Roles of C-terminal processing, and involvement in transacylation reaction of human group IVC phospholipase A2 (cPLA2gamma)

The phospholipase A2s (PLA2s) are a diverse group of enzymes that hydrolyze the sn-2 fatty acid from phospholipids and play a role in a wide range of physiological functions. A 61-kDa calcium-independent PLA2, termed cPLA2gamma, was identified as an ortholog of cPLA2alpha with approximately 30% overall sequence identity. cPLA2gamma contains a potential prenylation motif at its C terminus, and is known to have PLA2 and lysophospholipase activities, but its physiological roles have not been clarified. In the present study, we expressed various forms of recombinant cPLA2gamma, including non-prenylated and non-cleaved forms, in order to investigate the effects of C-terminal processing. We examined the expression of the wild type and non-prenylated (SCLA) forms of cPLA2gamma, and found that the SCLA form was expressed normally and retained almost full activity. Expression of the prenylated and non-cleaved form of cPLA2gamma using yeast mutants lacking prenyl protein proteases AFC1 (a-factor-converting enzyme) and RCE1 (Ras-converting enzyme) revealed decreased expression in the mutant strain compared to that in the wild type yeast, suggesting that complete C-terminal processing is important for the functional expression of cPLA2gamma. In addition, cPLA2gamma was found to have coenzyme A (CoA)-independent transacylation and lysophospholipid (LPL) dismutase (LPLase/transacylase) activities, suggesting that it may be involved in fatty acid remodeling of phospholipids and the clearance of toxic lysophospholipids in cells.     

Phospholipase A2

Phospholipase A2 (PLA2) catalyzes the hydrolysis of the sn-2 position of membrane glycerophospholipids to liberate arachidonic acid (AA), a precursor of eicosanoids including prostaglandins (PGs) and leukotrienes (LTs). The same reaction also produces lysophosholipids, which represent another class of lipid mediators. So far, at least 19 enzymes that possess PLA2 activity have been identified in mammals. The secretory PLA2 (sPLA2) family, in which 10 isozymes have been identified, consists of low-molecular-weight, Ca2+-requiring, secretory enzymes that have been implicated in a number of biological processes, such as modification of eicosanoid generation, inflammation, host defense, and atherosclerosis. The cytosolic PLA2 (cPLA2) family consists of 3 enzymes, among which cPLA2alpha plays an essential role in the initiation of AA metabolism. Intracellular activation of cPLA2alpha is tightly regulated by Ca2+ and phosphorylation. The Ca2+-independent PLA2 (iPLA2) family contains 2 enzymes and may play a major role in membrane phospholipid remodeling. The platelet-activating factor (PAF) acetylhydrolase (PAF-AH) family represents a unique group of PLA2 that contains 4 enzymes exhibiting unusual substrate specificity toward PAF and/or oxidized phospholipids. In this review, we will overview current understanding of the properties and functions of each enzyme belonging to the sPLA2, cPLA2, and iPLA2 families, which have been implicated in signal transduction.     

Biochemical properties and pathophysiological roles of cytosolic phospholipase A2s

Phospholipase A(2) (PLA(2)) (EC 3.1.1.4) catalyzes hydrolysis of the sn-2 ester bond of glycerophospholipids. The enzyme is essential for the production of two classes of lipid mediators, fatty acid metabolites and lysophospholipid-related lipids, as well as being involved in the remodeling of membrane phospholipids. Among many mammalian PLA(2)s, cytosolic PLA(2)alpha (cPLA(2)alpha) plays a critical role in various physiological and pathophysiological conditions through generating lipid mediators. Here, we summarize the in vivo significance of cPLA(2)alpha, revealed from the phenotypes of cPLA(2)alpha-null mice, and properties of newly discovered cPLA(2) family enzymes. We also briefly introduce a quantitative lipidomics strategy using liquid chromatography-mass spectrometry, a powerful tool for the comprehensive analysis of lipid mediators.     

