The importance of the production of phosphatidic acid for the release of arachidonic acid in stimulated platelets

Phosphatidic acid is formed by platelets as a result of the combined activities of phospholipase C and diacylglycerol kinase. The initial thrombin-activated phospholipase C-mediated reaction is quinacrine insensitive and is followed by quinacrine-sensitive phospholipase A₂ activities. The phosphatidic acid released probably results in cellular calcium gating, which among other actions, might release arachidonic acid by activating phospholipases A₂: Indeed, we have detected a phosphatidic acid-specific phospholipase A₂ in platelets, which might have an important role in the liberation of arachidonic acid.     

A rapid phospholipase A2 bioassay using14C-oleate-labelledE. coli bacterias

Summary Two methods of phospholipase A₂ determination using¹⁴C-labelledE. coli bacterias as substrate were compared. One method works with a filter membrane for separation of cleaved¹⁴C-oleate from remaining phospholipids, the other uses the well-known thin-layer chromatography for lipid analysis. Some features of human serum phospholipase A₂ regarding pH and Ca²⁺ dependency were investigated. Possible sources of errors were discussed. It was shown that either method can differentiate between normal and pathologically elevated phospholipase A₂ levels, but that the filter method is superior in terms of sensitivity and workload.Abbreviations PLA₂ phospholipase A₂ - BSA bovine serum albumin - (F)FA (free) fatty acid - TLC thin-layer chromatography     

The coumarin derivative AD6 inhibits the release of arachidonic acid by interfering with phospholipase A2 activity in human platelets stimulated with thrombin

AD₆ is a coumarin derivative which is able to inhibit platelet aggregation and release due to various agonists as adrenaline, PAF, Ca⁺⁺ ionophore and others. It has been demonstrated that this compound reduces the production of free arachidonate and diglyceride from human platelets pulse-labeled with radioactive arachidonic acid thus suggesting a possible interference with the activity of phospholipase A₂ and/or phospholipase C. The present report indicates that the drug has no effect on the increase of the labeling of phosphatidic acid which takes place when platelets pulse-labeled with arachidonic acid are stimulated with thrombin. Furthermore, AD₆ is not able to cause changes on the metabolism of phosphoinositides monitored using platelets pre-labeled with [³H] inositol. These observations exclude the possibility that AD₆ interferes with phospholipase C activity. Experiments with platelets pulse-labeled with arachidonate suggest that AD₆ inhibits phospholipase(s) A₂ activity or modulate negatively one or more processes involved in its activation.     

Eicosanoid production by mouse peritoneal macrophages during Toxoplasma gondii penetration: role of parasite and host cell phospholipases.

The metabolism of endogenous arachidonic acid by mouse resident peritoneal macrophages infected in vitro with Toxoplasma gondii was studied. Prelabeling of macrophages with [5,6,8,9,11,12,14,15-3H]arachidonic acid and challenge with tachyzoites for 15 min resulted in a high mobilization of free labeled arachidonic acid (178%) in the culture medium. The parasites also triggered the synthesis of 6-keto-prostaglandin F1 alpha (47%), prostaglandin E2 (44%), leukotrienes C4 and D4 (33%) and 5-, 12-hydroxyeicosatetraenoic acids (155%). The study indicated that during the intracellular development phase of the parasites, 6-keto-prostaglandin F1 alpha (38%), prostaglandin E2 (31%) leukotrienes C4 and D4 (15%), hydroxyeicosatetraenoic acids (43%), and free arachidonic acid (110%) were secreted into the culture medium. Pretreatment of tachyzoites with phospholipase A2 inhibitors (4-p-bromophenacyl bromide and quinacrine) and no calcium in the culture medium resulted in inhibition of tachyzoite penetration into the macrophages and a decrease of the arachidonic acid metabolism. The triggering of the arachidonic acid cascade by T. gondii was dependent on the active penetration of the parasites into the macrophages, whereas preincubation of the macrophages with phospholipase A2 inhibitors did not affect penetration or free arachidonic acid release, thereby supporting a role for parasite phospholipase in the penetration process and in arachidonic acid mobilization from macrophage membrane phospholipids. Moreover, treatment of macrophages with phospholipase A2 inhibitors decreased the activities of the cyclooxygenase and lipoxygenase pathways, also suggesting an activation of host cell phospholipase A2 by the parasite.     

