Peroxynitrite stimulates the activity of cytosolic phospholipase A2 in U937 cells: the extent of arachidonic acid formation regulates the balance between cell survival or death

Peroxynitrite stimulates in U937 cells release of arachidonic acid (AA) sensitive to various phospholipase A(2) (PLA(2)) inhibitors, including arachidonyl trifluoromethyl ketone (AACOCF(3)), which specifically inhibits cytosolic PLA(2) (cPLA(2)). This response linearly increases using non toxic concentrations of the oxidant, and reaches a plateau at levels at which toxicity becomes apparent. Three separate lines of evidence are consistent with the notion that AA generated by cPLA(2) promotes survival in cells exposed to peroxynitrite. Firstly, toxicity was suppressed by nanomolar levels of exogenous AA, or by AA generated by the direct PLA(2) activator melittin. Secondly AACOCF(3), or other PLA(2) inhibitors, promoted cell death after exposure to otherwise non toxic concentrations of peroxynitrite; exogenous AA abolished the enhancing effects mediated by the PLA(2) inhibitors. Finally, U937 cells transfected with cPLA(2) antisense oligonucleotides were killed by concentrations of peroxynitrite that were non-toxic for cells transfected with nonsense oligonucleotides. This lethal response was insensitive to AACOCF(3) and prevented by exogenous AA.     

Differential effects of arachidonoyl trifluoromethyl ketone on arachidonic acid release and lipid mediator biosynthesis by human neutrophils. Evidence for different arachidonate pools.

The goal of this study was to determine the effects of a putative specific cytosolic phospholipase A2 inhibitor, arachidonyl trifluoromethyl ketone (AACOCF3), on arachidonic acid (AA) release and lipid mediator biosynthesis by ionophore-stimulated human neutrophils. Initial studies indicated that AACOCF3 at concentrations 0-10 micro m did not affect AA release from neutrophils. In contrast, AACOCF3 potently inhibited leukotriene B4 formation by ionophore-stimulated neutrophils (IC50 approximately 2.5 micro m). Likewise, AACOCF3 significantly inhibited the biosynthesis of platelet activating factor. In cell-free assay systems, 10 micro m AACOCF3 inhibited 5-lipoxygenase and CoA-independent transacylase activities. [3H]AA labeling studies indicated that the specific activities of cell-associated AA mimicked that of leukotriene B4 and PtdCho/PtdIns, while the specific activities of AA released into the supernatant fluid closely mimicked that of PtdEtn. Taken together, these data argue for the existence of segregated pools of arachidonate in human neutrophils. One pool of AA is linked to lipid mediator biosynthesis while another pool provides free AA that is released from cells. Additionally, the data suggest that AACOCF3 is also an inhibitor of CoA-independent transacylase and 5-lipoxygenase. Thus, caution should be exercised in using AACOCF3 as an inhibitor of cytosolic phospholipase A2 in whole cell assays because of the complexity of AA metabolism.     

Interleukin-1beta-induced substance P release from rat cultured primary afferent neurons driven by two phospholipase A2 enzymes: secretory type IIA and cytosolic type IV

We previously described that recombinant interleukin-1beta (IL-1beta) induced the significant release of substance P (SP) via a cyclooxygenase (COX) pathway in primary cultured rat dorsal root ganglion (DRG) cells. In the present study, we examined the involvement of two types of phospholipase A2 (PLA2) enzymes, which lie upstream of COX in the prostanoid-generating pathway, in the IL-1beta-induced release of SP from DRG cells. The expression of type IIA secretory PLA2 (sPLA2 -IIA) mRNA was undetectable by ribonuclease protection assay in non-treated DRG cells, while in DRG cells incubated with 1 ng/mL of IL-1beta, the expression was induced in a time-dependent manner. On the other hand, type IV cytosolic PLA2 (cPLA2 ) mRNA was constitutively expressed in the non-treated DRG cells, and treatment with 1 ng/mL of IL-1beta for 3 h significantly increased the levels of cPLA2 mRNA. The IL-1beta-induced SP release was significantly inhibited by the sPLA2 inhibitor, thioetheramide phosphorylcholine (TEA-PC), and the cPLA2 inhibitor, arachidonyl trifluoromethyl ketone (AACOCF3 ). Furthermore AACOCF3 suppressed the induction of sPLA2 -IIA mRNA expression induced by IL-1beta. These observations suggested that two types of PLA2, sPLA2 -IIA and cPLA2, were involved in the IL-1beta-induced release of SP from DRG cells, and that the functional cross-talk between the two enzymes might help to control their activity in the prostanoid-generating system in DRG cells. These events might be key steps in the inflammation-induced hyperactivity in primary afferent neurons of spinal cord.     

