Phospholipase A2, reactive oxygen species, and lipid peroxidation in cerebral ischemia

Ischemic stroke is caused by obstruction of blood flow to the brain, resulting in energy failure that initiates a complex series of metabolic events, ultimately causing neuronal death. One such critical metabolic event is the activation of phospholipase A2 (PLA2), resulting in hydrolysis of membrane phospholipids and release of free fatty acids including arachidonic acid, a metabolic precursor for important cell-signaling eicosanoids. PLA2 enzymes have been classified as calcium-dependent cytosolic (cPLA2) and secretory (sPLA2) and calcium-independent (iPLA2) forms. Cardiolipin hydrolysis by mitochondrial sPLA2 disrupts the mitochondrial respiratory chain and increases production of reactive oxygen species (ROS). Oxidative metabolism of arachidonic acid also generates ROS. These two processes contribute to formation of lipid peroxides, which degrade to reactive aldehyde products (malondialdehyde, 4-hydroxynonenal, and acrolein) that covalently bind to proteins/nucleic acids, altering their function and causing cellular damage. Activation of PLA2 in cerebral ischemia has been shown while other studies have separately demonstrated increased lipid peroxidation. To the best of our knowledge no study has directly shown the role of PLA2 in lipid peroxidation in cerebral ischemia. To date, there are very limited data on PLA2 protein by Western blotting after cerebral ischemia, though some immunohistochemical studies (for cPLA2 and sPLA2) have been reported. Dissecting the contribution of PLA2 to lipid peroxidation in cerebral ischemia is challenging due to multiple forms of PLA2, cardiolipin hydrolysis, diverse sources of ROS arising from arachidonic acid metabolism, catecholamine autoxidation, xanthine oxidase activity, mitochondrial dysfunction, activated neutrophils coupled with NADPH oxidase activity, and lack of specific inhibitors. Although increased activity and expression of various PLA2 isoforms have been demonstrated in stroke, more studies are needed to clarify the cellular origin and localization of these isoforms in the brain, their responses in cerebral ischemic injury, and their role in oxidative stress.     

Secretory phospholipase A2 mediates cooperative prostaglandin generation by growth factor and cytokine independently of preceding cytosolic phospholipase A2 expression in rat gastric epithelial cells

Transforming growth factor (TGF)-alpha and interleukin (IL)-1beta are responsible for the healing of gastric lesions through, in part, prostaglandin (PG) generation. We examined the contribution of cytosolic and secretory phospholipase A(2)s (cPLA(2) and sPLA(2)) to the PG generation by rat gastric epithelial cells in response to both stimuli. Stimulation with TGF-alpha for 24 h increased cPLA(2) and cyclooxygenase (COX)-2 markedly, PGE(2) slightly, and type IIA sPLA(2) and COX-1 not at all, whereas IL-1beta increased sPLA(2) only. Both stimuli synergistically increased PGE(2), sPLA(2), and the two COXs but not cPLA(2). The onset of the PGE(2) generation paralleled the sPLA(2) release but was apparently preceded by increases in cPLA(2) and the two COXs. The increase in PGE(2) was impaired by inhibitors for sPLA(2) and COX-2 but not COX-1. cPLA(2) inhibitors suppressed PGE(2) generation by TGF-alpha alone but not augmentation of PGE(2) generation or sPLA(2) release by IL-1beta in combination with TGF-alpha. Furthermore, despite an increase in cPLA(2) including its phosphorylated form (phosphoserine), -induced arachidonic acid liberation was impaired in the TGF-alpha/IL-1beta-stimulated cells, in which p11, a putative cPLA(2) inhibitory molecule, was also increased and co-immunoprecipitated with cPLA(2). These results suggest that synergistic stimulation of sPLA(2) and COX-2 expression by TGF-alpha and IL-1beta results in an increase in PGE(2). Presumably, the preceding cPLA(2) expression is not involved in the PGE(2) generation, because of impairment of its hydrolytic activity in the stimulated cells.     