Inhibition of platelet phospholipase-A2 as a mechanism for the anti-aggregating effect of linoleic acid

Linoleic acid was incorporated into platelet phospholipids and then released after activation of phospholipase A₂ (PL-A₂) with thrombin or ionophore A23187. The rate of this release was tenfold lower for linoleic than for arachidonic acid. This observation strongly suggests that incorporation of linoleic acid in platelet phospholipids might inhibit platelet PL-A₂ and might explain the anti-aggregating effect of linoleic acid. In fact it has also been shown that linoleic acid inhibits PL-A₂ activity at concentrations which antagonize platelet aggregation. Moreover, in experimental conditions where platelet cyclo-oxygenase is totally inhibited, aggregation does occur and can still be blocked by linoleic acid. This latter observation led to the conclusion that the anti-aggregating effect of linoleic acid is independent from prostaglandin pathway and is probably related to the phospholipid metabolism.     

Characterization of the reaction sequence involved in phospholipid labeling and deacylation and prostaglandin synthesis and actions

The synthesis and release of prostaglandins (PG) primarily involve a series of enzymatic transformations of the fatty acid arachidonic acid. The source of this free fatty acid is thought to be the cleavage by phospholipase A₂ of the acyl bond on the 2-position of phospholipids. The liberated arachidonic acid is primarily converted by cyclo-oxygenase (PG-synthetase) into the PG-endoperoxides (PGG₂ and PGH₂). These compounds represent a critical branch point in PG metabolism since they serve as substrates for the enzymatic synthesis of PGE₂, PGD₂, thromboxane A₂, PGI₂ (prostacyclin), as well as some likely products yet to be identified. The ultimate fate of arachidonate in a given tissue is determined by the repertoire of enzymes present. The prostaglandins have a very broad sprectrum of identified biological activities and the newly discovered arachidonate metabolites (endoperoxides, thromboxane A₂ and PGI₂) are labile and extremely potent.     

Pasteurella haemolytica A1-Derived Leukotoxin and Endotoxin Induce Intracellular Calcium Elevation in Bovine Alveolar Macrophages by Different Signaling Pathways

Leukotoxin and endotoxin derived from Pasteurella haemolytica serotype 1 are the primary virulence factors contributing to the pathogenesis of lung injury in bovine pneumonic pasteurellosis. Activation of bovine alveolar macrophages with endotoxin or leukotoxin results in the induction of cytokine gene expression, with different kinetics (H. S. Yoo, S. K. Maheswaran, G. Lin, E. L. Townsend, and T. R. Ames, Infect. Immun. 63:381–388, 1995; H. S. Yoo, B. S. Rajagopal, S. K. Maheswaran, and T. R. Ames, Microb. Pathog. 18:237–252, 1995). Furthermore, extracellular Ca²⁺ is required for leukotoxin-induced cytokine gene expression. However, the involvement of Ca²⁺ in endotoxin effects and the precise signaling mechanisms in the regulation of intracellular Ca²⁺ by leukotoxin and endotoxin are not known. In fura-2-acetoxymethyl ester-loaded alveolar macrophages, intracellular Ca²⁺ regulation by leukotoxin and endotoxin was studied by video fluorescence microscopy. Leukotoxin induced a sustained elevation of intracellular Ca²⁺ in a concentration-dependent fashion by influx of extracellular Ca²⁺ through voltage-gated channels. In the presence of fetal bovine serum, endotoxin elevated intracellular Ca²⁺ even in the absence of extracellular Ca²⁺. Leukotoxin-induced intracellular Ca²⁺ elevation was inhibited by pertussis toxin, inhibitors of phospholipases A₂ and C, and the arachidonic acid analog 5,8,11,14-eicosatetraynoic acid. Intracellular Ca²⁺ elevation by endotoxin was inhibited by inhibitors of phospholipase C and protein tyrosine kinase, but not by pertussis toxin, or the arachidonic acid analog. To the best of our knowledge, this is the first report of Ca²⁺ signaling by leukotoxin through a G-protein-coupled mechanism involving activation of phospholipases A₂ and C and release of arachidonic acid in bovine alveolar macrophages. Ca²⁺ signaling by endotoxin, on the other hand, involves activation of phospholipase C and requires tyrosine phosphorylation. The differences in the Ca²⁺ signaling mechanisms may underlie the reported temporal differences in gene expression during leukotoxin and endotoxin activation.     

