Involvement of cytosolic phospholipase A2 and secretory phospholipase A2 in arachidonic acid release from human neutrophils

The purpose of this study was to define the role of secretory phospholipase A2 (sPLA2), calcium-independent PLA2, and cytosolic PLA2 (cPLA2) in arachidonic acid (AA) release from fMLP-stimulated human neutrophils. While fMLP induced the release of extracellular sPLA2 activity and AA, 70% of sPLA2 activity remained associated with the cell. Treatment with the cell-impermeable sPLA2 inhibitors DTT or LY311-727, or the anti-sPLA2 Ab 3F10 all inactivated extracellular sPLA2 activity, but had minimal effect on neutrophil AA mass release. In contrast, coincubation of streptolysin-O toxin-permeabilized neutrophils with DTT, LY311-727, or 3F10 all decreased [3H8]AA release from [3H8]AA-labeled, fMLP-stimulated cells. Exposure to fMLP resulted in a decrease in the electrophoretic mobility of cPLA2, a finding consistent with cPLA2 phosphorylation, and stimulated the translocation of cPLA2 from cytosolic to microsomal and nuclear compartments. The role of cPLA2 was further evaluated with the cPLA2 inhibitor methyl arachidonyl fluorophosphonate, which attenuated cPLA2 activity in vitro and decreased fMLP-stimulated AA mass release by intact neutrophils, but had no effect on neutrophil sPLA2 activity. Inhibition of calcium-independent PLA2 with haloenol lactone suicide substrate had no effect on neutrophil cPLA2 activity or AA mass release. These results indicate a role for cPLA2 and an intracellular or cell-associated sPLA2 in the release of AA from fMLP-stimulated human neutrophils.     

Role of secretory and cytosolic phospholipase A(2) enzymes in lysophosphatidylcholine-stimulated monocyte arachidonic acid release

To determine if lysophosphatidylcholine (lysoPC) is able to induce proinflammatory changes in monocytes, its ability to stimulate arachidonic acid (AA) release, a product of phospholipase A2 (PLA(2)) activity, has been analyzed. LysoPC increased AA release in THP-1 and Mono Mac6 cells in a time- and concentration-dependent manner. The monocytes expressed both secretory and cytosolic PLA(2) enzymes and AA release was strongly reduced by cellular pretreatment with different PLA(2) inhibitors and by pertussis toxin, an inhibitor of G(i)-protein activation. This indicates that both cytosolic and secretory PLA(2) enzymes regulate specific lysoPC receptor-induced AA release, suggesting lysoPC participation in monocyte proinflammatory activation.     

Cross-talk between cytosolic phospholipase A2 alpha (cPLA2 alpha) and secretory phospholipase A2 (sPLA2) in hydrogen peroxide-induced arachidonic acid release in murine mesangial cells: sPLA2 regulates cPLA2 alpha activity that is responsible for arachidonic acid release

Oxidant stress and phospholipase A2 (PLA2) activation have been implicated in numerous proinflammatory responses of the mesangial cell (MC). We investigated the cross-talk between group IValpha cytosolic PLA2 (cPLA2alpha) and secretory PLA2s (sPLA2s) during H2O2-induced arachidonic acid (AA) release using two types of murine MC: (i). MC+/+, which lack group IIa and V PLA2s, and (ii). MC-/-, which lack groups IIa, V, and IValpha PLA2s. H2O2-induced AA release was greater in MC+/+ compared with MC-/-. It has been argued that cPLA2alpha plays a regulatory role enhancing the activity of sPLA2s, which act on phospholipids to release fatty acid. Group IIa, V, or IValpha PLA2s were expressed in MC-/- or MC+/+ using recombinant adenovirus vectors. Expression of cPLA2alpha in H2O2-treated MC-/- increased AA release to a level approaching that of H2O2-treated MC+/+. Expression of either group IIa PLA2 or V PLA2 enhanced AA release in MC+/+ but had no effect on AA release in MC-/-. When sPLA2 and cPLA2alpha are both present, the effect of H2O2 is manifested by preferential release of AA compared with oleic acid. Inhibition of the ERK and protein kinase C signaling pathways with the MEK-1 inhibitor, U0126, and protein kinase C inhibitor, GF 1092030x, respectively, and chelating intracellular free calcium with 1,2-bis(2-aminophenoyl)ethane-N,N,N',N'-tetraacetic acid-AM, which also reduced ERK1/2 activation, significantly reduced H2O2-induced AA release in MC+/+ expressing either group IIa or V PLA2s. By contrast, H2O2-induced AA release was not enhanced when ERK1/2 was activated by infection of MC+/+ with constitutively active MEK1-DD. We conclude that the effect of group IIa and V PLA2s on H2O2-induced AA release is dependent upon the presence of cPLA2alpha and the activation of PKC and ERK1/2. Group IIa and V PLA2s are regulatory and cPLA2alpha is responsible for AA release.     

