Arachidonic Acid Release In Response Research Articles

Phospholipid Arachidonic Acid Remodeling During Phagocytosis in Mouse Peritoneal Macrophages.

Macrophages contain large amounts of arachidonic acid (AA), which distributes differentially across membrane phospholipids. This is largely due to the action of coenzyme A-independent transacylase (CoA-IT), which transfers the AA primarily from diacyl choline-containing phospholipids to ethanolamine-containing phospholipids. In this work we have comparatively analyzed glycerophospholipid changes leading to AA mobilization in mouse peritoneal macrophages responding to either zymosan or serum-opsonized zymosan (OpZ). These two phagocytic stimuli promote the cytosolic phospholipase A2-dependent mobilization of AA by activating distinct surface receptors. Application of mass spectrometry-based lipid profiling to identify changes in AA-containing phospholipids during macrophage exposure to both stimuli revealed significant decreases in the levels of all major choline phospholipid molecular species and a major phosphatidylinositol species. Importantly, while no changes in ethanolamine phospholipid species were detected on stimulation with zymosan, significant decreases in these species were observed when OpZ was used. Analyses of CoA-IT-mediated AA remodeling revealed that the process occurred faster in the zymosan-stimulated cells compared with OpZ-stimulated cells. Pharmacological inhibition of CoA-IT strongly blunted AA release in response to zymosan but had only a moderate effect on the OpZ-mediated response. These results suggest a hitherto undescribed receptor-dependent role for CoA-independent AA remodeling reactions in modulating the eicosanoid biosynthetic response of macrophages. Our data help define novel targets within the AA remodeling pathway with potential use to control lipid mediator formation

Modulation of the activity of cytosolic phospholipase A2α (cPLA2α) by cellular sphingolipids and inhibition of cPLA2α by sphingomyelin

We examined the effect of the cellular sphingolipid level on the release of arachidonic acid (AA) and activity of cytosolic phospholipase A2alpha (cPLA2alpha) using two Chinese hamster ovary (CHO)-K1-derived mutants deficient in sphingolipid synthesis: LY-B cells defective in the LCB1 subunit of serine palmitoyltransferase for de novo synthesis of sphingolipid species, and LY-A cells defective in the ceramide transfer protein CERT for SM synthesis. When LY-B and LY-A cells were cultured in Nutridoma medium and the sphingolipid level was reduced, the release of AA stimulated by the Ca(2+) ionophore A23187 increased 2-fold and 1.7-fold, respectively, compared with that from control cells. The enhancement in LY-B cells was decreased by adding sphingosine and treatment with the cPLA2alpha inhibitor. When CHO cells were treated with an acid sphingomyelinase inhibitor to increase the cellular SM level, the release of AA induced by A23187 or PAF was decreased. In vitro studies were then conducted to test whether SM interacts directly with cPLA2alpha. Phosphatidylcholine vesicles containing SM reduced cPLA2alpha activity. Furthermore, SM disturbed the binding of cPLA2alpha to glycerophospholipids. These results suggest that SM at the biomembrane plays important roles in regulating the cPLA2alpha-dependent release of AA by inhibiting the binding of cPLA2alpha to glycerophospholipids.

Regulation of cytosolic phospholipase A2α by hsp90 and a p54 kinase in okadaic acid-stimulated macrophages

In resident mouse peritoneal macrophages, group IVA cytosolic phospholipase A(2) (cPLA(2)alpha) mediates arachidonic acid (AA) release and eicosanoid production in response to diverse agonists such as A23187, phorbol myristate acetate, zymosan, and the enterotoxin, okadaic acid (OA). cPLA(2)alpha is regulated by phosphorylation and by calcium that binds to the C2 domain and induces translocation from the cytosol to membranes. In contrast, OA activates cPLA(2)alpha-induced AA release and translocation to the Golgi in macrophages without an apparent increase in calcium. Inhibitors of heat shock protein 90 (hsp90), geldanamycin, and herbimycin blocked AA release in response to OA but not to A23187, PMA, or zymosan. OA, but not the other agonists, induced activation of a cytosolic serine/threonine 54-kDa kinase (p54), which phosphorylated cPLA(2)alpha in in-gel kinase assays and was associated with cPLA(2)alpha in immunoprecipitates. Activation of the p54 kinase was inhibited by geldanamycin. The kinase coimmunoprecipitated with hsp90 in unstimulated macrophages, and OA induced its loss from hsp90, concomitant with its association with cPLA(2)alpha. The results demonstrate a role for hsp90 in regulating cPLA(2)alpha-mediated AA release that involves association of a p54 kinase with cPLA(2)alpha upon OA stimulation.