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 delayed prostaglandin production in activated P388D1 macrophages by group IV cytosolic and group V secretory phospholipase A2s

Group V secretory phospholipase A2 (sPLA2) rather than Group IIA sPLA2 is involved in short term, immediate arachidonic acid mobilization and prostaglandin E2 (PGE2) production in the macrophage-like cell line P388D1. When a new clone of these cells, P388D1/MAB, selected on the basis of high responsivity to lipopolysaccharide plus platelet-activating factor, was studied, delayed PGE2 production (6-24 h) in response to lipopolysaccharide alone occurred in parallel with the induction of Group V sPLA2 and cyclooxygenase-2 (COX-2). No changes in the level of cytosolic phospholipase A2 (cPLA2) or COX-1 were observed, and Group IIA sPLA2 was not detectable. Use of a potent and selective sPLA2 inhibitor, 3-(3-acetamide 1-benzyl-2-ethylindolyl-5-oxy)propanesulfonic acid (LY311727), and an antisense oligonucleotide specific for Group V sPLA2 revealed that delayed PGE2 was largely dependent on the induction of Group V sPLA2. Also, COX-2, not COX-1, was found to mediate delayed PGE2 production because the response was completely blocked by the specific COX-2 inhibitor NS-398. Delayed PGE2 production and Group V sPLA2 expression were also found to be blunted by the inhibitor methylarachidonyl fluorophosphonate. Because inhibition of Ca2+-independent PLA2 by an antisense technique did not have any effect on the arachidonic acid release, the data using methylarachidonyl fluorophosphonate suggest a key role for the cPLA2 in the response as well. Collectively, the results suggest a model whereby cPLA2 activation regulates Group V sPLA2 expression, which in turn is responsible for delayed PGE2 production via COX-2.     

Group X secretory phospholipase A2 can induce arachidonic acid release and eicosanoid production without activation of cytosolic phospholipase A2 alpha

Group X secretory phospholipase A2 (sPLA2-X) and cytosolic phospholipase A2 alpha (cPLA2alpha) are involved in the release of arachidonic acid (AA) from membrane phospholipids linked to the eicosanoid production in various pathological states. Recent studies have indicated the presence of various types of cross-talk between sPLA2s and cPLA2alpha resulting in effective AA release. Here we examined the dependence of sPLA2-X-induced potent AA release on the cPLA2alpha activation by using specific cPLA2alpha or sPLA2 inhibitors as well as cPLA2alpha-deficient mice. We found that Pyrrophenone, a cPLA2alpha-specific inhibitor, did not suppress the sPLA2-X-induced potent AA release and prostaglandin E2 formation in mouse spleen cells. Furthermore, the amount of AA released by sPLA2-X from spleen cells was not significantly altered by cPLA2alpha deficiency. These results suggest that sPLA2-X induces potent AA release without activation of cPLA2a, which might be relevant to eicosanoid production in some pathological states where cPLA2a is not activated.     

Type IIA secretory phospholipase A2 up-regulates cyclooxygenase-2 and amplifies cytokine-mediated prostaglandin production in human rheumatoid synoviocytes

Human type IIA secretory phospholipase A2 (sPLA2-IIA) is induced in association with several immune-mediated inflammatory conditions. We have evaluated the effect of sPLA2-IIA on PG production in primary synovial fibroblasts from patients with rheumatoid arthritis (RA). At concentrations found in the synovial fluid of RA patients, exogenously added sPLA2-IIA dose-dependently amplified TNF-alpha-stimulated PGE2 production by cultured synovial fibroblasts. Enhancement of TNF-alpha-stimulated PGE2 production in synovial cells was accompanied by increased expression of cyclooxygenase (COX)-2 and cytosolic phospholipase A2 (cPLA2)-alpha. Blockade of COX-2 enzyme activity with the selective inhibitor NS-398 prevented both TNF-alpha-stimulated and sPLA2-IIA-amplified PGE2 production without affecting COX-2 protein induction. However, both sPLA2-IIA-amplified PGE2 production and enhanced COX-2 expression were blocked by the sPLA2 inhibitor LY311727. Colocalization studies using triple-labeling immunofluorescence microscopy showed that sPLA2-IIA and cPLA2-alpha are coexpressed with COX-2 in discrete populations of CD14-positive synovial macrophages and synovial tissue fibroblasts from RA patients. Based on these findings, we propose a model whereby the enhanced expression of sPLA2-IIA by RA synovial cells up-regulates TNF-alpha-mediated PG production via superinduction of COX-2. Therefore, sPLA2-IIA may be a critical modulator of cytokine-mediated synovial inflammation in RA.     