Lipopolysaccharide-induced release of arachidonic acid and prostaglandins in liver macrophages: regulation by Group IV cytosolic phospholipase A2, but not by Group V and Group IIA secretory phospholipase A2.

Lipopolysaccharide (LPS) induces a delayed release (lag phase of 2-4 h) of arachidonic acid (AA) and prostaglandin (PG) D2 in rat liver macrophages. Group IV cytosolic phospholipase A2 (cPLA2) becomes phosphorylated within minutes after the addition of LPS. The phosphorylated form of cPLA2 shows an enhanced in vitro activity. The Ca2+ dependence of cPLA2 activity is not affected by phosphorylation of the enzyme. In addition, LPS induces an enhanced expression of cPLA2 mRNA (after 2-4 h) and an enhanced expression of cPLA2 protein (after 8 h). The cellular cPLA2 activity is enhanced about twofold 24 h after LPS treatment. Liver macrophages constitutively express mRNAs encoding Groups V and IIA secretory PLA2 (sPLA2). LPS has no effect on the levels of Groups V and IIA sPLA2 mRNA expression. Despite mRNA expression, Groups V and IIA sPLA2 protein and sPLA2 activity are not detectable in unstimulated or LPS-stimulated liver macrophages. Collectively, these and earlier [Mediators Inflammation 8 (1999) 295.] results suggest that in liver macrophages the LPS-induced delayed release of AA and prostanoids is mediated by phosphorylation and an enhanced expression of cPLA2, a de novo expression of cyclooxygenase (COX)-2, but not by the actions of Group V or Group IIA sPLA2.     

Regulation of the specific release of arachidonic acid by cytosolic phospholipase A2

Cytosolic phospholipase A(2) alpha (cPLA(2)alpha) is the only PLA(2) that exhibits specificity for sn-2 arachidonic acid consistent with its primary role in mediating the agonist-induced release of arachidonic acid for eicosanoid production. It is subject to complex mechanisms of regulation that ensure that levels of free arachidonic acid are tightly controlled. The calcium-induced translocation of cPLA(2)alpha from the cytosol to membrane regulates its interaction with phospholipid substrate. cPLA(2)alpha is additionally regulated by phosphorylation on sites in the catalytic domain. Because of its central position as the upstream regulatory enzyme for initiating production of several classes of bioactive lipid mediators (leukotrienes, prostaglandins and platelet-activating factor), it is a potentially important pharmacological target for the control of inflammatory diseases.     

Purified group X secretory phospholipase A(2) induced prominent release of arachidonic acid from human myeloid leukemia cells