Costimulation of Dectin-1 and DC-SIGN Triggers the Arachidonic Acid Cascade in Human Monocyte-Derived Dendritic Cells

Inflammatory mediators derived from arachidonic acid (AA) alter the function of dendritic cells (DC), but data regarding their biosynthesis resulting from stimulation of opsonic and nonopsonic receptors are scarce. To address this issue, the production of eicosanoids by human monocyte-derived DC stimulated via receptors involved in Ag recognition was assessed. Activation of FcgammaR induced AA release, short-term, low-grade PG biosynthesis, and IL-10 production, whereas zymosan, which contains ligands of both the mannose receptor and the human beta-glucan receptor dectin-1, induced a wider set of responses including cyclooxygenase 2 induction and biosynthesis of leukotriene C(4) and IL-12p70. The cytosolic phospholipase A(2) inhibitor pyrrolidine 1 completely inhibited AA release stimulated via all receptors, whereas the spleen tyrosine kinase (Syk) inhibitors piceatannol and R406 fully blocked AA release in response to immune complexes, but only partially blocked the effect of zymosan. Furthermore, anti-dectin-1 mAb partially inhibited the response to zymosan, and this inhibition was enhanced by mAb against DC-specific ICAM-3-grabbing nonintegrin (SIGN). Immunoprecipitation of DC lysates showed coimmunoprecipitation of DC-SIGN and dectin-1, which was confirmed using Myc-dectin-1 and DC-SIGN constructs in HEK293 cells. These data reveal a robust metabolism of AA in human DC stimulated through both opsonic and nonopsonic receptors. The FcgammaR route depends on the ITAM/Syk/cytosolic phospholipase A(2) axis, whereas the response to zymosan involves the interaction with the C-type lectin receptors dectin-1 and DC-SIGN. These findings help explain the distinct functional properties of DC matured by immune complexes vs those matured by beta-glucans.

CPLA2 phosphorylation at serine-515 and serine-505 is required for arachidonic acid release in vascular smooth muscle cells

Cytosolic phospholipase A(2) (cPLA(2)) is activated by phosphorylation at serine-505 (S505) by extracellular regulated kinase 1/2 (ERK1/2). However, rat brain calcium/calmodulin-dependent kinase II (CaMKII) phosphorylates recombinant cPLA(2) at serine-515 (S515) and increases its activity in vitro. We have studied the sites of cPLA(2) phosphorylation and their significance in arachidonic acid (AA) release in response to norepinephrine (NE) in vivo in rabbit vascular smooth muscle cells (VSMCs) using specific anti-phospho-S515- and -S505 cPLA(2) antibodies and by mutagenesis of S515 and S505 to alanine. NE increased the phosphorylation of cPLA(2) at S515, followed by phosphorylation of ERK1/2 and consequently phosphorylation of cPLA(2) at S505. The CaMKII inhibitor 2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzene-sulfonyl)]amino-N-(4-chlorocinnamyl)-methylbenzylamine attenuated cPLA(2) at S515 and S505, whereas the ERK1/2 inhibitor U0126 reduced phosphorylation at S505 but not at S515. NE in cells transduced with adenovirus carrying enhanced cyan fluorescent protein cPLA(2) wild type caused phosphorylation at S515 and S505 and increased AA release. Expression of the S515A mutant in VSMCs reduced the phosphorylation of S505, ERK1/2, and AA release in response to NE. Transduction with a double mutant (S515A/S505A) blocked the phosphorylation of cPLA(2) and AA release. These data suggest that the NE-stimulated phosphorylation of cPLA(2) at S515 is required for the phosphorylation of S505 by ERK1/2 and that both sites of phosphorylation are important for AA release in VSMCs.

