number of phospholipase activities, but the specific phospholipases and the mechanisms by which the CaR activates them are not defined. We investigated regulation of phospholipase A(2) (PLA(2)) by the Ca(2+)-sensing receptor (CaR) in human embryonic kidney 293 cells that express either the wild-type receptor or a nonfunctional mutant (R796W) CaR. The PLA(2) activity was attributable to cytosolic PLA(2) (cPLA(2)) based on its inhibition by arachidonyl trifluoromethyl ketone, lack of inhibition by bromoenol lactone, and enhancement of the CaR-stimulated phospholipase activity by coexpression of a cDNA encoding the 85-kDa human cPLA(2). No CaR-stimulated cPLA(2) activity was found in the cells that expressed the mutant CaR. Pertussis toxin treatment had a minimal effect on CaR-stimulated arachidonic acid release and the CaR-stimulated rise in intracellular Ca(2+) (Ca(2+)(i)), whereas inhibition of phospholipase C (PLC) with completely inhibited CaR-stimulated PLC and cPLA(2) activities. CaR-stimulated PLC activity was inhibited by expression of RGS4, an RGS (Regulator of G protein Signaling) protein that inhibits Galpha(q) activity. CaR-stimulated cPLA(2) activity was inhibited 80% by chelation of extracellular Ca(2+) and depletion of intracellular Ca(2+) with EGTA and inhibited 90% by treatment with W7, a calmodulin inhibitor, or with KN-93, an inhibitor of Ca(2+), calmodulin-dependent protein kinases. Chemical inhibitors of the ERK activator, MEK, and a dominant negative MEK, MEK(K97R), had no effect on CaR-stimulated cPLA(2) activity but inhibited CaR-stimulated ERK activity. These results demonstrate that the CaR activates cPLA(2) via a Galpha(q), PLC, Ca(2+)-CaM, and calmodulin-dependent protein kinase-dependent pathway that is independent the ERK pathway.

Topics: Arachidonic Acid; Arachidonic Acids; Benzylamines; Binding, Competitive; Calcium; Calcium-Calmodulin-Dependent Protein Kinases; Calmodulin; Cell Line; Cytosol; DNA, Complementary; Dose-Response Relationship, Drug; Egtazic Acid; Enzyme Activation; Enzyme Inhibitors; Estrenes; Genes, Dominant; GTP-Binding Protein alpha Subunits, Gq-G11; Heterotrimeric GTP-Binding Proteins; Humans; Immunoblotting; Inhibitory Concentration 50; Kinetics; Mitogen-Activated Protein Kinases; Mutation; Naphthalenes; Pertussis Toxin; Phosphodiesterase Inhibitors; Phospholipases A; Phospholipases A2; Protein Binding; Protein Kinase C; Pyrones; Pyrrolidinones; Receptors, Calcium-Sensing; Receptors, Cell Surface; RGS Proteins; Signal Transduction; Spectrometry, Fluorescence; Sulfonamides; Time Factors; Transfection; Type C Phospholipases; Virulence Factors, Bordetella

Monocyte chemoattractant protein 1 (MCP-1) has an important influence on monocyte migration into sites of inflammation. Our understanding of the signal transduction pathways involved in the response of monocytes to MCP-1 is quite limited yet potentially significant for understanding and manipulating the inflammatory response. Prior studies have demonstrated a crucial regulatory role for cytosolic phospholipase A(2) (cPLA(2)) in monocyte chemotaxis to MCP-1. In these studies we investigated the role for another PLA(2), calcium-independent PLA(2) (iPLA(2)) in comparison to cPLA(2). Pharmacological inhibitors of PLA(2) were found to substantially inhibit chemotaxis. Using antisense oligodeoxyribonucleotide treatment we found that iPLA(2) expression is required for monocyte migration to MCP-1. Complete blocking of the chemotactic response was observed with inhibition of either iPLA(2) or cPLA(2) expression by their respective antisense oligodeoxyribonucleotide. In reconstitution experiments, lysophosphatidic acid completely restored MCP-1-stimulated migration in iPLA(2)-deficient monocytes, whereas lysophosphatidic acid was without effect in restoring migration in cPLA(2)-deficient monocytes. To the contrary, arachidonic acid fully restored migration of cPLA(2)-deficient monocytes while having no effect on the iPLA(2)-deficient monocytes. Additional studies revealed that neither enzyme appears to be upstream of the other indicating that iPLA(2) and cPLA(2) represent parallel regulatory pathways. These data demonstrate novel and distinct roles for these two phospholipases in this critical step in inflammation.