Mapping the phospholipid-binding surface and translocation determinants of the C2 domain from cytosolic phospholipase A2.

Cytosolic phospholipase A2 (cPLA2) plays a key role in the generation of arachidonic acid, a precursor of potent inflammatory mediators. Intact cPLA2 is known to translocate in a calcium-dependent manner from the cytosol to the nuclear envelope and endoplasmic reticulum. We show here that the C2 domain of cPLA2 alone is sufficient for this calcium-dependent translocation in living cells. We have identified sets of exposed hydrophobic residues in loops known as calcium-binding region (CBR) 1 and CBR3, which surround the C2 domain calcium-binding sites, whose mutation dramatically decreased phospholipid binding in vitro without significantly affecting calcium binding. Mutation of a residue that binds calcium ions (D43N) also eliminated phospholipid binding. The same mutations that prevent phospholipid binding of the isolated C2 domain in vitro abolished the calcium-dependent translocation of cPLA2 to internal membranes in vivo, suggesting that the membrane targeting is driven largely by direct interactions with the phospholipid bilayer. Using fluorescence quenching by spin-labeled phospholipids for a series of mutants containing a single tryptophan residue at various positions in the cPLA2 C2 domain, we show that two of the calcium-binding loops, CBR1 and CBR3, penetrate in a calcium-dependent manner into the hydrophobic core of the phospholipid bilayer, establishing an anchor for docking the domain onto the membrane.     

Distinct roles of two intracellular phospholipase A2s in fatty acid release in the cell death pathway. Proteolytic fragment of type IVA cytosolic phospholipase A2alpha inhibits stimulus-induced arachidonate release, whereas that of type VI Ca2+-independent phospholipase A2 augments spontaneous fatty acid release

Cytosolic phospholipase A(2)alpha (cPLA(2)alpha; type IVA), an essential initiator of stimulus-dependent arachidonic acid (AA) metabolism, underwent caspase-mediated cleavage at Asp(522) during apoptosis. Although the resultant catalytically inactive N-terminal fragment, cPLA(2)(1-522), was inessential for cell growth and the apoptotic process, it was constitutively associated with cellular membranes and attenuated both the A23187-elicited immediate and the interleukin-1-dependent delayed phases of AA release by several phospholipase A(2)s (PLA(2)s) involved in eicosanoid generation, without affecting spontaneous AA release by PLA(2)s implicated in phospholipid remodeling. Confocal microscopic analysis revealed that cPLA(2)(1-522) was distributed in the nucleus. Pharmacological and transfection studies revealed that Ca(2+)-independent PLA(2) (iPLA(2); type VI), a phospholipid remodeling PLA(2), contributes to the cell death-associated increase in fatty acid release. iPLA(2) was cleaved at Asp(183) by caspase-3 to a truncated enzyme lacking most of the first ankyrin repeat, and this cleavage resulted in increased iPLA(2) functions. iPLA(2) had a significant influence on cell growth or death, according to cell type. Collectively, the caspase-truncated form of cPLA(2)alpha behaves like a naturally occurring dominant-negative molecule for stimulus-induced AA release, rendering apoptotic cells no longer able to produce lipid mediators, whereas the caspase-truncated form of iPLA(2) accelerates phospholipid turnover that may lead to apoptotic membranous changes.     

Activation of cytosolic phospholipase A2 and fatty acid transacylase is essential but not sufficient for thrombin-induced smooth muscle cell proliferation