Activation of Cytosolic Phospholipase A2α in Resident Peritoneal Macrophages by Listeria monocytogenes Involves Listeriolysin O and TLR2

Eicosanoid production by macrophages is an early response to microbial infection that promotes acute inflammation. The intracellular pathogen Listeria monocytogenes stimulates arachidonic acid release and eicosanoid production from resident mouse peritoneal macrophages through activation of group IVA cytosolic phospholipase A2 (cPLA2alpha). The ability of wild type L. monocytogenes (WTLM) to stimulate arachidonic acid release is partially dependent on the virulence factor listeriolysin O; however, WTLM and L. monocytogenes lacking listeriolysin O (DeltahlyLM) induce similar levels of cyclooxygenase 2. Arachidonic acid release requires activation of MAPKs by WTLM and DeltahlyLM. The attenuated release of arachidonic acid that is observed in TLR2-/- and MyD88-/- macrophages infected with WTLM and DeltahlyLM correlates with diminished MAPK activation. WTLM but not DeltahlyLM increases intracellular calcium, which is implicated in regulation of cPLA2alpha. Prostaglandin E2, prostaglandin I2, and leukotriene C4 are produced by cPLA2alpha+/+ but not cPLA2alpha-/- macrophages in response to WTLM and DeltahlyLM. Tumor necrosis factor (TNF)-alpha production is significantly lower in cPLA2alpha+/+ than in cPLA2alpha-/- macrophages infected with WTLM and DeltahlyLM. Treatment of infected cPLA2alpha+/+ macrophages with the cyclooxygenase inhibitor indomethacin increases TNFalpha production to the level produced by cPLA2alpha-/- macrophages implicating prostaglandins in TNFalpha down-regulation. Therefore activation of cPLA2alpha in macrophages may impact immune responses to L. monocytogenes.

The immunosuppressant drug FTY720 inhibits cytosolic phospholipase A2 independently of sphingosine-1-phosphate receptors

AbstractFTY720 is a potent immunomodulator drug that inhibits the egress of lymphocytes from secondary lymphoid tissues and thymus. FTY720 is phosphorylated in vivo by sphingosine kinase 2 to FTY720-phosphate, which acts as a potent sphingosine-1-phosphate (S1P) receptor agonist. However, in contrast to S1P, FTY720 has no effect on mast-cell degranulation, yet significantly reduces antigen-induced secretion of PGD2 and cysteinyl-leukotriene. Unexpectedly, this effect of FTY720 was independent of its phosphorylation and S1P receptor functions. The rate-limiting step in the biosynthesis of all eicosanoids is the phospholipase A2 (PLA2)–mediated release of arachidonic acid from glycerol phospholipids. Although FTY720 also reduced arachidonic acid release in response to antigen, it had no effect on translocation of cPLA2 or ERK1/2 activation, suggesting that it does not interfere with FcϵRI-mediated events leading to cPLA2 activation. Remarkably, however, FTY720 drastically inhibited recombinant cPLA2α activity, whereas FTY720-phosphate, sphingosine, or S1P had no effect. This study has uncovered a unique action of FTY720 as an inhibitor of cPLA2α and hence on production of all eicosanoids. Our results have important implications for the potential therapeutic mechanism of action of FTY720 in eicosanoid-driven inflammatory disorders such as asthma and multiple sclerosis.

Uric acid inhibits renal proximal tubule cell proliferation via at least two signaling pathways involving PKC, MAPK, cPLA2, and NF-κB