Topics: Aminobenzoates; Arachidonic Acid; Arachidonic Acids; Aristolochic Acids; Chemokine CCL2; Chemotaxis, Leukocyte; Chlorobenzoates; Cinnamates; Enzyme Inhibitors; Fatty Acids; Group IV Phospholipases A2; Group VI Phospholipases A2; Humans; Inflammation; Lysophospholipids; Monocytes; Naphthalenes; Oligodeoxyribonucleotides, Antisense; ortho-Aminobenzoates; Phenanthrenes; Phospholipases A; Pyrones; Signal Transduction

The oscillatory [Ca(2+)](i) signals typically seen following physiologically relevant stimulation of phospholipase C-linked receptors are associated with a receptor-activated entry of Ca(2+), which plays a critical role in driving the oscillations and influencing their frequency. We have recently shown that this receptor-activated entry of Ca(2+) does not conform to the widely accepted "capacitative" model and, instead, reflects the activity of a distinct, novel Ca(2+) entry pathway regulated by arachidonic acid (Shuttleworth, T. J., and Thompson, J. L. (1998) J. Biol. Chem. 273, 32636-32643). We now show that the generation of arachidonic acid under these conditions results from the activity of a type IV cytosolic phospholipase A(2) (cPLA(2)). Although cPLA(2) activation commonly involves a Ca(2+)-dependent translocation to the membrane, at these low agonist concentrations cPLA(2) activation was independent of increases in [Ca(2+)](i), and no detectable translocation to the membrane occurs. Nevertheless, stimulation of cPLA(2) activity was confined to the membrane fraction, where an increase in phosphorylation of the enzyme was observed. We suggest that, at the low agonist concentrations associated with oscillatory [Ca(2+)](i) signals, cPLA(2) activation involves an increased phosphorylation of a discrete pool of the total cellular cPLA(2) that is already localized within the membrane fraction at resting [Ca(2+)](i).

Topics: Arachidonic Acid; Arachidonic Acids; Biological Transport; Butanols; Calcium Signaling; Carbachol; Cell Compartmentation; Cell Line; Cytosol; Electric Conductivity; Enzyme Activation; Ethanol; Group IV Phospholipases A2; Humans; Muscarinic Agonists; Naphthalenes; Phosphodiesterase Inhibitors; Phospholipases A; Pyrones; Receptor, Muscarinic M3; Receptors, Muscarinic

Activation of lymphocytes induces blastogenesis and cell division which is accompanied by membrane lipid metabolism such as increased fatty acid turnover. To date little is known about the enzymatic mechanism(s) regulating this process. Release of fatty acids such as arachidonic acid requires sn-2-deacylation catalyzed by a class of enzymes known as phospholipases A(2) (PLA(2), EC ). Herein, we confirm that human peripheral blood B or T lymphocytes (PBL) do not possess measurable levels of 85-kDa PLA(2) as assessed by Western immunoblot. Low levels of 14-kDa PLA(2) protein and activity were detectable in the particulate fraction of PBL and Jurkat cells. Western immunoblot analysis indicates that PBLs possess the calcium-independent PLA(2) (iPLA(2)) protein. Calcium-independent sn-2-acylhydrolytic activity was measurable in PBL cytosols and could be inhibited by the selective iPLA(2) inhibitor bromoenol lactone. Mitogen activation of PBLs resulted in maintenance of activity levels which remained constant over 72 h suggesting an important role for iPLA(2) in this proliferative process. Indeed, evaluation of iPLA(2) activity in cell cycle-arrested Jurkat T cell fractions revealed the highest iPLA(2) levels occurring at the G(2)/M phase. Addition of the iPLA(2) inhibitors, bromoenol lactone, or arachidonyl trifluoromethyl ketone (AAOCF(3)), inhibited both mitogen-induced PBL as well as Jurkat T cell proliferation. Moreover, specific depletion of iPLA(2) protein by antisense treatment also resulted in marked suppression of cell division. Inhibition of Jurkat cell proliferation was not associated with arrest at a particular phase of the cell cycle nor was it associated with apoptosis as assessed by flow cytometry. These findings provide the first evidence that iPLA(2) plays a key role in the lymphocyte proliferative response.

Topics: Apoptosis; Arachidonic Acids; B-Lymphocytes; Cell Division; Cells, Cultured; Group VI Phospholipases A2; Humans; Immunoblotting; Indomethacin; Isoenzymes; Jurkat Cells; Lymphocytes; Monocytes; Naphthalenes; Oligonucleotides, Antisense; Phospholipases A; Phospholipases A2; Phytohemagglutinins; Pyrones; Sulfonamides; T-Lymphocytes