Thrombin is a potent stimulant of smooth muscle cell (SMC) proliferation in inflammatory conditions, leading to pathological thickening of vascular walls in atherosclerosis and airway remodeling in asthma. Cell proliferation requires the formation and remodeling of cell membrane phospholipids (PLs), involving the activation of PL-metabolizing enzymes. Yet, the role of specific PL-metabolizing enzymes in SMC proliferation has hardly been studied. To bridge this gap, in the present study, we investigated the role of key enzymes involved in PL metabolism, the PL-hydrolyzing enzyme phospholipase A2 (PLA2) and the PL-synthesizing enzyme lysophosphatidic acid-fatty acid transacylase (LPAAT), in thrombin-induced proliferation of bovine aortic SMCs (BASMCs). Concomitantly with the induction of BASMC proliferation, thrombin activated cytosolic PLA2 (cPLA2-alpha), expressed by selective release of arachidonic acid and mRNA expression, as well as LPAAT, expressed by nonselective incorporation of fatty acid and mRNA expression. Specific inhibitors of these enzymes, arachidonyl-trifluoromethyl-ketone for cPLA2 and thimerosal for LPAAT, suppressed their activities, concomitantly with suppression of BASMC proliferation, suggesting a mandatory requirement for cPLA2 and LPAAT activation in thrombin-induced SMC proliferation. Thrombin acts through the protease-activated receptor (PAR-1), and, accordingly, we found that thrombin-induced BASMC proliferation was suppressed by the PAR-1 inhibitor SCH-79797. However, the PAR-1 inhibitor did not prevent thrombin-induced mRNA expression of cPLA2 and LPAAT, implying that the activation of cPLA2 and LPAAT is essential but not sufficient for thrombin-induced proliferation of BASMCs.     

Differential mode of attack on membrane phospholipids by an acidic phospholipase A(2) (RVVA-PLA(2)-I) from Daboia russelli venom

An acidic phospholipase A(2) (RVVA-PLA(2)-I) purified from Daboia russelli venom demonstrated dose-dependent catalytic, mitochondrial and erythrocyte membrane damaging activities. RVVA-PLA(2)-I was non-lethal to mice at the tested dose, however, it affected the different organs of mice particularly the liver and cardiac tissues as deduced from the enzymatic activities measured in mice serum after injection of this PLA(2) enzyme. RVVA-PLA(2)-I preferentially hydrolyzed phospholipids (phosphatidylcholine) of erythrocyte membrane compared to the liver mitochondrial membrane. Interestingly, RVVA-PLA(2)-I failed to hydrolyze membrane phospholipids of HT-29 (colon adenocarcinoma) cells, which contain an abundance of phosphatidylcholine in its outer membrane, within 24h of incubation. The gas-chromatographic (GC) analysis of saturated/unsaturated fatty acids' release patterns from intact mitochondrial and erythrocyte membranes after the addition of RVVA-PLA(2)-I showed a distinctly different result. The results are certainly a reflection of differences in the outer membrane phospholipid composition of tested membranes owing to which they are hydrolyzed by the venom PLA(2)s to a different extent. The chemical modification of essential amino acids present in the active site, neutralization study with polyvalent antivenom and heat-inactivation of RVVA-PLA(2)-I suggested the correlation between catalytic and membrane damaging activities of this PLA(2) enzyme. Our study advocates that the presence of a large number of PLA(2)-sensitive phospholipid domains/composition, rather than only the phosphatidylcholine (PC) content of that particular membrane may determine the extent of membrane damage by a particular venom PLA(2) enzyme.     

Molecules in focus: cytosolic phospholipase A2-alpha

Cytosolic phospholipase A(2)-alpha (cPLA(2)-alpha) cleaves its preferred substrate, arachidonic acid, at the sn-2 position of membrane glycerophospholipids. Stimulation of cells with agents that mobilize intracellular calcium and/or promote the phosphorylation of cPLA(2)-alpha leads to (i) translocation of the enzyme from cytosol to endoplasmic reticulum, Golgi apparatus and perinuclear membranes-where it associates with the arachidonic acid in close proximity to downstream eicosanoid-producing enzymes; and (ii) the change in configuration induced by phosphorylation increases the phospholipid binding affinity and arachidonic acid release. As a mediator of growth factors, cytokines, chemokines, and hormones that modulate survival and growth in various cell types, cPLA(2)-alpha has attracted considerable attention as a potential therapeutic target in control of inflammation and cancer. The importance of the enzyme may have been underestimated by the relatively normal phenotype in the enzyme knockout animals. A clear phenotype has emerged when these knockout animals are used as models of various diseases.     