Group X secretory phospholipase A(2) (sPLA(2)-X) possesses several structural features characteristic of both group IB and IIA sPLA(2)s (sPLA(2)-IB and -IIA) and is postulated to be involved in inflammatory responses owing to its restricted expression in the spleen and thymus. Here, we report the purification of human recombinant COOH-terminal His-tagged sPLA(2)-X, the preparation of its antibody, and the purification of native sPLA(2)-X. The affinity-purified sPLA(2)-X protein migrated as various molecular species of 13-18 kDa on SDS-polyacrylamide gels, and N-glycosidase F treatment caused shifts to the 13- and 14-kDa bands. NH(2)-terminal amino acid sequencing analysis revealed that the 13-kDa form is a putative mature sPLA(2)-X and the 14-kDa protein possesses a propeptide of 11 amino acid residues attached at the NH(2) termini of the mature protein. Separation with reverse-phase high performance liquid chromatography revealed that N-linked carbohydrates are not required for the enzymatic activity and pro-sPLA(2)-X has a relatively weak potency compared with the mature protein. The mature sPLA(2)-X induced the release of arachidonic acid from phosphatidylcholine more efficiently than other human sPLA(2) groups (IB, IIA, IID, and V) and elicited a prompt and marked release of arachidonic acid from human monocytic THP-1 cells compared with sPLA(2)-IB and -IIA with concomitant production of prostaglandin E(2). A prominent release of arachidonic acid was also observed in sPLA(2)-X-treated human U937 and HL60 cells. Immunohistochemical analysis of human lung preparations revealed its expression in alveolar epithelial cells. These results indicate that human sPLA(2)-X is a unique N-glycosylated sPLA(2) that releases arachidonic acid from human myeloid leukemia cells more efficiently than sPLA(2)-IB and -IIA.     

A pyrrolidine-based specific inhibitor of cytosolic phospholipase A(2)alpha blocks arachidonic acid release in a variety of mammalian cells

We analyzed a recently reported (K. Seno, T. Okuno, K. Nishi, Y. Murakami, F. Watanabe, T. Matsuur, M. Wada, Y. Fujii, M. Yamada, T. Ogawa, T. Okada, H. Hashizume, M. Kii, S.-H. Hara, S. Hagishita, S. Nakamoto, J. Med. Chem. 43 (2000)) pyrrolidine-based inhibitor, pyrrolidine-1, against the human group IV cytosolic phospholipase A(2) alpha-isoform (cPLA(2)alpha). Pyrrolidine-1 inhibits cPLA(2)alpha by 50% when present at approx. 0.002 mole fraction in the interface in a number of in vitro assays. It is much less potent on the cPLA(2)gamma isoform, calcium-independent group VI PLA(2) and groups IIA, X, and V secreted PLA(2)s. Pyrrolidine-1 blocked all of the arachidonic acid released in Ca(2+) ionophore-stimulated CHO cells stably transfected with cPLA(2)alpha, in zymosan- and okadaic acid-stimulated mouse peritoneal macrophages, and in ATP- and Ca(2+) ionophore-stimulated MDCK cells.     

Modulation of the activity and arachidonic acid selectivity of group X secretory phospholipase A2 by sphingolipids

To investigate the role of sphingomyelin (SM) in the regulation of inflammatory reactions, we studied its effect on the activity and fatty acid specificity of group X secretory phospholipase A(2) (sPLA(2)X). Compared with other phospholipases, recombinant sPLA(2)X released more arachidonate from HDL. Pretreatment of HDL with sphingomyelinase (SMase) C activated the sPLA(2)X activity, but the release of arachidonate was stimulated less than that of linoleate. In liposomes containing synthetic phosphatidylcholines (PCs), sPLA(2)X showed no clear selectivity among the various sn-2 unsaturated fatty acids. However, when SM was incorporated into liposomes at 30 mol%, the enzyme exhibited strong preference for arachidonate, although its overall activity was inhibited. Degradation of liposomal SM by SMase C resulted in sPLA(2)X activation and loss of its arachidonate preference. Incorporation of ceramide into HDL or PC liposomes activated the enzyme activity, the release of arachidonate being stimulated more than that of linoleate. SM-deficient cells released more arachidonate than normal cells in response to exogenous sPLA(2)X. SMase pretreatment of normal cells stimulated the release of arachidonate by the exogenous sPLA(2)X. These results show that SM not only inhibits sPLA(2)X activity but also contributes to its selectivity for arachidonate, whereas ceramide stimulates the hydrolysis of arachidonate-containing PCs.     

fMLP-induced arachidonic acid release in db-cAMP-differentiated HL-60 cells is independent of phosphatidylinositol-4, 5-bisphosphate-specific phospholipase C activation and cytosolic phospholipase A(2) activation