Secretory phospholipase A2 receptor-mediated activation of cytosolic phospholipase A2 in murine bone marrow-derived mast cells

The current study examined the signal transduction steps involved in the selective release of arachidonic acid (AA) induced by the addition of secretory phospholipase A2 (sPLA2) isotypes to bone marrow-derived mast cells (BMMC). Overexpression of sPLA2 receptors caused a marked increase in AA and PGD2 release after stimulation of BMMC, implicating sPLA2 receptors in this process. The hypothesis that the release of AA by sPLA2 involved activation of cytosolic PLA2 (cPLA2) was next tested. Addition of group IB PLA2 to BMMC caused a transient increase in cPLA2 activity and translocation of this activity to membrane fractions. Western analyses revealed that these changes in cPLA2 were accompanied by a time-dependent gel shift of cPLA2 induced by phosphorylation of cPLA2 at various sites. A noncatalytic ligand of the sPLA2 receptor, p-amino-phenyl-alpha-D-mannopyranoside BSA, also induced an increase in cPLA2 activity in BMMC. sPLA2 receptor ligands induced the phosphorylation of p44/p42 mitogen-activated protein kinase. Additionally, an inhibitor of p44/p42 mitogen-activated protein kinase (PD98059) significantly inhibited sPLA2-induced cPLA2 activation and AA release. sPLA2 receptor ligands also increased Ras activation while an inhibitor of tyrosine phosphorylation (herbimycin) inhibited the increase in cPLA2 activation and AA release. Addition of partially purified sPLA2 from BMMC enhanced cPLA2 activity and AA release. Similarly, overexpression of mouse groups IIA or V PLA2 in BMMC induced an increase in AA release. These data suggest that sPLA2 mediate the selective release of AA by binding to cell surface receptors and then inducing signal transduction events that lead to cPLA2 activation.     

Dietary n-6 polyunsaturated fatty acid deprivation increases docosahexaenoic acid metabolism in rat brain

Dietary n-6 polyunsaturated fatty acid (PUFA) deprivation in rodents reduces brain arachidonic acid (20:4n-6) concentration and 20:4n-6-preferring cytosolic phospholipase A(2) (cPLA(2) -IVA) and cyclooxygenase (COX)-2 expression, while increasing brain docosahexaenoic acid (DHA, 22:6n-3) concentration and DHA-selective calcium-independent phospholipase A(2) (iPLA(2) )-VIA expression. We hypothesized that these changes are accompanied by up-regulated brain DHA metabolic rates. Using a fatty acid model, brain DHA concentrations and kinetics were measured in unanesthetized male rats fed, for 15 weeks post-weaning, an n-6 PUFA 'adequate' (31.4 wt% linoleic acid) or 'deficient' (2.7 wt% linoleic acid) diet, each lacking 20:4n-6 and DHA. [1-(14) C]DHA was infused intravenously, arterial blood was sampled, and the brain was microwaved at 5 min and analyzed. Rats fed the n-6 PUFA deficient compared with adequate diet had significantly reduced n-6 PUFA concentrations in brain phospholipids but increased eicosapentaenoic acid (EPA, 20:5n-3), docosapentaenoic acid n-3 (DPAn-3, 22:5n-3), and DHA (by 9.4%) concentrations, particularly in ethanolamine glycerophospholipid (EtnGpl). Incorporation rates of unesterified DHA from plasma, which represent DHA metabolic loss from brain, were increased 45% in brain phospholipids, as was DHA turnover. Increased DHA metabolism following dietary n-6 PUFA deprivation may increase brain concentrations of antiinflammatory DHA metabolites, which with a reduced brain n-6 PUFA content, likely promotes neuroprotection and alters neurotransmission.     

Amplification mechanisms of inflammation: paracrine stimulation of arachidonic acid mobilization by secreted phospholipase A2 is regulated by cytosolic phospholipase A2-derived hydroperoxyeicosatetraenoic acid