Recent work indicates that respiratory muscles generate superoxide radicals during contraction (M. B. Reid, K. E. Haack, K. M. Francik, P. A. Volberg, L. Kabzik, and M. S. West. J. Appl. Physiol. 73: 1797-1804, 1992). The intracellular pathways involved in this process are, however, unknown. The purpose of the present study was to test the hypothesis that contraction-related formation of reactive oxygen species (ROS) by skeletal muscle is linked to activation of the 14-kDa isoform of phospholipase A(2) (PLA(2)). Studies were performed by using an in vitro hemidiaphragm preparation submerged in an organ bath, and formation of ROS in muscles was assessed by using a recently described fluorescent indicator technique. We examined ROS formation in resting and contracting muscle preparations and then determined whether contraction-related ROS generation could be altered by administration of various PLA(2) inhibitors: manoalide and aristolochic acid, both inhibitors of 14-kDa PLA(2); arachidonyltrifluoromethyl ketone (AACOCF(3)), an inhibitor of 85-kDa PLA(2); and haloenol lactone suicide substrate (HELSS), an inhibitor of calcium-independent PLA(2). We found 1) little ROS formation [2.0 +/- 0.8 (SE) ng/mg] in noncontracting control diaphragms, 2) a high level of ROS (20.0 +/- 2.0 ng/mg) in electrically stimulated contracting diaphragms (trains of 20-Hz stimuli for 10 min, train rate 0.25 s(-1)), 3) near-complete suppression of ROS generation in manoalide (3.0 +/- 0.5 ng/mg, P < 0. 001)- and aristolochic acid-treated contracting diaphragms (4.0 +/- 1.0 ng/mg, P < 0.001), and 4) no effect of AACOCF(3) or HELSS on ROS formation in contracting diaphragm. During in vitro studies examining fluorescent measurement of ROS formation in response to a hypoxanthine/xanthine oxidase superoxide-generating solution, manoalide, aristolochic acid, AACOCF(3), and HELSS had no effect on signal intensity. These data indicate that ROS formation by contracting diaphragm muscle can be suppressed by the administration of inhibitors of the 14-kDa isoform of PLA(2) and suggest that this enzyme plays a critical role in modulating ROS formation during muscle contraction.

Topics: Animals; Arachidonic Acids; Aristolochic Acids; Diaphragm; Electric Stimulation; Enzyme Activation; Enzyme Inhibitors; Ethidium; Fluorescence; Free Radicals; Kinetics; Male; Muscle Contraction; Naphthalenes; Phenanthrenes; Phospholipases A; Pyrones; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Superoxides; Terpenes

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Arteries; Cyclohexanones; Enzyme Activation; Enzyme Inhibitors; Estrenes; In Vitro Techniques; Isoenzymes; Lipoprotein Lipase; Muscle, Smooth, Vascular; Naphthalenes; Norepinephrine; Phospholipases A; Pyrones; Pyrrolidinones; Rats; Terpenes; Type C Phospholipases

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Carcinogens; Cell Transformation, Neoplastic; Cells, Cultured; Cytosol; Eicosanoids; Enzyme Activation; Enzyme Inhibitors; Epidermal Cells; Fatty Acids; Isoenzymes; Keratinocytes; Membrane Lipids; Mice; Naphthalenes; Oligonucleotides, Antisense; Papilloma; Phosphatidylcholines; Phospholipases A; Phospholipids; Pyrones; Recombinant Fusion Proteins; Skin Neoplasms; Tetradecanoylphorbol Acetate; Transfection; Tumor Cells, Cultured

The predominant phospholipase activity present in rat hippocampus is a calcium-independent phospholipase A2 (302.9 +/- 19.8 pmol/mg.min for calcium-independent phospholipase A2 activity vs. 14.6 +/- 1.0 pmol/mg.min for calcium-dependent phospholipase A2 activity). This calcium-independent phospholipase A2 is exquisitely sensitive to inhibition by the mechanism-based inhibitor, (E)-6-(bromomethylene)-tetrahydro-3-(1-naphthalenyl)-2H-pyran -2-one (BEL). Moreover, treatment of hippocampal slices with BEL prior to tetanic stimulation prevents the induction of LTP (40.8 +/- 5.6% increase in excitatory post-synaptic potential (EPSP) slope for control slices (n = 6) vs. 5.8 +/- 8.5% increase in EPSP slope for BEL-treated slices (n = 8)). Importantly, LTP can be induced following mechanism-based inhibition of phospholipase A2 by providing the end product of the phospholipase A2 reaction, arachidonic acid, during the application of tetanic stimulation. Furthermore, the induction of LTP after treatment with BEL is dependent on the stereoelectronic configuration of the fatty acid provided since eicosa-5,8,11-trienoic acid, but not eicosa-8,11,14-trienoic acid, rescues LTP after BEL treatment (37.6 +/- 16.1% increase in EPSP slope for eicosa-5,8,11-trienoic acid vs. -3.7 +/- 5.2% increase in EPSP slope for eicosa-8,11,14-trienoic acid). Collectively, these results provide the first demonstration of the essential role of calcium-independent phospholipase A2 in synaptic plasticity.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Calcium; Enzyme Activation; Hippocampus; Long-Term Potentiation; Male; Naphthalenes; Phosphodiesterase Inhibitors; Phospholipases A; Phospholipases A2; Pyrones; Rats