In inflammatory cells, agonist-stimulated arachidonic acid (AA) release is thought to be induced by activation of group IV Ca(2+)-dependent cytosolic phospholipase A(2) (cPLA(2)) through mitogen-activated protein kinase (MAP kinase)- and/or protein kinase C (PKC)-mediated phosphorylation and Ca(2+)-dependent translocation of the enzyme to the membrane. Here we investigated the role of phospholipases in N-formylmethionyl-l-leucyl-l-phenylalanine (fMLP; 1 nM-10 microM)-induced AA release from neutrophil-like db-cAMP-differentiated HL-60 cells. U 73122 (1 microM), an inhibitor of phosphatidyl-inositol-4,5-biphosphate-specific phospholipase C, or the membrane-permeant Ca(2+)-chelator 1, 2-bis?2-aminophenoxy?thane-N,N,N',N'-tetraacetic acid (10 microM) abolished fMLP-mediated Ca(2+) signaling, but had no effect on fMLP-induced AA release. The protein kinase C-inhibitor Ro 318220 (5 microM) or the inhibitor of cPLA(2) arachidonyl trifluoromethyl ketone (AACOCF(3); 10-30 microM) did not inhibit fMLP-induced AA release. In contrast, AA release was stimulated by the Ca(2+) ionophore A23187 (10 microM) plus the PKC activator phorbol myristate acetate (PMA) (0.2 microM). This effect was inhibited by either Ro 318220 or AACOCF(3). Accordingly, a translocation of cPLA(2) from the cytosol to the membrane fraction was observed with A23187 + PMA, but not with fMLP. fMLP-mediated AA release therefore appeared to be independent of Ca(2+) signaling and PKC and MAP kinase activation. However, fMLP-mediated AA release was reduced by approximately 45% by Clostridium difficile toxin B (10 ng/ml) or by 1-butanol; both block phospholipase D (PLD) activity. The inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), D609 (100 microM), decreased fMLP-mediated AA release by approximately 35%. The effect of D609 + 1-butanol on fMLP-induced AA release was additive and of a magnitude similar to that of propranolol (0.2 mM), an inhibitor of phosphatidic acid phosphohydrolase. This suggests that the bulk of AA generated by fMLP stimulation of db-cAMP-differentiated HL-60 cells is independent of the cPLA(2) pathway, but may originate from activation of PC-PLC and PLD.     

Group IV cytosolic phospholipase A2 mediates arachidonic acid release in H9c2 rat cardiomyocyte cells in response to hydrogen peroxide

Damaging reactive oxygen species are released during episodes of ischemia and reperfusion. Some cellular adaptive responses are triggered to protect the injured organ, while other cascades are triggered which potentiate the damage. In these studies, we demonstrate that rat cardiomyocte H9c2 cells release arachidonic acid in response to hydrogen peroxide. In H9c2 cells, arachidonic acid release is attenuated by methyl arachidonyl fluorophosphonate (MAFP) and pyrrophenone, indicating that a phospholipase A2 Group IV enzyme mediates arachidonic acid mobilization. Moreover, hydrogen peroxide alters the cellular morphology of the H9c2 cells, causing drastic cell shrinkage. Because MAFP and pyrrophenone prevent the morphological alterations caused by hydrogen peroxide, these studies show that phospholipase A2 Group IV activity is likely integrally involved in the damage initiated by hydrogen peroxide.     

Downregulation of nitric oxide formation by cytosolic phospholipase A2-released arachidonic acid

Exposure of PC12 cells to A23187 or thapsigargin caused a concentration-dependent release of arachidonic acid (AA) mediated by cytosolic phospholipase A2 (PLA2). Under the same conditions, however, analysis of nitric oxide (NO) formation revealed that activation of NO synthase (NOS) is best described by a bell-shaped curve. Reduced detection of NO observed at increasing A23187 or thapsigargin concentrations was not due to formation of peroxynitrite or to activation of NO-consuming processes, but rather to AA-dependent inhibition of NOS activity. Furthermore, NO formation observed under optimal conditions for NOS activity was suppressed by AA as well as by the PLA2 activator melittin. Finally, the effects of AA were not the consequence of direct enzyme inhibition, because this lipid messenger failed to inhibit formation of NO by purified neuronal NOS, but were mediated by an AA-dependent signaling and not by downstream products of the cyclooxygenase and lipoxygenase pathways. In conclusion, the present study underscores a novel mechanism whereby endogenous, or exogenous, AA promotes inhibition of NOS activity. Because AA is generated in response to various agonists acting on membrane receptors and extensively released in inflammatory conditions, these findings have important physiopathological implications.     

