Involvement of group VI Ca2+-independent phospholipase A2 in protein kinase C-dependent arachidonic acid liberation in zymosan-stimulated macrophage-like P388D1 cells.

We investigated the possible involvement of group VI Ca2+-independent phospholipase A2 (iPLA2) in arachidonic acid (AA) liberation in zymosan-stimulated macrophage-like P388D1 cells. Zymosan-induced AA liberation was markedly inhibited by methyl arachidonoyl fluorophosphonate, a dual inhibitor of group IV cytosolic phospholipase A2 (cPLA2) and iPLA2. We found that a relatively specific iPLA2 inhibitor, bromoenol lactone, significantly decreased the zymosan-induced AA liberation in parallel with the decrease in iPLA2 activity, without an effect on diacylglycerol formation. Consistent with this, attenuation of iPLA2 activity by a group VI iPLA2 antisense oligonucleotide resulted in a decrease in zymosan-induced prostaglandin D2 generation. These findings suggest that zymosan-induced AA liberation may be, at least in part, mediated by iPLA2. A protein kinase C (PKC) inhibitor diminished zymosan-induced AA liberation, while a PKC activator, phorbol 12-myristate 13-acetate (PMA), enhanced the liberation. Bromoenol lactone suppressed the PMA-enhanced AA liberation without any effect on PMA-induced PKC activation. Down-regulation of PKCalpha on prolonged exposure to PMA also decreased zymosan-induced AA liberation. Under these conditions, the remaining AA liberation was insensitive to bromoenol lactone. Furthermore, the PKC depletion suppressed increases in iPLA2 proteins and the activity in the membrane fraction of zymosan-stimulated cells. In contrast, the zymosan-induced increases in iPLA2 proteins and the activity in the fraction were facilitated by simultaneous addition of PMA. Although intracellular Ca2+ depletion prevented zymosan-induced AA liberation, the translocation of PKCalpha to membranes was also inhibited. Taken together, we propose that zymosan may stimulate iPLA2-mediated AA liberation, probably through a PKC-dependent mechanism.     

Involvement of calcium-independent phospholipase A2 in uterine stromal cell phospholipid remodelling.

The role of Ca2+-independent phospholipase A2 (iPLA2) in arachidonic (AA) and docosahexaenoic (DHA) acid incorporation and phospholipid remodelling in rat uterine stromal cells (UIII cells) was studied. Incorporation of AA and DHA into UIII cell phospholipids was Ca2+-independent. Bromoenollactone (BEL), a potent inhibitor of iPLA2, reduced lysophosphatidylcholine level and AA incorporation into phospholipids by approximately 20%. DHA incorporation was not affected by BEL, indicating that the pathways for AA and DHA incorporation are partially different. In control cells, the transfer of AA occurred mainly from diacyl-glycerophosphocholine (GroPCho) to alkenylacyl-glycerophosphoethanolamine (GroPEtn) and to a lesser extent from diacyl-GroPCho to diacyl-GroPEtn. [3H]DHA was redistributed from diacyl-GroPCho and alkylacyl-GroPEtn to alkenylacyl-GroPEtn. BEL treatment inhibited completely the redistributrion of AA within diacyl-GroPCho and diacyl -GroPEtn and reduced the [3H]DHA content of diacyl-GroPEtn, indicating that a BEL-sensitive iPLA2 controls the redistribution of polyunsaturated fatty acids to diacyl-GroPEtn. In contrast the redistribution of radioactive AA and DHA to alkenylacyl-GroPEtn was almost insensitive to BEL. The analysis of substrate specificity and BEL sensitivity of iPLA2 activity indicates that UIII cells exhibit at least two isoforms of iPLA2, one of which is BEL-sensitive and quite selective of diacyl species, and another one that is insensitive to BEL and selective for alkenylacyl-GroPEtn. Taken together, these results suggest that several iPLA2 participate independently in the remodelling of UIII cell phospholipids.     

Studies of the role of group VI phospholipase A2 in fatty acid incorporation, phospholipid remodeling, lysophosphatidylcholine generation, and secretagogue-induced arachidonic acid release in pancreatic islets and insulinoma cells.