Enzymatic properties of human cytosolic phospholipase A(2)gamma

The enzymatic properties of cytosolic phospholipase A(2)gamma (cPLA(2)gamma), an isoform of 85-kDa group IV cPLA(2)alpha (cPLA(2)alpha) were studied in vitro and when the enzyme was expressed in cells. cPLA(2)gamma expressed in Sf9 cells is associated with membrane. Membranes isolated from [(3)H]arachidonic acid-labeled Sf9 cells expressing cPLA(2)gamma, constitutively release [(3)H]arachidonic acid. The membrane-associated activity is inhibited by the group IV PLA(2) inhibitor methylarachidonyl fluorophosphonate, but not effectively by the group VI PLA(2) inhibitor (E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one. cPLA(2)gamma has higher lysophospholipase activity than PLA(2) activity. Purified His-cPLA(2)gamma does not exhibit phospholipase A(1) activity, but sequentially hydrolyzes fatty acid from the sn-2 and sn-1 positions of phosphatidylcholine. cPLA(2)gamma overexpressed in HEK293 cells is constitutively active in isolated membranes, releasing large amounts of oleic, arachidonic, palmitic, and stearic acids; however, basal fatty acid release from intact cells is not increased. cPLA(2)gamma overexpressed in lung fibroblasts from cPLA(2)alpha-deficient mice is activated by mouse serum resulting in release of arachidonic, oleic, and palmitic acids, whereas overexpression of cPLA(2)alpha results primarily in arachidonic acid release.     

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.     

Naegleria fowleri amoebae express a membrane-associated calcium-independent phospholipase A(2)

Naegleria fowleri, a free-living amoeba, is the causative agent of primary amoebic meningoencephalitis. Previous reports have demonstrated that N. fowleri expresses one or more forms of phospholipase A(2) (PLA(2)) and that a secreted form of this enzyme is involved in pathogenesis. However, the molecular nature of these phospholipases remains largely unknown. This study was initiated to determine whether N. fowleri expresses analogs of the well-characterized PLA(2)s that are expressed by mammalian macrophages. Amoeba cell homogenates contain a PLA(2) activity that hydrolyzes the substrate that is preferred by the 85 kDa calcium-dependent cytosolic PLA(2), cPLA(2). However, unlike the cPLA(2) enzyme in macrophages, this activity is largely calcium-independent, is constitutively associated with membranes and shows only a modest preference for phospholipids that contain arachidonate. The amoeba PLA(2) activity is sensitive to inhibitors that block the activities of cPLA(2)-alpha and the 80 kDa calcium-independent PLA(2), iPLA(2), that are expressed by mammalian cells. One of these compounds, methylarachidonyl fluorophosphonate, partially inhibits the constitutive release of [(3)H]arachidonic acid from pre-labeled amoebae. Together, these data suggest that N. fowleri expresses a constitutively active calcium-independent PLA(2) that may play a role in the basal phospholipid metabolism of these cells.     

Studies on a mechanism by which cytosolic phospholipase A2 regulates the expression and function of type IIA secretory phospholipase A2

Although it has been proposed that arachidonate release by several secretory phospholipase A2 (sPLA2) isozymes is modulated by cytosolic PLA2 (cPLA2), the cellular component(s) that intermediates between these two signaling PLA2s remains unknown. Here we provide evidence that 12- or 15-lipoxygenase (12/15-LOX), which lies downstream of cPLA2, plays a pivotal role in cytokine-induced gene expression and function of sPLA2-IIA. The sPLA2-IIA expression and associated PGE2 generation induced by cytokines in rat fibroblastic 3Y1 cells were markedly attenuated by antioxidants that possess 12/15-LOX inhibitory activity. 3Y1 cells expressed 12/15-LOX endogenously, and forcible overexpression of 12/15-LOX in these cells greatly enhanced cytokine-induced expression of sPLA2-IIA, with a concomitant increase in delayed PG generation. Moreover, studies using 293 cells stably transfected with sPLA2-IIA revealed that stimulus-dependent hydrolysis of membrane phospholipids by sPLA2-IIA was enhanced by overexpression of 12/15-LOX. These results indicate that the product(s) generated by the cPLA2-12/15-LOX pathway following cell activation may play two roles: enhancement of sPLA2-IIA gene expression and membrane sensitization that leads to accelerated sPLA2-IIA-mediated hydrolysis.     