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).     

Kinetics of phospholipase A2, arachidonic acid, and eicosanoid appearance in mouse zymosan peritonitis

Intraperitoneal injection of zymosan into mice induces a peritonitis characterized by cellular influx, plasma leakage and the appearance of arachidonic acid (AA) metabolites. We report that zymosan injection also stimulates the accumulation of AA, docosahexaenoic acid, linoleic acid, and phospholipase A2 (PLA2) activity. The amount of the unsaturated fatty acids (UnFA) varies both with the zymosan dose and time. Significantly increased levels of UnFA were first detected 15 min after zymosan injection. Maximal levels of the UnFA were reached 1 to 2 h post zymosan injection (AA: 725 +/- 29 ng/mouse, docosahexaenoic acid: 296 +/- 23 ng/mouse, linoleic acid: 4489 +/- 179 ng/mouse) and declined to saline control levels by 8 h. PLA2 activity was significantly increased 5 to 15 min after zymosan injection. Maximal levels of PLA2 activity occurred 15 to 30 min after zymosan injection (31.8 +/- 9.1 nmol phospholipid/mg protein/h) and then decreased by 30% through 24 h. Neither the appearance of UnFA nor PLA2 activity correlated with cellular influx, but both were coincident with plasma exudation at 5 to 15 min after zymosan. However, maximal exudation occurred 1 to 2 h post zymosan injection similar to that seen with the UnFA but not PLA2. These latter results suggest that a significant portion of the UnFA found in the peritoneal cavity of zymosan-injected mice originates from the plasma. PLA2 activity at the early time points (5 to 15 min) may also contribute to the levels of UnFA via hydrolysis of tissue and/or cellular phospholipids.     

Group X secretory phospholipase A2 enhances TLR4 signaling in macrophages

Secretory phospholipase A(2)s (sPLA(2)) hydrolyze glycerophospholipids to liberate lysophospholipids and free fatty acids. Although group X (GX) sPLA(2) is recognized as the most potent mammalian sPLA(2) in vitro, its precise physiological function(s) remains unclear. We recently reported that GX sPLA(2) suppresses activation of the liver X receptor in macrophages, resulting in reduced expression of liver X receptor-responsive genes including ATP-binding cassette transporters A1 (ABCA1) and G1 (ABCG1), and a consequent decrease in cellular cholesterol efflux and increase in cellular cholesterol content (Shridas et al. 2010. Arterioscler. Thromb. Vasc. Biol. 30: 2014-2021). In this study, we provide evidence that GX sPLA(2) modulates macrophage inflammatory responses by altering cellular cholesterol homeostasis. Transgenic expression or exogenous addition of GX sPLA(2) resulted in a significantly higher induction of TNF-α, IL-6, and cyclooxygenase-2 in J774 macrophage-like cells in response to LPS. This effect required GX sPLA(2) catalytic activity, and was abolished in macrophages that lack either TLR4 or MyD88. The hypersensitivity to LPS in cells overexpressing GX sPLA(2) was reversed when cellular free cholesterol was normalized using cyclodextrin. Consistent with results from gain-of-function studies, peritoneal macrophages from GX sPLA(2)-deficient mice exhibited a significantly dampened response to LPS. Plasma concentrations of inflammatory cytokines were significantly lower in GX sPLA(2)-deficient mice compared with wild-type mice after LPS administration. Thus, GX sPLA(2) amplifies signaling through TLR4 by a mechanism that is dependent on its catalytic activity. Our data indicate this effect is mediated through alterations in plasma membrane free cholesterol and lipid raft content.     