An 84-kDa group VI phospholipase A2 (iPLA2) that does not require Ca2+ for catalysis has been cloned from Chinese hamster ovary cells, murine P388D1 cells, and pancreatic islet beta-cells. A housekeeping role for iPLA2 in generating lysophosphatidylcholine (LPC) acceptors for arachidonic acid incorporation into phosphatidylcholine (PC) has been proposed because iPLA2 inhibition reduces LPC levels and suppresses arachidonate incorporation and phospholipid remodeling in P388D1 cells. Because islet beta-cell phospholipids are enriched in arachidonate, we have examined the role of iPLA2 in arachidonate incorporation into islets and INS-1 insulinoma cells. Inhibition of iPLA2 with a bromoenol lactone (BEL) suicide substrate did not suppress and generally enhanced [3H]arachidonate incorporation into these cells in the presence or absence of extracellular calcium at varied time points and BEL concentrations. Arachidonate incorporation into islet phospholipids involved deacylation-reacylation and not de novo synthesis, as indicated by experiments with varied extracellular glucose concentrations and by examining [14C]glucose incorporation into phospholipids. BEL also inhibited islet cytosolic phosphatidate phosphohydrolase (PAPH), but the PAPH inhibitor propranolol did not affect arachidonate incorporation into islet or INS-1 cell phospholipids. Inhibition of islet iPLA2 did not alter the phospholipid head-group classes into which [3H]arachidonate was initially incorporated or its subsequent transfer from PC to other lipids. Electrospray ionization mass spectrometric measurements indicated that inhibition of INS-1 cell iPLA2 accelerated arachidonate incorporation into PC and that inhibition of islet iPLA2 reduced LPC levels by 25%, suggesting that LPC mass does not limit arachidonate incorporation into islet PC. Gas chromatography/mass spectrometry measurements indicated that BEL but not propranolol suppressed insulin secretagogue-induced hydrolysis of arachidonate from islet phospholipids. In islets and INS-1 cells, iPLA2 is thus not required for arachidonate incorporation or phospholipid remodeling and may play other roles in these cells.     

Potassium regulates IL-1 beta processing via calcium-independent phospholipase A2

We report that potassium leakage from cells leads to activation of the Ca2+-independent phospholipase A2 (iPLA2), and the latter plays a pivotal role in regulating the cleavage of pro-IL-1 beta by the IL-converting enzyme caspase-1 in human monocytes. K+ efflux led to increases of cellular levels of glycerophosphocholine, an unambiguous indicator of phospholipase A2 activation. Both maturation of IL-1 beta and formation of glycerophosphocholine were blocked by bromoenol lactone, the specific iPLA2 inhibitor. Bromoenol lactone-dependent inhibition of IL-1 beta processing was not due to perturbation of the export machinery for pro-IL-1 beta and IL-1 beta or to caspase-1 suppression. Conspicuously, activation of Ca2+-dependent phospholipase A2 did not support but rather suppressed IL-1 beta processing. Thus, our findings reveal a specific role for iPLA2 activation in the sequence of events underlying IL-1 beta maturation.     

Mechanism of arachidonic acid liberation in platelet-activating factor-stimulated human polymorphonuclear neutrophils

Upon stimulation of human polymorphonuclear neutrophils with platelet-activating factor (PAF), arachidonic acid (AA) is released from membrane phospholipids. The mechanism for AA liberation, a key step in the synthesis of biologically active eicosanoids, was investigated. PAF was found to elicit an increase in the cytoplasmic level of free Ca2+ as monitored by fluorescent indicator fura 2. When [3H] AA-labeled neutrophils were exposed to PAF, the enhanced release of AA was observed with a concomitant decrease of radioactivity in phosphatidylinositol and phosphatidylcholine fractions. The inhibitors of phospholipase A2, mepacrine and 2-(p-amylcinnamoyl)-amino-4-chlorobenzoic acid, effectively suppressed the liberation of [3H]AA from phospholipids, indicating that liberation of AA is mainly catalyzed by the action of phospholipase A2. The extracellular Ca2+ is not required for AA release. However, intracellular Ca2+ antagonists, TMB-8 and high dose of quin 2/AM drastically reduced the liberation of AA induced by PAF, indicating that Ca2+ is an essential factor for phospholipase A2 activation. PAF raised the fluorescence of fura 2 at concentrations as low as 8 pM which reached a maximal level about 8 nM, whereas more than nM order concentrations of PAF was required for the detectable release of [3H]AA. Pretreatment of neutrophils with pertussis toxin resulted in complete abolition of AA liberation in response to PAF. However, the fura 2 response to PAF was not effectively inhibited by toxin treatment. In human neutrophil homogenate and membrane preparations, guanosine 5'-O-(thiotriphosphate) stimulated AA release and potentiated the action of PAF. Guanosine 5'-O-(thiodiphosphate) inhibited the effects of guanosine 5'-O-(thiotriphosphate). These results suggest several points: 1) PAF stimulates human polymorphonuclear neutrophils to liberate AA mainly by the action of phospholipase A2; 2) Ca2+ mobilization alone is not sufficient to stimulate AA release, although Ca2+ is the important factor for phospholipase A2 activation; and 3) a pertussis toxin-sensitive GTP-binding protein may be implicated in activation of phospholipase A2.     