The accumulation of uric acid, an end-product of purine metabolism, is responsible for the many deleterious effects observed in gouty arthritis, including renal injury. Here, we present evidence that under conditions of hyperuricemia (>10(-4) M uric acid) [(3)H]thymidine incorporation into primary renal proximal tubule cells (PTCs) is inhibited, and we delineate the signaling pathways involved. Elevated uric acid was observed to stimulate MAPK phosphorylation. The uric acid induced p38 MAPK phosphorylation was also blocked by H-7 (a PKC inhibitor), indicating that p38 MAPK was a downstream target of PKC. Evidence that cytoplasmic phospholipase A(2) (cPLA(2)) was involved further downstream included 1) the stimulatory effect of uric acid on [(3)H]-labeled arachidonic acid (AA) release; 2) the stimulation of AA release in response to uric acid was blocked by the PKC inhibitor H-7 as well as by the p38 MAPK inhibitor SB 203580; and 3) the uric acid-induced inhibition of [(3)H]thymidine incorporation was prevented by SB 203580, as well as by the cPLA(2) inhibitor arachidonyl trifluoromethyl ketone, and mepacrine (another PLA(2) inhibitor). Evidence of a uric acid-induced activation of NF-kappaB as well as PLA(2) was obtained. Moreover the uric acid-induced inhibition of [(3)H]thymidine incorporation was also blocked by two NF-kappaB inhibitors, pyrrolidine dithiocarbamate and SN 50. However, SN 50 did not block the uric acid induced [(3)H]AA release. Thus the inhibition of [(3)H]thymidine incorporation caused by uric acid can be explained by two distinct mechanisms, the activation of NF-kappaB as well as the activation of PLA(2).

Expression of Cytosolic Phospholipase A2α in Murine C12 Cells, a Variant of L929 Cells, Induces Arachidonic Acid Release in Response to Phorbol Myristate Acetate and Ca2+ Ionophores, but Not to Tumor Necrosis Factor-α

Tumor necrosis factor-α (TNFα)-induced cell death is regulated through the release of arachidonic acid (AA) by group IVA cytosolic phospholipase A2 (cPLA2α) in the murine fibroblast cell line L929. However, the signaling pathway by which TNFα activates cPLA2α remained to be solved. We examined AA release in L929 cells, in a variant of L929 (C12 cells) lacking cPLA2α, and in C12 cells transfected with cPLA2α expression vectors. In transient and stable clones of C12 cells expressing cPLA2α, Ca2+ ionophore A23187 and phorbol myristate acetate (PMA) stimulated AA release within 90 min, although no response to TNFα was observed within 6 h. These results suggest that C12 cells may lack the components necessary for TNFα-induced AA release, in addition to cPLA2α. PMA is known to stimulate AA release via phosphorylation of Ser505 in cPLA2α by activating extracellular signal-regulated kinases (ERK1/2). However, PMA-induced AA release from C12 cells expressing mutant cPLA2αS505A (mutation of Ser505 to Ala), which is not phosphorylated by ERK1/2, was similar to that from L929 cells and C12 cells expressing wild-type cPLA2α. The role of Ser505 phosphorylation in AA release induced by PMA is also discussed.

Signal transduction pathways involved in proteolysis-inducing factor induced proteasome expression in murine myotubes.

The proteolysis-inducing factor (PIF) is produced by cachexia-inducing tumours and initiates protein catabolism in skeletal muscle. The potential signalling pathways linking the release of arachidonic acid (AA) from membrane phospholipids with increased expression of the ubiquitin–proteasome proteolytic pathway by PIF has been studied using C2C12 murine myotubes as a surrogate model of skeletal muscle. The induction of proteasome activity and protein degradation by PIF was blocked by quinacrine, a nonspecific phospholipase A2 (PLA2) inhibitor and trifluroacetyl AA, an inhibitor of cytosolic PLA2. PIF was shown to increase the expression of calcium-independent cytosolic PLA2, determined by Western blotting, at the same concentrations as those inducing maximal expression of 20S proteasome α-subunits and protein degradation. In addition, both U-73122, which inhibits agonist-induced phospholipase C (PLC) activation and D609, a specific inhibitor of phosphatidylcholine-specific PLC also inhibited PIF-induced proteasome activity. This suggests that both PLA2 and PLC are involved in the release of AA in response to PIF, and that this is important in the induction of proteasome expression. The two tyrosine kinase inhibitors genistein and tryphostin A23 also attenuated PIF-induced proteasome expression, implicating tyrosine kinase in this process. PIF induced phosphorylation of p44/42 mitogen-activated protein kinase (MAPK) at the same concentrations as that inducing proteasome expression, and the effect was blocked by PD98059, an inhibitor of MAPK kinase, as was also the induction of proteasome expression, suggesting a role for MAPK activation in PIF-induced proteasome expression.