Phosphatidylinositol 4,5-bisphosphate anchors cytosolic group IVA phospholipase A2 to perinuclear membranes and decreases its calcium requirement for translocation in live cells

The eicosanoids are centrally involved in the onset and resolution of inflammatory processes. A key enzyme in eicosanoid biosynthesis during inflammation is group IVA phospholipase A2 (also known as cytosolic phospholipase A2alpha, cPLA2alpha). This enzyme is responsible for generating free arachidonic acid from membrane phospholipids. cPLA2alpha translocates to perinuclear membranes shortly after cell activation, in a process that is governed by the increased availability of intracellular Ca2+. However, cPLA2alpha also catalyzes membrane phospholipid hydrolysis in response to agonists that do not mobilize intracellular Ca2+. How cPLA2alpha interacts with membranes under these conditions is a major, still unresolved issue. Here, we report that phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] promotes translocation of cPLA2alpha to perinuclear membranes of intact cells in a manner that is independent of rises in the intracellular Ca2+ concentration. PtdIns(4,5)P2 anchors the enzyme to perinuclear membranes and allows for a proper interaction with its phospholipid substrate to release arachidonic acid.     

Group IVA phospholipase A2 is necessary for the biogenesis of lipid droplets

Lipid droplets (LD) are organelles present in all cell types, consisting of a hydrophobic core of triacylglycerols and cholesteryl esters, surrounded by a monolayer of phospholipids and cholesterol. This work shows that LD biogenesis induced by serum, by long-chain fatty acids, or the combination of both in CHO-K1 cells was prevented by phospholipase A(2) inhibitors with a pharmacological profile consistent with the implication of group IVA cytosolic phospholipase A(2) (cPLA(2)alpha). Knocking down cPLA(2)alpha expression with short interfering RNA was similar to pharmacological inhibition in terms of enzyme activity and LD biogenesis. A Chinese hamster ovary cell clone stably expressing an enhanced green fluorescent protein-cPLA(2)alpha fusion protein (EGFP-cPLA(2)) displayed higher LD occurrence under basal conditions and upon LD induction. Induction of LD took place with concurrent phosphorylation of cPLA(2)alpha at Ser(505). Transfection of a S505A mutant cPLA(2)alpha showed that phosphorylation at Ser(505) is key for enzyme activity and LD formation. cPLA(2)alpha contribution to LD biogenesis was not because of the generation of arachidonic acid, nor was it related to neutral lipid synthesis. cPLA(2)alpha inhibition in cells induced to form LD resulted in the appearance of tubulo-vesicular profiles of the smooth endoplasmic reticulum, compatible with a role of cPLA(2)alpha in the formation of nascent LD from the endoplasmic reticulum.     

A novel role of group VIB calcium-independent phospholipase A2 (iPLA2gamma) in the inducible expression of group IIA secretory PLA2 in rat fibroblastic cells