Regulatory functions of protein kinase c in glomerular mesangial cells

Summary Protein kinase C is a family of isozymes that are activated by hormone-stimulated phosphoinositide hydrolysis and participate in the signalling process by phosphorylating certain target proteins. In glomerular mesangial cells protein kinase C fulfills two major functions: it contributes to hormone-induced prostaglandin formation, and it acts as a negative feedback regulator of the inositol lipid signalling cascade. Furthermore, protein kinase C activates a phosphatidylcholine-degrading phospholipase D activity with as-yet-unknown cellular function.Abbreviations PKC protein kinase C - PLA₂ phospholipase A₂ - PLC phospholipase C - PLD phospholipase D - IP₃ inositol 1,4,5-trisphosphate - DAG 1,2-diacylglycerol     

Further study on the roles of the effector molecules of immunosuppressive macrophages induced by mycobacterial infection in expression of their suppressor function against mitogen-stimulated T cell proliferation

Previously, we found that phospholipids and reactive nitrogen intermediates (RNI) collaborated in expression of the T cell mitogenesis-inhibitory activity of immunosuppressive macrophages induced by Mycobacterium avium-intracellularecomplex (MAIC) infection. In this study, we examined the roles of free fatty acids (FFA) and prostaglandins (PG) as effectors of MAIC-induced macrophages, and moreover, their collaborating effects with RNI. First, treatment of MAIC-induced macrophages with quinacrine (phospholipase A₂ (PLA₂) inhibitor), dexamethasone (inhibitor of PLA₂ and PG synthesis) or indomethacin (PG synthesis inhibitor) attenuated their suppressor activity against concanavalin A (Con A)-induced mitogenesis of splenocytes (SPC), indicating important roles of FFA liberated from membrane phospholipids and PG, as effectors. Second, oleic acid, PGE₂, RNI generated from NOR 4 (a new nitric oxide (NO) donor), and phosphatidylserine (PS) exhibited suppressor activity against SPC mitogenesis without showing significant cytotoxicity, in an irreversible manner. Third, the suppressor activities of RNI and PGE₂ were potentiated by combined use with oleic acid in a synergistic manner. Fourth, a dual-chamber experiment in which target SPC were separated from MAIC-induced macrophages by a Millipore filter revealed a requirement for cell-to-cell contact for expression of the suppressor function of MAIC-induced macrophages. These findings indicate that RNI, FFA, PG, and phospholipids (presumably PS) and their collaboration play central roles in expression of the T cell mitogenesis-inhibitory function of MAIC-induced suppressor macrophages.     

Positive inotropic effect of interleukin-2. Role of phospholipases and protein kinase C