Ceramide Kinase Mediates Cytokine- and Calcium Ionophore-induced Arachidonic Acid Release

Despite the importance of prostaglandins, little is known about the regulation of prostanoid synthesis proximal to the activation of cytosolic phospholipase A2, the initial rate-limiting step. In this study, ceramide-1-phosphate (C-1-P) was shown to be a specific and potent inducer of arachidonic acid (AA) and prostanoid synthesis in cells. This study also demonstrates that two well established activators of AA release and prostanoid synthesis, the cytokine, interleukin-1beta (IL-1beta), and the calcium ionophore, A23187, induce an increase in C-1-P levels within the relevant time-frame of AA release. Furthermore, the enzyme responsible for the production of C-1-P in mammalian cells, ceramide kinase, was activated in response to IL-1beta and A23187. RNA interference targeted to ceramide kinase specifically down-regulated ceramide kinase mRNA and activity with a concomitant decrease of AA release in response to IL-1beta and A23187. Down-regulation of ceramide kinase had no effect on AA release induced by exogenous C-1-P. Collectively, these results indicate that ceramide kinase, via the formation of C-1-P, is an upstream modulator of phospholipase A2 activation. This study identifies previously unknown roles for ceramide kinase and its product, C-1-P, in AA release and production of eicosanoids and provides clues for potential new targets to block inflammatory responses.

Oxidative stress induces arachidonate release from human lung cells through the epithelial growth factor receptor pathway.

Oxidative stress is thought to be a factor influencing many inflammatory responses, including arachidonic acid (AA) release. We have studied the effect of hydrogen peroxide on AA and prostaglandin E(2) release, cytosolic phospholipase (cPLA(2)) steady-state mRNA, cPLA(2) protein levels, cPLA(2) enzyme activity, and cPLA(2) phosphorylation in a human lung epithelial cell line: A549 cells. Hydrogen peroxide caused a dose-dependent increase of A23187-stimulated AA and prostaglandin E(2) release, with a maximum effect at 1 h. This effect is associated with a maximum specific cPLA(2) activity at 1 h, and with a significant increase in cPLA(2) Serine 505 phosphorylation. All these effects were abolished, in a dose-related manner, by the epithelial growth factor receptor kinase inhibitor, AG 1478. To further investigate the pathway leading to the increase cPLA(2) phosphorylation, we used cells transfected with a Ras dominant negative vector and mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) and p38 kinase inhibitors. Cells transfected with the Ras dominant negative vector exhibited diminished hydrogen peroxide-induced AA release and cPLA(2) phosphorylation as compared with cells transfected with the Ras expression vector. Both MEK and p38 kinase inhibitors inhibited the hydrogen peroxide effect on AA release and specific cPLA(2) activity. Finally, cells stably transfected with an antisense cPLA(2) vector exhibited diminished A23187-stimulated AA release in response to hydrogen peroxide as compared with cells stably transfected with empty expression vector. Collectively, these data show that hydrogen peroxide increases cPLA(2) activity through its phosphorylation utilizing an epithelial growth factor/Ras/extracellular signal-regulated kinase and p38 pathway.

Regulation of kinin receptors in airway epithelial cells by inflammatory cytokines and dexamethasone

The two kinin receptors, B 1 and B 2, are upregulated in inflammation and may play a role in diseases such as asthma. In pulmonary A549 cells, TNF-α or interleukin-1β dramatically increased bradykinin B 1 and B 2 receptor mRNA expression and this response was prevented by dexamethasone. In primary human bronchial epithelial cells, bradykinin B 1 receptor mRNA expression showed a similar trend, whereas bradykinin B 2 receptor showed almost constitutive expression. Radioligand-binding studies revealed significant increases in bradykinin B 2 receptor protein expression following both interleukin-1β and TNF-α treatment of A549 cells; however, no evidence was found for bradykinin B 1 receptor. Functionally, the bradykinin B 2 receptor ligand, bradykinin, but not the B 1 ligand, des-Arg 10-kallidin, produced a marked increase in prostaglandin E 2 release when administered following interleukin-1β treatment. Arachidonic acid release in response to bradykinin was markedly enhanced by prior incubation with interleukin-1β and this was prevented by the prior addition of dexamethasone.