Group IIA secretory phospholipase A(2) (sPLA(2)-IIA) is a prototypic sPLA(2) enzyme that may play roles in modification of eicosanoid biosynthesis as well as antibacterial defense. In several cell types, inducible expression of sPLA(2) by pro-inflammatory stimuli is attenuated by group IVA cytosolic PLA(2) (cPLA(2)alpha) inhibitors such as arachidonyl trifluoromethyl ketone, leading to the proposal that prior activation of cPLA(2)alpha is required for de novo induction of sPLA(2). However, because of the broad specificity of several cPLA(2)alpha inhibitors used so far, a more comprehensive approach is needed to evaluate the relevance of this ambiguous pathway. Here, we provide evidence that the induction of sPLA(2)-IIA by pro-inflammatory stimuli requires group VIB calcium-independent PLA(2) (iPLA(2)gamma), rather than cPLA(2)alpha, in rat fibroblastic 3Y1 cells. Results with small interfering RNA unexpectedly showed that the cytokine induction of sPLA(2)-IIA in cPLA(2)alpha knockdown cells, in which cPLA(2)alpha protein was undetectable, was similar to that in replicate control cells. By contrast, knockdown of iPLA(2)gamma, another arachidonyl trifluoromethyl ketone-sensitive intracellular PLA(2), markedly reduced the cytokine-induced expression of sPLA(2)-IIA. Supporting this finding, the R-enantiomer of bromoenol lactone, an iPLA(2)gamma inhibitor, suppressed the cytokine-induced sPLA(2)-IIA expression, whereas (S)-bromoenol lactone, an iPLA(2)beta inhibitor, failed to do so. Moreover, lipopolysaccharide-stimulated sPLA(2)-IIA expression was also abolished by knockdown of iPLA(2)gamma. These findings open new insight into a novel regulatory role of iPLA(2)gamma in stimulus-coupled sPLA(2)-IIA expression.     

Genetic and structural analysis of Hmg2p-induced endoplasmic reticulum remodeling in Saccharomyces cerevisiae

The endoplasmic reticulum (ER) is highly plastic, and increased expression of distinct single ER-resident membrane proteins, such as HMG-CoA reductase (HMGR), can induce a dramatic restructuring of ER membranes into highly organized arrays. Studies on the ER-remodeling behavior of the two yeast HMGR isozymes, Hmg1p and Hmg2p, suggest that they could be mechanistically distinct. We examined the features of Hmg2p required to generate its characteristic structures, and we found that the molecular requirements are similar to those of Hmg1p. However, the structures generated by Hmg1p and Hmg2p have distinct cell biological features determined by the transmembrane regions of the proteins. In parallel, we conducted a genetic screen to identify HER genes (required for Hmg2p-induced ER Remodeling), further confirming that the mechanisms of membrane reorganization by these two proteins are distinct because most of the HER genes were required for Hmg2p but not Hmg1p-induced ER remodeling. One of the HER genes identified was PSD1, which encodes the phospholipid biosynthetic enzyme phosphatidylserine decarboxylase. This direct connection to phospholipid biosynthesis prompted a more detailed examination of the effects of Hmg2p on phospholipid mutants and composition. Our analysis revealed that overexpression of Hmg2p caused significant and specific growth defects in nulls of the methylation pathway for phosphatidylcholine biosynthesis that includes the Psd1p enzyme. Furthermore, increased expression of Hmg2p altered the composition of cellular phospholipids in a manner that implied a role for PSD1. These phospholipid effects, unlike Hmg2p-induced ER remodeling, required the enzymatic activity of Hmg2p. Together, our results indicate that, although related, Hmg2p- and Hmg1p-induced ER remodeling are mechanistically distinct.     

Negative feedback between secretory and cytosolic phospholipase A2 and their opposing roles in ovalbumin-induced bronchoconstriction in rats