NS-398: cyclooxygenase-2 independent inhibition of leukocyte priming for lipid body formation and enhanced leukotriene generation

Because the induction of new lipid body formation in leukocytes correlates with and likely contributes to their enhanced 'primed' prostaglandin and leukotriene formation, we evaluated two selective cyclooxygenase (COX)-2 inhibitors. Three types of stimuli, cis -unsaturated fatty acids, platelet activating factor and protein kinase C activators, stimulate lipid body formation. NS-398 (0.1-10 microM), but not another COX-2 inhibitor, SC58125 (0.1- 10 microM), blocked leukocyte lipid body formation elicited by all three types of stimuli and also blocked priming for enhanced LTB(4) production and PGE(2) production. The effect of NS-398 on lipid body formation was independent of its inhibitory effects on COX-2 since arachidonate-induced lipid body formation in COX-2-deficient mouse leukocytes was also inhibited by NS-398. By means of its ability to inhibit leukocyte lipid body formation, NS-398 may exert actions independent of its COX-2 inhibition and more broadly contribute to the suppression of formation of COX-1 and lipoxygenase-derived eicosanoids.     

Regulation of lung surfactant secretion by phospholipase A2

Arachidonic acid has been shown to stimulate lung surfactant secretion from alveolar epithelial type II cells. To identify the (phospho)lipases responsible for generating arachidonic acid during lung surfactant secretion, the effects of various (phospho)lipase inhibitors on phosphatidylcholine (PC) secretion from rat alveolar type II cells were investigated. N-(p-amylcinnamoyl)anthranilic acid (ACA), a general inhibitor of phsopholipase A2 (PLA2), inhibited ATP-stimulated PC secretion in a dose-dependent manner. ACA also blocked PC secretion from type II cells stimulated by other secretagogues including phorbol 12-myristate 13-acetate, Ca2+ ionophore A23187 and terbutaline, indicating that PLA2 acts at a late step distal to the generation of second messengers. To determine which PLA2 isoform(s) is involved in lung surfactant secretion, selective inhibitors to different types of PLA2 were used to inhibit PLA2 activity in type II cells. The cytosolic PLA2 (cPLA2) inhibitor, arachidonyl trifluoromethyl ketone, was found to inhibit ATP-stimulated PC secretion, whereas the secretory PLA2 inhibitors, oleoyloxyethylphosphocholine, aristolochic acid, or p-bromophenacyl bromide, and the Ca2+-independent PLA2 inhibitors, palmitoyl trifluoromethyl ketone, or haloenol lactone suicide substrate, had no effect. In addition to PLA2, arachidonic acid is released from diacylglycerol (DAG) by DAG and monoacylglycerol lipases. The DAG lipase inhibitor, RHC-80267 also blocked ATP-stimulated PC secretion. The results suggest that both pathways for generating arachidonic acid via cPLA2 and DAG lipase may participate in lung surfactant secretion.     

Purification and characterization of a cytosolic phospholipase A2 from rat liver

A cytosolic phospholipase A2 (PLA2) was purified 640-fold from rat liver by sequential anion-exchange chromatography, Ca2+-precipitation/KCl-solubilization, gel filtration chromatography, and affinity chromatography. A single peak of PLA2 activity was eluted at an apparent molecular mass of 197 kDa from a Superdex 200HR gel filtration column. In the presence of Ca2+, the purified enzyme catalyzed the hydrolysis of 81.8 nmol of phosphatidylethanolamine per hour per mg of protein. The apparent Km was 1.83 nM. The enzyme was inhibited by arachidonyl trifluoromethyl ketone (AACOCF3), an inhibitor of cPLA2. However, it was not inhibited by bromoenol lactone (BEL), an inhibitor of iPLA2, and p-bromophenacyl bromide (p-BPB), an inhibitor of sPLA2. These data suggest that the purified enzyme is a novel Ca2+-dependent cytosolic PLA2.     