Vitamin E up-regulates arachidonic acid release and phospholipase A2 in megakaryocytes

The alteration in calcium transport in the liver nuclei of rats orally administered carbon tetrachloride (CCl4) was investigated. Rats received a single oral administration of CCl₄(5, 10, and 25%, 1.0ml/100 g body weight), and 5, 24 and 48 h later the animals were sacrificed. The administration of CCl₄ (25%) caused a remarkable elevetion of calcium content in the liver tissues and the nuclei of rats. Liver nuclear Ca2+-ATPase activity was markedly decreased by CCl₄ (25%) administration. The presence of dibutyryl cyclic AMP(10^-4 and 10^-3 M) or inositol 1,4,5-trisphosphate (10^-6 and 10^-5 M) in the enzyme reaction mixture caused a significant decrease in Ca²⁺-ATPase activity in the liver nuclei obtained from normal rat, while the enzyme activity was significantly increased by calmodulin (1.0 and 2.0 g/ml). These signaling factor's effects were completely impaired in the liver nuclei obtained from CCl₄ (25%)-administered rats. DNA fragmentation in the liver nuclei obtained from CCl₄ -administered rats was significantly decreased by the presence of EGTA (2 mM) in the reaction mixture, suggesting that the endogenous calcium activates nuclear DNA fragmentation. The present study demonstrates that calcium transport system in the liver nuclei is impaired by liver injury with CCl₄ administration in rats.     

Amyloid beta enhances cytosolic phospholipase A2 level and arachidonic acid release via nitric oxide in APP-transfected PC12 cells

Cytosolic phospholipase A2 (cPLA2) preferentially liberates arachidonic acid (AA), which is known to be elevated in Alzheimer's disease (AD). The aim of this study was to investigate the possible relationship between enhanced nitric oxide (NO) generation observed in AD and cPLA2 protein level, phosphorylation, and AA release in rat pheochromocytoma cell lines (PC12) differing in amyloid beta secretion. PC12 control cells, PC12 cells bearing the Swedish double mutation in amyloid beta precursor protein (APPsw), and PC12 cells transfected with human APP (APPwt) were used. The transfected APPwt and APPsw PC12 cells showed an about 2.8- and 4.8-fold increase of amyloid beta (Abeta) secretion comparing to control PC12 cells. An increase of NO synthase activity, cGMP and free radical levels in APPsw and APPwt PC12 cells was observed. cPLA2 protein level was higher in APPsw and APPwt PC12 cells comparing to PC12 cells. Moreover, phosphorylated cPLA2 protein level and [3H]AA release were also higher in APP-transfected PC12 cells than in the control PC12 cells. An NO donor, sodium nitroprusside, stimulated [3H]AA release from prelabeled cells. The highest NO-induced AA release was observed in control PC12 cells, the effect in the other cell lines being statistically insignificant. Inhibition of cPLA2 by AACOCF3 significantly decreased the AA release. Inhibitors of nNOS and gamma-secretase reduced AA release in APPsw and APPwt PC12 cells. The basal cytosolic [Ca2+](i) and mitochondrial Ca2+ concentration was not changed in all investigated cell lines. Stimulation with thapsigargin increased the cytosolic and mitochondrial Ca2+ level, activated NOS and stimulated AA release in APP-transfected PC12 cells. These results indicate that Abeta peptides enhance the protein level and phosphorylation of cPLA2 and AA release by the NO signaling pathway.     

Ceramide binds to the CaLB domain of cytosolic phospholipase A2 and facilitates its membrane docking and arachidonic acid release.