In macrophages and other major immunoinflammatory cells, two phospholipase A(2) (PLA(2)) enzymes act in concert to mobilize arachidonic acid (AA) for immediate PG synthesis, namely group IV cytosolic phospholipase A(2) (cPLA(2)) and a secreted phospholipase A(2) (sPLA(2)). In this study, the molecular mechanism underlying cross-talk between the two PLA(2)s during paracrine signaling has been investigated. U937 macrophage-like cells respond to Con A by releasing AA in a cPLA(2)-dependent manner, and addition of exogenous group V sPLA(2) to the activated cells increases the release. This sPLA(2) effect is abolished if the cells are pretreated with cPLA(2) inhibitors, but is restored by adding exogenous free AA. Inhibitors of cyclooxygenase and 5-lipoxygenase have no effect on the response to sPLA(2). In contrast, ebselen strongly blocks it. Reconstitution experiments conducted in pyrrophenone-treated cells to abolish cPLA(2) activity reveal that 12- and 15-hydroperoxyeicosatetraenoic acid (HPETE) are able to restore the sPLA(2) response to levels found in cells displaying normal cPLA(2) activity. Moreover, 12- and 15-HPETE are able to enhance sPLA(2) activity in vitro, using a natural membrane assay. Neither of these effects is mimicked by 12- or 15-hydroxyeicosatetraenoic acid, indicating that the hydroperoxy group of HPETE is responsible for its biological activity. Collectively, these results establish a role for 12/15-HPETE as an endogenous activator of sPLA(2)-mediated phospholipolysis during paracrine stimulation of macrophages and identify the mechanism that connects sPLA(2) with cPLA(2) for a full AA mobilization response.     

Disturbed brain phospholipid and docosahexaenoic acid metabolism in calcium-independent phospholipase A(2)-VIA (iPLA(2)β)-knockout mice

Calcium-independent phospholipase A(2) group VIA (iPLA(2)β) releases docosahexaenoic acid (DHA) from phospholipids in vitro. Mutations in the iPLA(2)β gene, PLA2G6, are associated with dystonia-parkinsonism and infantile neuroaxonal dystrophy. To understand the role of iPLA(2)β in brain, we applied our in vivo kinetic method using radiolabeled DHA in 4 to 5-month-old wild type (iPLA(2)β(+/+)) and knockout (iPLA(2)β(-/-)) mice, and measured brain DHA kinetics, lipid concentrations, and expression of PLA(2), cyclooxygenase (COX), and lipoxygenase (LOX) enzymes. Compared to iPLA(2)β(+/+) mice, iPLA(2)β(-/-) mice showed decreased rates of incorporation of unesterified DHA from plasma into brain phospholipids, reduced concentrations of several fatty acids (including DHA) esterified in ethanolamine- and serine-glycerophospholipids, and increased lysophospholipid fatty acid concentrations. DHA turnover in brain phospholipids did not differ between genotypes. In iPLA(2)β(-/-) mice, brain levels of iPLA(2)β mRNA, protein, and activity were decreased, as was the iPLA(2)γ (Group VIB PLA(2)) mRNA level, while levels of secretory sPLA(2)-V mRNA, protein, and activity and cytosolic cPLA(2)-IVA mRNA were increased. Levels of COX-1 protein were decreased in brain, while COX-2 protein and mRNA were increased. Levels of 5-, 12-, and 15-LOX proteins did not differ significantly between genotypes. Thus, a genetic iPLA(2)β deficiency in mice is associated with reduced DHA metabolism, profound changes in lipid-metabolizing enzyme expression (demonstrating lack of redundancy) and of phospholipid fatty acid content of brain (particularly of DHA), which may be relevant to neurologic abnormalities in humans with PLA2G6 mutations.     

GTP-binding protein mediated phospholipase A2 activation in rat liver during the progression of sepsis

Effects of GTP-binding proteins on the activation of secretory phospholipase A2 (sPLA2) and cytosolic phospholipase A2 (cPLA2) in rat liver during two different phases of sepsis were studied. Sepsis was induced by cecal ligation and puncture (CLP). Experiments were divided into three groups: control, early sepsis, and late sepsis. Early and late sepsis refers to those animals sacrificed at 9 and 18 h, respectively, after CLP. The results show that in the absence of G-protein modulator, hepatic sPLA2 and cPLA2 activities were activated by 40.8-46 and 91.6-105.8%, respectively, during early and late phases of sepsis. GTPS and fluoroaluminate (A1F4-) stimulated sPLA2 and cPLA2 activities within each experimental group, i.e., control, early sepsis, and late sepsis. The GTPS and A1F4--stimulated sPLA2 and cPLA2 activities remained significantly elevated during early phase (22.3-65.6% increase) and late phase (32.5-109.1% increase) of sepsis. Further analyses demonstrate that cholera toxin significantly stimulated sPLA2 and cPLA2 activities within each experimental group, and that the cholera toxin stimulated sPLA2 and cPLA2 activities remained significantly higher during early phase (23.5-37%increase) and late phase (56.7-70% increase) of sepsis. In contrast, pertussis toxin significantly inhibited sPLA2 and cPLA2 activities within each experimental group, and that the pertussis toxin-inhibited sPLA2 and cPLA2 activities remained significantly higher in early septic (57-68.5% increase) and late septic (34.6-45.5% increase) experiments. These data demonstrate that cholera toxin-sensitive Gs and pertussis toxin-sensitive Gi were both involved in the activation of sPLA2 and cPLA2 activities in rat liver during the progression of sepsis.     