Inhibition of the MEK1/ERK pathway reduces arachidonic acid release independently of cPLA2 phosphorylation and translocation

BackgroundThe 85-kDa cytosolic phospholipase A2 (cPLA2) mediates arachidonic acid (AA) release in MDCK cells. Although calcium and mitogen-activated protein kinases regulate cPLA2, the correlation of cPLA2 translocation and phosphorylation with MAPK activation and AA release is unclear.ResultsMEK1 inhibition by U0126 inhibited AA release in response to ATP and ionomycin. This directly correlated with inhibition of ERK activation but not with phosphorylation of cPLA2 on Ser505, which was only partially inhibited by ERK inhibition. Inhibition of AA release by U0126 was still observed when stoichiometric phosphorylation of cPLA2 on Ser505 was maintained by activating p38 with anisomycin. Translocation kinetics of wild-type cPLA2 and cPLA2 containing S505A or S727A mutations to Golgi were similar in response to ATP and ionomycin and were not affected by U0126.ConclusionsThese results suggest that the ability of cPLA2 to hydrolyze membrane phospholipid is reduced by inhibition of the MEK1/ERK pathway and that the reduction in activity is independent of cPLA2 phosphorylation and translocation to membrane. The results also demonstrate that cPLA2 mutated at the phosphorylation sites Ser505 and Ser727 translocated with similar kinetic as wild-type cPLA2.

Evidence for a Role for Phospholipase C, but not Phospholipase A2, in Platelet Activation in Response to Low Concentrations of Collagen

The release of arachidonic acid is a key component in platelet activation in response to low concentrations (1-20 microg/ml) of collagen. The precise mechanism remains elusive although a variety of pathways have been implicated. In the present study the effects of inhibitors of several potentially key enzymes in these pathways have been examined. Collagen 1-10 microg/ml) caused maximal platelet aggregation which was accompanied by the release of arachidonic acid, the synthesis of thromboxane A2, and p38MAPK phosphorylation. Preincubation with the dual cyclooxygenase/lipoxygenase inhibitor BW755C inhibited aggregation and thromboxane production, and reduced p38MAPK phosphorylation. A phospholipase C inhibitor, U73122, blocked collagen-induced aggregation and reduced arachidonic acid release, thromboxane synthesis and p38MAPK phosphorylation. Pretreatment with a cytosolic phospholipase A2 inhibitor, AACOCF3, blocked collagen-induced aggregation, reduced the levels of thromboxane formation and p38MAPK phosphorylation but had no significant effect on arachidonic acid release. In contrast inhibition of PKC by Ro31-8220 inhibited collagen-induced aggregation. did not affect p38MAPK phosphorylation but significantly potentiated arachidonic acid release and thromboxane formation. Collagen caused the tyrosine phosphorylation of phospholipase Cgamma2 which was inhibited by pretreatment with U73122, unaffected by AACOCF3 and enhanced by Ro31-8220. These results suggest that cytosolic phospholipase A2 plays no role in the arachidonic acid release in response to collagen. In contrast, the data are consistent with phospholipase Cgamma2 playing a role in an intricately controlled pathway, or multiple pathways, mediating the release of arachidonic acid in collagen-stimulated platelets.

N-terminal and C-terminal plasma membrane anchoring modulate differently agonist-induced activation of cytosolic phospholipase A2.

The 85 kDa cytosolic phospholipase A2 (cPLA2) plays a key role in liberating arachidonic acid from the sn-2 position of membrane phospholipids. When activated by extracellular stimuli, cPLA2 undergoes calcium-dependent translocation from cytosol to membrane sites which are still a matter of debate. In order to evaluate the effect of plasma membrane association on cPLA2 activation, we constructed chimeras of cPLA2 constitutively targeted to the plasma membrane by the N-terminal targeting sequence of the protein tyrosine kinase Lck (Lck-cPLA2) or the C-terminal targeting signal of K-Ras4B (cPLA2-Ras). Constitutive expression of these chimeras in Chinese hamster ovary cells overproducing the alpha2B adrenergic receptor (CHO-2B cells) did not affect the basal release of [3H]arachidonic acid, indicating that constitutive association of cPLA2 with cellular membranes did not ensure the hydrolysis of membrane phospholipids. However, Lck-cPLA2 increased [3H]arachidonic acid release in response to receptor stimulation and to increased intracellular calcium, whereas cPLA2-Ras inhibited it, compared with parental CHO-2B cells and CHO-2B cells producing comparable amounts of recombinant wild-type cPLA2. The lack of stimulation of cPLA2-Ras was not due to a decreased enzymatic activity as measured using an exogenous substrate, or to a decreased phosphorylation of the protein. These results show that the plasma membrane is a suitable site for cPLA2 activation when orientated correctly.