Formation of reactive oxygen species by the contracting diaphragm is PLA(2) dependent.

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.     

Secretory and cytosolic phospholipase A(2)regulate the long-term cytokine-induced eicosanoid production in human keratinocytes

The involvement of cytosolic phospholipase A(2)(cPLA(2)) and secretory non-pancreatic PLA(2)(npPLA(2)) in release of arachidonic acid (AA) preceding eicosanoid formation in the human keratinocyte cell line HaCaT was examined. Interleukin 1beta (IL-1beta) and tumour necrosis factor-alpha (TNF), phorbol myristate acetate (PMA) and calcium ionophore A(23187)increased the extracellular AA release, and stimulated eicosanoid synthesis as determined by HPLC analysis. The main metabolites after stimulation with IL-1beta, PMA or A(23187)were PGE(2), an unidentified PG and LTB(4), while TNF stimulated HETE-production. Both cPLA(2)and npPLA(2)message and enzyme activity were detected in unstimulated HaCaT cells. IL-1beta, PMA and TNF increased both cPLA(2)enzyme activity and expression, but did not lead to any increase in npPLA(2)expression or activity. The selective npPLA(2)inhibitors LY311727 and 12-epi-scalaradial, or the cPLA(2)inhibitor arachidonyl trifluoro methyl ketone (AACOCF(3)) reduced IL-1beta-induced eicosanoid production in a concentration dependent manner. The results presented strongly suggest that both cPLA(2)and npPLA(2)contribute to the long-term generation of AA preceding eicosanoid production in differentiated, human keratinocytes. Inhibitors against npPLA2 or cPLA2 enzymes should be useful in treating inflammatory skin diseases, such as psoriasis.     

Role of mitogen-activated protein kinase-mediated cytosolic phospholipase A2 activation in arachidonic acid metabolism in human eosinophils.

The objective of this investigation was to determine the role of secretory and cytosolic isoforms of phospholipase A(2) (PLA(2)) in the induction of arachidonic acid (AA) and leukotriene synthesis in human eosinophils and the mechanism of PLA(2) activation by mitogen-activated protein kinase (MAPK) isoforms in this process. Pharmacological activation of eosinophils with fMLP caused increased AA release in a concentration (EC(50) = 8.5 nM)- and time-dependent (t(1/2) = 3.5 min) manner. Both fMLP-induced AA release and leukotriene C(4) (LTC(4)) secretion were inhibited concentration dependently by arachidonic trifluoromethyl ketone, a cytosolic PLA(2) (cPLA(2)) inhibitor; however, inhibition of neither the 14-kDa secretory phospholipase A(2) by 3-(3-acetamide-1-benzyl-2-ethylindolyl-5-oxy)propanephosphonic acid nor cytosolic Ca(2+)-independent phospholipase A(2) inhibition by bromoenol lactone blocked hydrolysis of AA or subsequent leukotriene synthesis. Pretreatment of eosinophils with a mitogen-activated protein/extracellular signal-regulated protein kinase (ERK) kinase inhibitor, U0126, or a p38 MAPK inhibitor, SB203580, suppressed both AA production and LTC(4) release. fMLP induced phosphorylation of MAPK isoforms, ERK1/2 and p38, which were evident after 30 s, maximal at 1-5 min, and declined thereafter. fMLP stimulation also increased cPLA(2) activity in eosinophils, which was inhibited completely by 30 microM arachidonic trifluoromethyl ketone. Preincubation of eosinophils with U0126 or SB203580 blocked fMLP-enhanced cPLA(2) activity. Furthermore, inhibition of Ras, an upstream GTP-binding protein of ERK, also suppressed fMLP-stimulated AA release. These findings demonstrate that cPLA(2) activation causes AA hydrolysis and LTC(4) secretion. We also find that cPLA(2) activation caused by fMLP occurs subsequent to and is dependent upon ERK1/2 and p38 MAPK activation. Other PLA(2) isoforms native to human eosinophils possess no significant activity in the stimulated production of AA or LTC(4).     