Excessive production of eicosanoids is characteristic of many inflammatory diseases. In this study we show that ceramide, which is an early messenger of inflammatory cytokine action, exerts a dual effect on the cytosolic phospholipase A2 (cPLA2), the rate-limiting enzyme in arachidonic acid release and subsequent eicosanoid formation. Stimulation of renal mesangial cells with exogenous short-chain ceramide analogs for 30 and 60 min leads to a concentration-dependent increase in arachidonic acid release that is not blocked by specific inhibitors of mitogen-activated protein kinase pathways. This suggests that these established upstream activators of cPLA2 are not involved in ceramide-induced arachidonic acid release. By use of photoactivatable ceramide analogs, D- and L-[125I]3-trifluoromethyl-3-(m-iodophenyl)diazirine-ceramides (TID-ceramides), we observed a direct interaction of ceramide with cPLA2. This interaction was independent of the absolute configuration as D- and L-TID-ceramide were equally effective in binding to cPLA2. Moreover, recombinant CaLB domain of cPLA2 as well as a mutant deficient in the connecting 'hinge' domain of cPLA2, efficiently bound D- and L-TID-ceramides, whereas the catalytic domain did not interact with TID-ceramides. In vitro binding assays reveal that stearoyl-arachidonyl-phosphatidylcholine (SAPC)-liposomes containing increasing mol% of ceramide lead to an increased association of recombinant cPLA2 to the liposomes. Furthermore, measurement of cPLA2 activity in vitro shows that the presence of SAPC-liposomes resulted in only weak cPLA2 activity. However, the activity dramatically increases by addition of ceramide to the liposomes. Furthermore, liposomes containing SAPC and sphingomyelin resulted in no better substrate than SAPC liposomes, unless bacterial sphingomyelinase was added to generate ceramide, which then causes a marked increase in cPLA2 activity. These results demonstrate that ceramide can interact directly with cPLA2 via the CaLB domain and thereby serves as a membrane-docking device that facilitates cPLA2 action in inflammatory diseases.     

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.     

Type II phospholipase A2 recombinant overexpression enhances stimulated arachidonic acid release

The coding sequence of type II phospholipase A2 from human placenta was cloned in a bovine papilloma virus-derived eukaryotic expression vector under the control of the metallothionein promoter. Stably transfected C127 mouse fibroblast lines were obtained with this vector. These transfected cells overexpressed a functional 14 kDa phospholipase A2, which was bulky secreted. However, a significant phospholipase A2 activity was measured in cell homogenates. The involvement of this 14 kDa phospholipase A2 in mechanisms related to stimulated arachidonic acid release was investigated. We could parallel the overexpression of phospholipase A2 with an increase in phorbol ester and fluoroaluminate-stimulated arachidonic acid release. Pertussis toxin inhibited this stimulation. These results suggest that the 14 kDa type II phospholipase A2 might contribute to stimulation of arachidonic acid release, and therefore to eicosanoid production.     

Hyperforin induces Ca-independent arachidonic acid release in human platelets by facilitating cytosolic phospholipase A2 activation through select phospholipid interactions

Here, we investigated the modulation of cytosolic phospholipase A₂ (cPLA₂)-mediated arachidonic acid (AA) release by the polyprenylated acylphloroglucinol hyperforin. Hyperforin increased AA release from human platelets up to 2.6 fold (maximal effect at 10 µM) versus unstimulated cells, which was blocked by cPLA₂α-inhibition, and induced translocation of cPLA₂ to a membrane compartment. Interestingly, these stimulatory effects of hyperforin were even more pronounced after depletion of intracellular Ca²⁺ by EDTA plus BAPTA/AM. Hyperforin induced phosphorylation of cPLA₂ at Ser505 and activated p38 mitogen-activated protein kinase (MAPK), and inhibition of p38 MAPK by SB203580 prevented cPLA₂ phosphorylation. However, neither AA release nor translocation of cPLA₂ was abrogated by SB203580. In cell-free assays using liposomes prepared from different lipids, hyperforin failed to stimulate phospholipid hydrolysis by isolated cPLA₂ in the presence of Ca²⁺. However, when Ca²⁺ was omitted, hyperforin caused a prominent increase in cPLA₂ activity using liposomes composed of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphoethanolamine but not of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine (PAPC) unless the PAPC liposomes were enriched in cholesterol (20 to 50%). Finally, two-dimensional ¹H-MAS-NMR analysis visualized the directed insertion of hyperforin into POPC liposomes. Together, hyperforin, through insertion into phospholipids, may facilitate cPLA₂ activation by enabling its access towards select lipid membranes independent of Ca²⁺ ions. Such Ca²⁺- and phosphorylation-independent mechanism of cPLA₂ activation may apply also to other membrane-interfering molecules.