Mechanisms of the priming effect of lipopolysaccharides on the biosynthesis of leukotriene B4 in chemotactic peptide-stimulated human neutrophils.

The goal of this study was to explain the priming effect of lipopolysaccharides (LPS) in human polymorphonuclear leukocytes on leukotriene B4 (LTB4) biosynthesis after stimulation with the receptor-mediated agonist formyl-methionyl-leucyl-phenylalanine (fMLP). This priming effect for LTB4 biosynthesis was maximal after a 30 min preincubation with LPS but was lost when incubations were extended to 90 min or longer. Priming with LPS resulted in an enhanced maximal activation of 5-lipoxygenase (5- to15-fold above unprimed cells) as well as a prolonged activation of the enzyme after stimulation with fMLP compared to that measured in unprimed cells. The activation of 5-lipoxygenase was associated with its translocation to the nuclear fraction of the cell after stimulation of LPS-primed cells but not of unprimed cells. Priming of cells with LPS also resulted in an enhanced capacity (fivefold increase) for arachidonic acid (AA) release after stimulation with fMLP compared to unprimed cells as measured by mass spectrometry. This release of AA was very efficiently blocked in a dose-dependent manner by the 85 kDa cytosolic phospholipase A2 (PLA2) inhibitor MAFP (IC50=10nM) but not by the 14 kDa secretory PLA2 inhibitor SB 203347 (up to 5 microM), indicating that the 85 kDa cPLA2 is the PLA2 responsible for AA release in response to receptor-mediated agonists. In accord with inhibitor studies, the LPS-mediated phosphorylation of cPLA2 followed the same kinetics as the priming for AA release, and a measurable fMLP-induced translocation of cPLA2 was observed only in primed cells. As with AA release and LTB4 biosynthesis, both the phosphorylation and capacity to translocate cPLA2 were reversed when the preincubation period with LPS was extended to 120 min. These results explain some of the cellular events responsible for the potentiation and subsequent decline of functional responses of human polymorphonuclear leukocytes recruited to inflammatory foci.

Modulatory actions of progesterone on gonadotropin-releasing hormone-induced arachidonic acid liberation from perifused rat pituitary cells.

The stimulatory action of gonadotropin-releasing hormone (GnRH) on gonadotropin secretion from cultured rat pituitary cells is modulated by estradiol and progesterone. Recent studies provided evidence that both steroids exert effects on different pathways of GnRH signal transduction, which might be responsible for their actions on luteinizing hormone (LH) release. Here we investigated whether the steroids are able to modulate GnRH-induced liberation of arachidonic acid, which is thought to be involved in GnRH signal transduction. Pituitary cells obtained from female rats were treated for 48 h with vehicle, 1 nmol/l estradiol or 1 nmol/l estradiol + 100 nmol/l progesterone, 48 h with 1 nmol/l estradiol and 2 h with 100 nmol/l progesterone. In addition, these cells were prelabeled with [3H]arachidonic acid. Then the cells were transferred to a perifusion system and challenged with a 6-min pulse of 100 nmol/l GnRH. Estradiol treatment enhanced the LH secretory response while GnRH-induced [3H]arachidonic acid liberation remained unaffected. However, progesterone modulated both LH secretion and [3H]arachidonic acid release in response to the GnRH stimulus. The short-term progesterone treatment paradigm enhanced the LH and arachidonic acid responses by up to 160 +/- 13 and 204 +/- 18%, respectively, while long-term treatment was inhibitory (59 +/- 9 and 63 +/- 4% vs control). Because arachidonic acid has been shown to be involved in GnRH signal transduction, it seems reasonable to speculate that the actions of progesterone described in the present study are related to its modulatory effect on GnRH-induced LH secretion.