Recently, we have shown that soluble factors released by human lymphocytes after lectin stimulation could increase the contractile tension of rat atria “in vitro” and that interleukin-2 (IL-2) could be part of this reaction. The effect of IL-2 was potentiated by the Ca²⁺ ionophore A23187 or free arachidonic acid (AA). In this study we demonstrate that the action of IL-2 can be prevented by pre-incubation of the heart tissue with monoclonal anti-IL-2 receptor (anti-p55), suggesting that binding to the IL-2 receptor is necessary for the induction of the biologic effect. In the presence of A23187 or AA, the effect of the synthetic diacylglyceride oleoyl-acetyl-glycerol (OAG) was similar to that of IL-2. Elimination of phospholipase C activity by pre-incubation of the atria with 2-nitro-carboxyphenyl,N,N-diphenylcarbamate (NCDC) abrogated the effects of IL-2 in the presence of A23187 or AA, but was ineffective when OAG + A23187 or OAG + AA was used. Inhibition of atrial phospholipase A₂ activity with p-bromo-phenacylbromide (BPB) blocked the response of atria to either IL-2 + A23187 or OAG + A23187 but was not effective when AA was used as second signal (IL-2 + AA or OAG + AA). Both the OAG and the IL-2 positive inotropic effects could be prevented by the protein kinase C inhibitor 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine (H7) but were poorly inhibited by N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA1004), an inhibitor of the cyclic nucleotide-dependent protein kinases. The atrial cyclo-oxygenase and lipoxygenase pathways of AA metabolism were required for the development of the IL-2 or OAG effects, indicating a role for the oxidative metabolites of AA in the production of the positive inotropic effect. These observations suggest that in this experimental system, IL-2 may trigger phospholipid turnover, generating metabolites of the phospholipase A₂ and phospholipase C pathways that can in turn favor the activation of protein kinase C and the positive inotropic response of the heart.     

Andrology: Modulation of prostaglandin synthesis in mammalian sperm acrosome reaction

Exogenous arachidonic acid induces the acrosome reaction andthe production of the prostaglandins PGE₂ and PGF₂ in bovinespermatozoa. Exogenous PGE₂ also induces the acrosome reactionand PGF₂ synthesis. To understand better the role of PGE₂ inthe induction of PGF₂ synthesis through modulation of phospholipaseA₂, inhibitors of this enzyme were used. The effects of PGE₂were blocked by phospholipase A₂ inhibitors and this inhibitionwas reversed by addition of arachidonic acid. These data indicatethat PGE₂ activates phospholipase A₂ to produce arachidonicacid. To determine whether protein kinase C modulates phospholipaseA₂ activity in this process, staurosporin, an inhibitor of proteinkinase C, was used. The effect of PGE₂ on PGF₂ production isinhibited by staurosporin and this inhibition was reversed byaddition of arachidonic acid indicating that protein kinaseC is involved in phospholipase A₂ activation. The effect ofexogenous arachidonic acid or PGE₂ on the acrosome reactionis blocked by lipoxygenase inhibitors but not by inhibitorsof cyclo-oxygenase, indicating that lipoxygenase products areinvolved in the mechanism of the acrosome reaction. The presenteddata shed light on the cross-talk between cyclo-oxygenase andlipoxygenase and their involvement in the sperm acrosome reaction.It is suggested that cyclo-oxygenase products modulate the activityof lipoxygenase which is a key enzyme in the mechanism leadingto the acrosome reaction. Stimulation of cyclo-oxygenase tosynthesize PGE₂ activates phospholipase A₂ to release arachidonicacid which is the substrate for lipoxygenase activity. Thus,the role of cyclo-oxygenase products is probably to reactivatethe phospholipase A₂ which was initially activated via the signaltransduction cascade.     

Phospholipase C activates protein kinase C during induction of slow Na current in Xenopus oocytes

Protein phosphorylation by protein kinase C (PKC) has recently been shown to be a key event in the induction of the slow inward Na current observed during sustained depolarization of the Xenopus oocyte membrane. The present work investigates the possible pathways leading to PKC activation. PKC is activated by a series of phospholipid metabolites, such as diacylglycerol (DAG) and arachidonic acid produced by phospholipases C (PLC) and A₂(PLA₂) respectively. To test whether PKC activation was dependent upon the phospholipid metabolites produced either by PLC or by PLA₂, enzyme activity was reduced using selective inhibitors. Results indicated that inhibition of PLA₂ activity and inhibition of the enzymes involved in the arachidonic acid cascade failed to affect Na current amplitude. On the other hand, PLC inhibition caused a marked decrease of Na current amplitude. In another series of experiments, Na current was fully restored, in spite of PLC inhibition, by directly enhancing PKC activity with a powerful activator phorbol 12-myristate 13-acetate. These data strongly suggest that PLC is involved in PKC activation during Na channel induction.     