Ergosterol elicits oxidative burst in tobacco cells via phospholipase A2 and protein kinase C signal pathway.

Ergosterol, a typical fungal sterol, induced in tobacco (Nicotiana tabacum L. cv. Xanthi) suspension cells the synthesis of reactive oxygen species and alkalization of the external medium that are dependent on the mobilization of calcium from internal stores. We used specific inhibitors to elucidate the signal pathway triggered by ergosterol compared with cryptogein, a proteinaceous elicitor of Phytophthora cryptogea. Herbimycin A and genistein, inhibitors of tyrosine protein kinases, had no effect on the oxidative burst and pH changes induced by both elicitors. Similarly, H-89, an inhibitor of protein kinase A, had no effect on the induction of these defense reactions. However, the response to both elicitors was completely blocked by NPC-15437, a specific inhibitor of animal protein kinase C (PKC). The responses induced by cryptogein but not those induced by ergosterol were inhibited by U73122 and neomycin, inhibitors of phospholipase C (PLC). On the other hand, the activity of phospholipase A2 (PLA2) measured using a fluorogenic substrate was stimulated by ergosterol and not by cholesterol and cryptogein. A specific inhibitor of PLA2, arachidonic acid trifluoromethyl ketone (AACOCF3), inhibited the pathway stimulated by ergosterol but not that induced by cryptogein. These results suggest that the cryptogein-induced signal pathway leading to the oxidative burst and DeltapH changes includes PLC and PKC, whereas this response induced by ergosterol includes PLA2 and PKC.     

Human calcium-independent phospholipase A2 mediates lymphocyte proliferation

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.     

Arachidonyltrifluoromethy ketone, a phospholipase A(2) antagonist, induces dispersal of both Golgi stack- and trans Golgi network-resident proteins throughout the cytoplasm

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.     

Effect of insulin on ketogenesis and fatty acid synthesis in rat hepatocytes incubated with dichloroacetate

In parenchymal liver cells isolated from fed rats, insulin increased the formation of 14CO2 from [1-14C]pyruvate (and presumably the flux through pyruvate dehydrogenase) by 14%. Dichloroacetate, an activator of the pyruvate dehydrogenase complex, stimulated this process by 133%. As judged from the conversion of [2-14C]pyruvate to 14CO2, the tricarboxylic acid cycle activity was not affected by insulin, but it was depressed by dichloroacetate. In hepatocytes from fed rats, incubated with glucose as the only carbon source, dichloroacetate caused a stimulation (31%) of fatty acid synthesis, measured as 3H incorporation from 3H2O into fatty acid, and an increased (134%) accumulation of ketone bodies (acetoacetate + D-3-hydroxybutyrate). Dichloroacetate did not affect ketone body formation from [14C]palmitate, suggesting that the increased accumulation of ketone bodies resulted from acetyl-CoA derived from pyruvate. Insulin stimulated fatty acid synthesis in hepatocytes from fed rats. In the combined presence of insulin plus dichloroacetate, fatty acid synthesis was more rapid than in the presence of either insulin or dichloroacetate, whereas the accumulation of ketone bodies was smaller than in the presence of dichloroacetate alone. Although pyruvate dehydrogenase activity, which is rate-limiting for fatty acid synthesis in hepatocytes from fed rats, is stimulated both by insulin and by dichloroacetate, the reciprocal changes in fatty acid synthesis and ketone body accumulation brought about by insulin in the presence of dichloroacetate suggest that insulin is also involved in the regulation of fatty acid synthesis at a mitochondrial site after pyruvate dehydrogenase, possibly at the partitioning of acetyl-CoA between citrate and ketone body formation.     