Phospholipase A2 (PLA2) hydrolyzes cell membrane phospholipids (PL) to produce arachidonic acid and lyso-PL. The PLA2 enzymes include the secretory (sPLA2) and cytosolic (cPLA2) isoforms, which are assumed to act synergistically in production of eicosanoids that are involved in inflammatory processes. However, growing evidence raises the possibility that in airways and asthma-related inflammatory cells (eosinophils, basophils), the production of the bronchoconstrictor cysteinyl leukotrienes (CysLT) is linked exclusively to sPLA2, whereas the bronchodilator prostaglandin PGE2 is produced by cPLA2. It has been further reported that the capacity of airway epithelial cells to produce CysLT is inversely proportional to PGE2 production. This seems to suggest that sPLA2 and cPLA2 play opposing roles in asthma pathophysiology and the possibility of a negative feedback between the two isoenzymes. To test this hypothesis, we examined the effect of a cell-impermeable extracellular sPLA2 inhibitor on bronchoconstriction and PLA2 expression in rats with ovalbumin (OVA)-induced asthma. It was found that OVA-induced bronchoconstriction was associated with elevation of lung sPLA2 expression and CysLT production, concomitantly with suppression of cPLA2 expression and PGE2 production. These were reversed by treatment with the sPLA2 inhibitor, resulting in amelioration of bronchoconstriction and reduced CysLT production and sPLA2 expression, concomitantly with enhanced PGE2 production and cPLA2 expression. This study demonstrates, for the first time in vivo, a negative feedback between sPLA2 and cPLA2 and assigns opposing roles for these enzymes in asthma pathophysiology: sPLA2 activation induces production of the bronchoconstrictor CysLT and suppresses cPLA2 expression and the subsequent production of the bronchodilator PGE2.     

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.     

The cytosolic phospholipase A2 catalytic domain modulates association and residence time at Golgi membranes

Cytosolic phospholipase A2 (cPLA2) catalyzes release of arachidonic acid from membranes following translocation to Golgi and endoplasmic reticulum. In response to an intracellular calcium concentration ([Ca2+]i) increase, the C2 domain binds Ca2+ and brings the catalytic domain into proximity with its phospholipid substrate. Because membrane residence is important in the regulation of cPLA2 activity, we explored the contributions of the C2 and catalytic domains in mediating membrane residence using an imaging approach in live cells with fluorescent protein chimeras of cPLA2. The isolated cPLA2 C2 domain associated with Golgi membranes rapidly in proportion to the [Ca2+]i, allowing for its use as a [Ca2+]i indicator. cPLA2 association with Golgi was slower than the isolated C2 domain in response to a [Ca2+]i increase. After [Ca2+]i decrease, cPLA2 remained associated with membrane in a Ca(2+)-independent fashion whereas C2 domain rapidly dissociated. Ca(2+)-independent membrane association was greatly reduced by mutation of Trp464, located at the membrane-exposed face of the catalytic domain, to Gly or Ala. Mutation of Trp464 to Phe supported Ca(2+)-independent association similar to wild type. These results demonstrate a role for the cPLA2 catalytic domain in regulating membrane association and membrane residence time.     

Induction of Ca-independent PLA(2) and conservation of plasmalogen polyunsaturated fatty acids in diabetic heart

Diabetes-induced changes in phospholipase A(2) (PLA(2)) activity have been measured in several tissues but are undefined in diabetic myocardium. We measured ventricular PLA(2) activity in control, streptozotocin-induced diabetic, and insulin-treated diabetic rats and characterized myocardial phospholipids to determine whether diabetes altered myocardial phospholipid metabolism. Increased membrane-associated Ca(2+)-independent PLA(2) (iPLA(2)) activity was observed in diabetes that was selective for arachidonylated phospholipids. Increased iPLA(2) activity was accompanied by an increase in choline lysophospholipids. Diabetes was associated with marked alterations in the phospholipid composition of the myocardium, characterized by decreases in esterified arachidonic and docosahexaenoic acids and increases in linoleic acid. The decrease in polyunsaturated fatty acids was confined to diacylphospholipids, whereas the relative amount of these fatty acids in plasmalogens was increased. Diabetes-induced changes in PLA(2) activity, lysophospholipid production, and alterations in phospholipid composition were all reversed by insulin treatment of diabetic animals. Diabetes-induced changes in membrane phospholipid content and phospholipid hydrolysis may contribute to some of the alterations in myocardial function that are observed in diabetic patients.