Flavonoids differentially inhibit guinea pig epidermal cytosolic phospholipase A2

Cytosolic phospholipase A2 (cPLA2) is believed to involve the regulation of essential cellular processes. Like other cell types, epidermal cPLA2 may participate in various metabolic processes including eicosanoid generation. In this investigation, we demonstrated the presence of cPLA2 in guinea pig epidermis. The epidermal cPLA2 is Ca2+-dependent, active at micromolar concentration of Ca2+ and resistant to disulfide-reducing agents. Furthermore, it is inhibited by methyl arachidonyl fluorophosphonate (MAFP), a selective inhibitor of cPLA2, while 12-epi-scalardial (a sPLA2 inhibitor) did not cause inhibition. A test of several flavonoids revealed that quercetin (flavonol) weakly inhibited cPLA2, while flavanone had negligible inhibitory activity. In contrast, amentoflavone and ginkgetin (biflavones) markedly inhibited cPLA2 activity in the epidermis. These results underscore that different flavonoids do vary in their capability to exert differential effects on arachidonate metabolism in the skin via modulation of epidermal cPLA2 activity.     

Cytosolic phospholipase A2alpha regulates induction of brain cyclooxygenase-2 in a mouse model of inflammation

The products of arachidonic acid metabolism are key mediators of inflammatory responses in the central nervous system, and yet we do not know the mechanisms of their regulation. The phospholipase A(2) enzymes are sources of cellular arachidonic acid, and the enzymes cyclooxygenase-2 (COX-2) and microsomal PGE synthase-1 (mPGES-1) are essential for the synthesis of inflammatory PGE(2) in the brain. These studies seek to determine the function of cytosolic phospholipase A(2)alpha (cPLA(2)alpha) in inflammatory PGE(2) production in the brain. We wondered whether cPLA(2)alpha functions in inflammation to produce arachidonic acid or to modulate levels of COX-2 or mPGES-1. We investigated these questions in the brains of wild-type mice and mice deficient in cPLA(2)alpha (cPLA(2)alpha(-/-)) after systemic administration of LPS. cPLA(2)alpha(-/-) mice had significantly less brain COX-2 mRNA and protein expression in response to LPS than wild-type mice. The reduction in COX-2 was most apparent in the cells of the cerebral blood vessels and the leptomeninges. The brain PGE(2) concentration of untreated cPLA(2)alpha(-/-) mice was equal to their wild-type littermates. After LPS treatment, however, the brain concentration of PGE(2) was significantly less in cPLA(2)alpha(-/-) than in cPLA(2)alpha(+/+) mice (24.4 +/- 3.8 vs. 49.3 +/- 11.6 ng/g). In contrast to COX-2, mPGES-1 RNA levels increased equally in both mouse genotypes, and mPGES-1 protein was unaltered 6 h after LPS. We conclude that cPLA(2)alpha regulates COX-2 levels and modulates inflammatory PGE(2) levels. These results indicate that cPLA(2)alpha inhibition is a novel anti-inflammatory strategy that modulates, but does not completely prevent, eicosanoid responses.     