Role of Phospholipase A2 in Activation of Isolated Cardiomyocyte Respiration in Postinfarction Cardiosclerosis

The rate of oxygen consumption by isolated cardiomyocytes was studied in rats with experimental postinfarction cardiosclerosis. The increase in oxygen consumption under these condition was comparable to that in melittin- and arachidonic acid-induced activation of phospholipase A₂ in cardiomyocytes of intact animals. Bromophenacyl bromide inhibition of phospholipase A₂ in cardiomyocytes of rats with postinfarction cardiosclerosis led to reduction of oxygen consumption rate to values characteristic of intact animal cardiomyocytes. The results confirm the hypothesis according to which high oxygen consumption in postinfarction cardiosclerosis is related to increased activity of phospholipase A₂. Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 146, No. 12, pp. 631–633, December, 2008     

Methylmercury-induced IL-6 release requires phospholipase C activities

Methylmercury (MeHg) is a neurotoxin capable of causing severe damage to the CNS, especially in the developing fetus. Glia in the CNS release a number of cytokines that are important for proper CNS development and function. We reported earlier that MeHg could induce interleukin-6 (IL-6) release in primary mouse glia. This finding is significant considering previous reports indicating that sustained IL-6 exposure could be detrimental to cerebellar granule neurons, one of the major cellular targets of MeHg cytotoxicity. By using pharmacological antagonists against phophatidycholine- and phosphoinositol-specific phospholipase C, the current study indicated that phospholipase C activity was necessary for MeHg-induced IL-6 release. Results from pharmacological antagonists further suggested that the calcium signaling initiated by phospholipase C appeared essential for this event. In contrast, protein kinase C activity did not appear to be important. Even though mitogen-activated protein kinases were important for IL-6 release in some experimental systems, these enzymes did not appear to be required for MeHg-induced IL-6 release in glia. Based on these data and those reported by us and others, there is a possibility that MeHg-induced phospholipase C activation initiates a calcium signaling that causes phospholipase A₂ activation. This, in turn, leads to arachidonic acid and lysophosphatidyl choline generation, both of which are potent inducers for IL-6 release.     

Physiological and pharmacological regulation of prostaglandin and leukotriene production by macrophages

The synthesis and secretion of prostaglandins and leukotrienes by mouse peritoneal macrophages is under several regulatory controls. Arachidonic acid must first be released from phospholipid stores by the action of phospholipases. Macrophages have the capacity to deacylate arachidonic acid directly from the SN2 position of phospholipids via the action of a phospholipase A2. In addition, these cells contain a phospholipase C capable of removing inositol-phosphate from phosphatidylinositol generating diacylglycerol. Another enzyme, diacylglycerol lipase is present to then generate arachidonic acid. The free arachidonic acid then enters the cyclooxygenase pathway to generate prostaglandins, the lipoxygenase pathway to generate leukotrienes or both pathways. The nature of the inflammatory stimulus added to these cells determines which of the above pathways become operative. Zymosan and the Ca++ ionophore, A23187 stimulate the synthesis of both prostaglandins and leukotrienes whereas phorbol myristate acetate and lipopolysaccharide induce only the synthesis of prostaglandins. In addition, the synthesis of these two products by macrophages can be regulated by certain antiinflammatory compounds. Indomethacin, aspirin, ibuprofen and benoxaprofen are only inhibitors of the prostaglandin pathway, whereas BW755C, 5,8,11-ETYA, NDGA and sulindac sulfide (high doses) are inhibitors of the synthesis of both prostaglandins and leukotrienes. Dapsone, an effective drug for leprosy, also inhibits the synthesis of both of these products.     