Cytosolic phospholipase A2 alpha inhibition prevents neuronal NMDA receptor-stimulated arachidonic acid mobilization and prostaglandin production but not subsequent cell death

Phospholipase A(2) (PLA(2)) enzymes encompass a superfamily of at least 13 extracellular and intracellular esterases that hydrolyze the sn-2 fatty acyl bonds of phospholipids to yield fatty acids and lysophospholipids. The purpose of this study was to characterize which phospholipase paralog regulates NMDA receptor-mediated arachidonic acid (AA) release. Using mixed cortical cell cultures containing both neurons and astrocytes, we found that [(3)H]-AA released into the extracellular medium following NMDA receptor stimulation (100 microM) increased with time and was completely prevented by the addition of the NMDA receptor antagonist MK-801 (10 microM) or by removal of extracellular Ca(2+). Neither diacylglycerol lipase inhibition (RHC-80267; 10 microM) nor selective inhibition of Ca(2+)-independent PLA(2) [bromoenol lactone (BEL); 10 microM] alone had an effect on NMDA receptor-stimulated release of [(3)H]-AA. Release was prevented by methyl arachidonyl fluorophosphonate (MAFP) (5 microM) and AACOCF(3) (1 microM), inhibitors of both cytosolic PLA(2) (cPLA(2)) and Ca(2+)-independent PLA(2) isozymes. This inhibition effectively translated to block of NMDA-induced prostaglandin (PG) production. An inhibitor of p38MAPK, SB 203580 (7.5 microM), also significantly reduced NMDA-induced PG production providing suggestive evidence for the role of cPLA(2)alpha. Its involvement in release was confirmed using cultures derived from mice deficient in cPLA(2)alpha, which failed to produce PGs in response to NMDA receptor stimulation. Interestingly, neither MAFP, AACOCF(3) nor cultures derived from cPLA(2)alpha null mutant animals showed any protection against NMDA-mediated neurotoxicity, indicating that inhibition of this enzyme may not be a viable protective strategy in disorders of the cortex involving over-activation of the NMDA receptor.     

Cytosolic and mitochondrial ROS in staurosporine-induced retinal cell apoptosis

In this study, we investigated the involvement of reactive oxygen species (ROS) and calcium in staurosporine (STS)-induced apoptosis in cultured retinal neurons, under conditions of maintained membrane integrity. The antioxidants idebenone (IDB), glutathione-ethylester (GSH/EE), trolox, and Mn(III)tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP) significantly reduced STS-induced caspase-3-like activity and intracellular ROS generation. Endogenous sources of ROS production were investigated by testing the effect of the following inhibitors: 7-nitroindazole (7-NI), a specific inhibitor of the neuronal isoform of nitric oxide synthase (nNOS); arachidonyl trifluoromethyl ketone (AACOCF(3)), a phospholipase A(2) (PLA(2)) inhibitor; allopurinol, a xanthine oxidase inhibitor; and the mitochondrial inhibitors rotenone and oligomycin. All these compounds decreased caspase-3-like activity and ROS generation, showing that both mitochondrial and cytosolic sources of ROS are implicated in this mechanism. STS induced a significant increase in intracellular calcium concentration ([Ca(2+)](i)), which was partially prevented in the presence of IDB and GSH/EE, indicating its dependence on ROS generation. These two antioxidants and the inhibitors allopurinol and 7-NI also reduced the number of TdT-mediated dUTP nick-end labeling-positive cells. Thus, endogenous ROS generation and the rise in intracellular calcium are important inter-players in STS-triggered apoptosis. Furthermore, the antioxidants may help to prolong retinal cell survival upon apoptotic cell death.     

Human fetal skin fibroblast migration stimulated by the autocrine growth factor bFGF is mediated by phospholipase A(2) via arachidonic acid without the involvement of pertussis toxin-sensitive G-protein