Cellular components that functionally interact with signaling phospholipase A(2)s

Accumulating evidence has suggested that cytosolic phospholipase A(2) (cPLA(2)) and several secretory PLA(2) (sPLA(2)) isozymes are signaling PLA(2)s that are functionally coupled with downstream cyclooxygenase (COX) isozymes for prostaglandin (PG) biosynthesis. Arachidonic acid (AA) released by cPLA(2) and sPLA(2)s is supplied to both COX-1 and COX-2 in the immediate, and predominantly to COX-2 in the delayed, PG-biosynthetic responses. Vimentin, an intermediate filament component, acts as a functional perinuclear adapter for cPLA(2), in which the C2 domain of cPLA(2) associates with the head domain of vimentin in a Ca(2+)-sensitive manner. The heparin-binding signaling sPLA(2)-IIA, IID and V bind the glycosylphosphatidylinositol-anchored heparan sulfate proteoglycan glypican, which plays a role in sorting of these isozymes into caveolae and perinuclear compartments. Phospholipid scramblase, which facilitates transbilayer movement of anionic phospholipids, renders the cellular membranes more susceptible to signaling sPLA(2)s. There is functional cooperation between cPLA(2) and signaling sPLA(2)s in that prior activation of cPLA(2) is required for the signaling sPLA(2)s to act properly. cPLA(2)-derived AA is oxidized by 12/15-lipoxygenase, the products of which not only augment the induction of sPLA(2) expression, but also cause membrane perturbation, leading to increased cellular susceptibility to the signaling sPLA(2)s. sPLA(2)-X, a heparin-non-binding sPLA(2) isozyme, is capable of releasing AA from intact cells in the absence of cofactors. This property is attributed to its ability to avidly hydrolyze zwitterionic phosphatidylcholine, a major phospholipid in the outer plasma membrane. sPLA(2)-V can also utilize this route in several cell types. Taken together, the AA-releasing function of sPLA(2)s depends on the presence of regulatory cofactors and interfacial binding to membrane phospholipids, which differ according to cell type, stimuli, secretory processes, and subcellular distributions.     

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.     

sPLA(2) cooperates with cPLA(2)alpha to regulate prostacyclin synthesis in human endothelial cells

The first step in prostacyclin (PGI(2)) synthesis involves the generation of arachidonic acid (AA) from membrane phospholipids mediated by the 85 kDa cytosolic phospholipase A(2) (cPLA(2)alpha). The current study examined the effects of secretory PLA(2)s (sPLA(2)s) on PGI(2) production by human umbilical vein endothelial cells (HUVEC). We demonstrate that exposure of HUVEC to sPLA(2) dose- and time-dependently enhances AA release and PGI(2) generation. sPLA(2)-stimulated AA mobilisation was blocked by AACOCF(3), an inhibitor of cPLA(2)alpha, suggesting cross-talk between the two classes of PLA(2). sPLA(2) induced the phosphorylation of cPLA(2)alpha and enhanced the phosphorylation states of p42/44(mapk), p38(mapk), and JNK, concomitant with elevated AA and PGI(2) release. The MEK inhibitor PD98059 attenuated sPLA(2)-stimulated cPLA(2)alpha phosphorylation and PGI(2) release. These data show that sPLA(2) cooperates with cPLA(2)alpha in a MAPK-dependent manner to regulate PGI(2) generation and suggests that cross-talk between sPLA(2) and cPLA(2)alpha is a physiologically important mechanism for enhancing prostanoid production in endothelial cells.     

Cyclooxygenases-1 and 2 couple to cytosolic but not group IIA phospholipase A2 in COS-1 cells

Phospholipases A2 (PLA2) and cyclooxygenases (COX) are important enzymes responsible for production of potent lipid mediators, including prostaglandins (PG) and thromboxane A2. We investigated coupling between PLA2 and COX isoforms by using transient transfection in COS-1 cells. Untransfected cells, incubated with or without phorbol ester + the Ca2+ ionophore ionomycin, generated trivial amounts of PGE2. In cells co-transfected with cytosolic PLA2 (cPLA2) and COX-1 or COX-2, phorbol ester + ionomycin markedly stimulated PGE2 production. There was no preferential coupling of cPLA2 to either of the COX isoforms. In contrast, group IIA secretory PLA2 (sPLA2) co-transfected with COX-1 or COX-2 did not lead to an increase in PGE2 production, despite high levels of sPLA2 enzymatic activity. Transfection of cPLA2 did not affect basal free arachidonic acid (AA) levels. Phorbol ester + ionomycin stimulated release of AA in cPLA2-transfected COS-1 cells, but not in untransfected cells, whereas sPLA2 transfection (without stimulation) led to high basal free AA. Thus, AA released by cPLA2 is accessible to both COX isoforms for metabolism to PG, whereas AA released by sPLA2 is not metabolized by COX.