Phospholipase A2 activation by interleukin 1: Release and metabolism of arachidonic acid by IL 1-stimulated rabbit chondrocytes

Treatment of rabbit chondrocytes with natural or recombinant human IL 1 results in a dramatic dose-dependent increase in intracellular phospholipase A₂ (PLA₂) activity and subsequent secretion of this enzyme into the intracellular millieu. PLA₂ activity is detectable as early as 1 hr after IL 1 stimulation and is maximal by 24 hr. In the present study, we have characterized more fully the relationship between PLA₂ activation and arachidonate metabolism in these cells. IL 1 treatment of chondrocytes which had been prelabeled with [¹⁴C] arachidonic acid resulted in an enhanced release of free arachidonic acid identified by thin-layer chromatography. Moreover, the arachidonic acid liberated was subsequently metabolized exclusively to PGE₂; no significant increases in the production of 6-keto PGF₁, LTB₄, LTC₄, 12-HETE or 15-HETE were observed following IL 1 stimulation.     

Mitogen-Activated Protein Kinase Is Activated During IgG-Mediated Phagocytosis, But Is Not Required for Target Ingestion

Arachidonic acid (AA) release is required for IgG-mediated phagocytosis in human monocytes. AA release is mediated by a calcium-independent phospholipase A₂ (PPL) that is in turn regulated by protein kinase C (PKC). As mitogen-activated protein kinase (MAPK) activates cytosolic phospholipase A₂, we examined the activation and involvement of MAPK in IgG-mediated phagocytosis. MAPK activity was assessed in immunoprecipitates; tyrosine phosphorylation was detected by immunoblotting. Ingestion of IgG-opsonized glass beads, or treatment with phorbol myristate acetate, increased enzymatic activity and tyrosine phosphorylation of p42 MAPK. This MAPK activation was attenuated by PKC inhibitors staurosporine or calphostin C. Treatment with PD98059, a p42/p44 MAPK kinase (MEK) inhibitor, decreased BIgG-stimulated p42 MAPK activity by >90% with no significant effect on phagocytosis or pPL activity. These results suggest that p42 MAPK is activated in a PKC-dependent manner during IgG-dependent phagocytosis but is not required for target ingestion.     

Role of phospholipase A2 and pertussis toxin-sensitive G proteins in histamine secretion induced by substance P

Incubation of mast cells with substance P or ionophore A23187 resulted in histamine release and arachidonic acid liberation from purified rat peritoneal mast cells. The treatment of mast cells with 100 ng/ml of pertussis toxin for 2h inhibited the effect of substance P on both histamine release and arachidonic acid liberation, but the response to the ionophore A23187 was not affected. Para-bromophenacyl bromide, a selective inhibitor of phospholipase A₂, inhibited similarly histamine and arachidonate release induced by substance P but not that evoked by ionophore A23187. These results suggest that phospholipase A₂ plays a key role in the histamine secretion induced by substance P.

     

Production of prostaglandin E₂ in response to infection with modified vaccinia Ankara virus

Prostaglandin E₂ (PGE₂) is an arachidonic acid (AA)-derived signaling molecule that can influence host immune responses to infection or vaccination. In this study, we investigated PGE₂ production in vitro by cells infected with the poxvirus vaccine strain, modified vaccinia Ankara virus (MVA). Human THP-1 cells, murine bone marrow-derived dendritic cells, and murine C3HA fibroblasts all accumulated PGE₂ to high levels in culture supernatants upon infection with MVA. We also demonstrated that MVA induced the release of AA from infected cells, and this was, most unusually, independent of host cytosolic phospholipase A₂ activity. The accumulation of AA and PGE₂ was dependent on viral gene expression, but independent of canonical NF-κB signaling via p65/RelA. The production of PGE₂ required host cyclooxygenase-2 (COX-2) activity, and COX-2 protein accumulated during MVA infection. The results of this study provide insight into a novel aspect of MVA biology that may affect the efficacy of MVA-based vaccines.