Basic fibroblast growth factor-stimulated arachidonic acid release in rat pancreatic acini: Sequential action of tyrosine kinase, phospholipase C, protein kinase C and diacylglycerol lipase

This study was performed to evaluate the effect of human recombinant basic fibroblast growth factor on arachidonic acid release from rat pancreatic acini and to determine the cellular mechanism involved. From enzymatic assays, basic fibroblast growth factor did not significantly stimulate phospholipase A 2 activity, whereas it significantly increased diacylglycerol lipase activity. Validity of phospholipase A 2 or diacylglycerol lipase inhibitors was confirmed by their ability to inhibit phospholipase A2 or diacylglycerol lipase activities. Basic fibroblast growth factor increased intracellular accumulation and extracellular release of arachidonic acid from metabolically labelled acinar cells in a concentration- and time-dependent manner. This effect was maximal with 50 pM basic fibroblast growth factor and became significant after a 5-min incubation period. The protein tyrosine kinase inhibitor, 0.5 mM genistein, inhibited arachidonic acid release in basic fibroblast growth factor-stimulated acini, whereas 100 μM vanadate, a protein tyrosine phosphatase inhibitor, enhanced arachidonic acid release. Two phospholipase A 2 inhibitors, mepacrine and aristolochic acid, failed to attenuate basic fibroblast growth factor-stimulated arachidonic acid release. A diacylglycerol lipase inhibitor RHC 80267 at 150 μM and 50 μM completely inhibited 50 pM basic fibroblast growth factor-induced intracellular accumulation and extracellular release of arachidonic acid, respectively. Furthermore, basic fibroblast growth factor stimulated arachidonic acid release was also inhibited by 10 μM U73122 and by 100 nM staurosporine, phospholipase C and protein kinase C respective inhibitors. Wortmannin, an inhibitor of basic fibroblast growth factor-stimulated phospholipase D, did not affect arachidonic acid release. 100 nM 4β-phorbol 12-myristate 13-acetate also increased arachidonic acid release, an effect also inhibited by staurosporine. Taken together, these data demonstrate activation of diacylglycerol lipase and arachodonic acid release in pancreatic acini upon stimulation by basic fibroblast growth factor, and strongly indicate that arachidonic acid release in response to basic fibroblast growth factor depends upon the sequential action of tyrosine kinase, phospholipase C, protein kinase C and diacylglycerol lipase but not from phospholipase A 2 nor phospholipase D activation.

Role of cytosolic phospholipase A2 in arachidonic acid release of rat-liver macrophages: regulation by Ca2+ and phosphorylation

In this study we have verified the existence of a cytosolic phospholipase A2 (cPLA2) in rat-liver macrophages. Stimulation of these cells with phorbol 12-myristate 13-acetate (PMA), zymosan and lipopolysaccharide (LPS), but not with the Ca(2+)-ionophore A23187, leads to phosphorylation of cPLA2 and activation of mitogen-activated protein (MAP) kinase, supporting the hypothesis that MAP kinase is involved in cPLA2 phosphorylation. We show furthermore, that the tyrosine kinase inhibitor genistein prevents the LPS- but not the PMA- or zymosan-induced phosphorylation of cPLA2 and activation of MAP kinase, indicating that tyrosine kinases participate in LPS- but not in PMA- and zymosan-induced cPLA2 phosphorylation and MAP kinase activation. Phosphorylation of cPLA2 does not strongly correlate with stimulation of the arachidonic acid (AA) cascade: (1) A23187, a potent stimulator of AA release, fails to induce cPLA2 phosphorylation; (2) withdrawal of extracellular Ca2+, which inhibits PMA-stimulated AA release (Dieter, Schulze-Specking and Decker (1988) Eur. J. Biochem. 177, 61-67), has no effect on PMA-induced phosphorylation of cPLA2; (3) LPS induces cPLA2 phosphorylation within minutes, whereas increased AA release upon treatment with LPS is detectable for the first time after 4 h; and (4) genistein, which prevents LPS-induced cPLA2 phosphorylation, does not inhibit AA release in response to LPS. From these data we suggest that a rise in intracellular Ca2+, but not phosphorylation of cPLA2, is essential for activation of the AA cascade in rat-liver macrophages.