We reported previously that human fetal skin fibroblast migration into a denuded area was stimulated by an autocrine factor, basic fibroblast growth factor (bFGF). Since the signal transduction pathway of this migration is unknown, we attempted to clarify it by comparing this fibroblast migration with a previously reported bovine endothelial cell migration into a wounded area stimulated by an addition of bFGF, in which the bFGF signal was mediated by phospholipase A(2)-coupled G-protein and phospholipase A(2) (PLA(2)) via arachidonic acid. Our study demonstrated that pertussis toxin, a specific inhibitor of PLA(2)-coupled G-protein, did not suppress human fetal skin fibroblast migration, but 2-(p-amylcinnamyl)amino-4-chlorobensoic acid (ONO-RS-082), a PLA(2) inhibitor, did. Since ONO-RS-082 is a non-specific PLA(2) inhibitor, a cytoplasmic, Ca-dependent PLA(2) (cPLA(2)) inhibitor, AACOCF3, was examined. AACOCF3 suppressed cell migration in certain concentrations. The PLA(2) inhibitor-suppressed cell migration was restored by adding arachidonic acid, and cell migration suppressed by anti-bFGF antibodies was restored by adding arachidonic acid. In addition, pertussis toxin did not suppress arachidonic acid release, which shows an action of PLA(2), but AACOCF3 did. These results indicate that human fetal skin fibroblast migration stimulated by an autocrine factor, bFGF, was mediated by PLA(2) via arachidonic acid without the involvement of PLA(2)-coupled G-protein.     

Amyloid beta(1-42) and its beta(25-35) fragment induce activation and membrane translocation of cytosolic phospholipase A2 in bovine retina capillary pericytes

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.     

Involvement of protein kinase A activation and phospholipase A(2) inhibition in the adenosine-activated basolateral 50 pS K(+) channels in the thick ascending limb of the rat kidney

The present study was designed to investigate the role of protein kinase A (PKA) and phospholipase A(2) (PLA(2)) in the stimulating effect of adenosine on the basolateral 50 pS K(+) channels in the thick ascending limb (TAL) of the rat kidney. Under the anatomic microscope, the TAL was dissected. The current of 50 pS K(+) channels were recorded by patch clamp technology. The protein expression of phosphorylated PKA and phosphorylated PLA(2) were examined by Western blot. The results showed that cyclohexyladenosine (CHA), an analog of adenosine, increased the 50 pS K(+) channel activity (P < 0.05). In the presence of H8, an antagonist of PKA, CHA did not affect the 50 pS K(+) channel activity. In the presence of AACOCF3 (an antagonist of PLA(2)), CHA did not further increase the 50 pS K(+) channel activity. CHA increased phosphorylation level of PKA, whereas inhibited phosphorylation of PLA(2) in the TAL of the rat kidney (P < 0.01). Furthermore, after blocking the PLA(2) with AACOCF3, CHA still increased the expression of phosphorylated PKA. On the contrary, CHA did not obviously change the expression of phosphorylated PLA(2) after H8 pretreatment. The results suggest that the stimulation of basolateral 50 pS K(+) channels by CHA is mediated by the activation of PKA followed by the inhibition of PLA(2) in the TAL of the rat kidney.     

Group IVA phospholipase A2 regulates testosterone biosynthesis by murine Leydig cells and is required for timely sexual maturation

In the present paper, we report that PLA2G4A (Group IVA phospholipase A2) is important in the development and function of rodent testes. Interstitial cells of rat testes had high PLA2 (phospholipase A2) activity that was very sensitive to the PLA2G4A-preferential inhibitor AACOCF3 (arachidonyl trifluoromethyl ketone). PLA2G4A protein was expressed primarily in the interstitial cells of wild-type mouse testes throughout maturation. Although Pla2g4a knockout (Pla2g4a-/-) male mice are fertile, their sexual maturation was delayed, as indicated by cauda epididymal sperm count and seminal vesicle development. Delayed function of Pla2g4a-/- mice testes was associated with histological abnormalities including disorganized architecture, swollen appearance and fewer interstitial cells. Basal secretion of testosterone was attenuated significantly and steroidogenic response to hCG (human chorionic gonadotropin) treatment was reduced in Pla2g4a-/- mice compared with their Pla2g4a+/+ littermates during the sexual maturation period. Chemical inhibition of PLA2G4A activity by AACOCF3 or pyrrophenone significantly reduced hCG-stimulated testosterone production in cultured rat interstitial cells. AACOCF3 inhibited forskolin- and cAMP analogue-stimulated testosterone production. These results provide the first evidence that PLA2G4A plays a role in male testes physiology and development. These results may have implications for the potential clinical use of PLA2G4A inhibitors.