Modulation of 32Pi incorporation into phospholipids of polymorphonuclear leukocytes by ionophore A23187.

The effects of ionophore A23187 on the incorporation of 32Pi into phospholipids and on 45Ca2+ uptake and release by polymorphonuclear leukocytes were examined. A23187 increased 32Pi incorporation into phosphatidic acid, phosphatidylglycerol, phosphatidylserine, and the phosphoinositides. It also promoted a rapid burst uptake and release of 45Ca2+ by leukocytes. External Ca2+, but not Mg2+, was required for full stimulation of 32Pi incorporation into phosphatidic acid and the phosphoinositides. In the absence of external Ca2+, the increased radiophosphorus activity of phosphatidic acid, phosphatidylserine and the phosphoinositides was grossly reduced but not eliminated, and the decreased radiophosphorus activity of phosphatidylcholine became pronounced. In addition, the ionophore effect on 32Pi incorporation into leukocyte phospholipids was not abolished by ethyleneglycol bis(beta-amino-ethylether)-N,N'-tetraacetic acid. ATP radiophosphorus activity was also enhanced by the presence of A23187, but the enhancement was much less than that of the acidic phospholipids. Based on these findings, it is suggested that the increased 32Pi incorporation into the acidic phospholipids of leukocytes induced by A23187 was not solely derived from the higher radioactivity of ATP, increased Ca2+ fluxes and perturbation of cellular Ca2+ distribution of leukocytes exposed to A 23187 may trigger part of the altered 32Pi incorporation into phospholipids.     

Bromoenol lactone, an inhibitor of Group V1A calcium-independent phospholipase A2 inhibits antigen-stimulated mast cell exocytosis without blocking Ca2+ influx

Calcium-independent phospholipase A2 (iPLA2beta) has recently been suggested to regulate Ca2+ entry by activating store-operated Ca2+ channels. These studies have been conducted in mast cells using thapsigargin to deplete intracellular stores. In RBL 2H3 and bone marrow-derived mast cells (BMMCs), Ca2+ entry is critical for exocytosis and therefore we have examined whether the proposed mechanism would be relevant when a physiological stimulus is applied to these cells. Using an iPLA2beta antibody, we demonstrate that the 84kDa iPLA2beta is expressed in these mast cells. As bromoenol lactone (BEL) is a suicide-based irreversible inhibitor of iPLA2beta it was used to probe this potential mechanism. We observe inhibition of exocytosis stimulated either with antigen or with thapsigargin. However, BEL also inhibits exocytosis when stimulated using a Ca2+ ionophore A23187, which passively transports Ca2+ down a concentration gradient and also in permeabilised mast cells where Ca2+ entry is no longer relevant. Moreover, BEL has only a minor effect on antigen- or thapsigargin-stimulated Ca2+ signalling, both the release from internal stores and sustained elevation due to Ca2+ influx. These results cast doubt on the proposed mechanism involving iPLA2beta required for Ca2+ entry. Although inhibition of exocytosis by BEL could imply a requirement for iPLA2beta activation for exocytosis, an alternative explanation is that BEL inactivates other target proteins required for exocytosis.     

Group VIB Ca2+-independent phospholipase A2gamma promotes cellular membrane hydrolysis and prostaglandin production in a manner distinct from other intracellular phospholipases A2

Although group VIA Ca2+-independent phospholipase A2beta (iPLA2beta) has been implicated in various cellular events, the functions of other iPLA2 isozymes remain largely elusive. In this study, we examined the cellular functions of group VIB iPLA2gamma. Lentiviral transfection of iPLA2gamma into HEK293 cells resulted in marked increases in spontaneous, stimulus-coupled, and cell death-associated release of arachidonic acid (AA), which was converted to prostaglandin E2 with preferred cyclooxygenase (COX)-1 coupling. Conversely, treatment of HEK293 cells with iPLA2gamma small interfering RNA significantly reduced AA release, indicating the participation of endogenous iPLA2gamma. iPLA2gamma protein appeared in multiple sizes according to cell types, and a 63-kDa form was localized mainly in peroxisomes. Electrospray ionization mass spectrometry of cellular phospholipids revealed that iPLA2gamma and other intracellular PLA2 enzymes acted on different phospholipid subclasses. Transfection of iPLA2gamma into HCA-7 cells also led to increased AA release and prostaglandin E2 synthesis via both COX-1 and COX-2, with a concomitant increase in cell growth. Immunohistochemistry of human colorectal cancer tissues showed elevated expression of iPLA2gamma in adenocarcinoma cells. These results collectively suggest distinct roles for iPLA2beta and iPLA2gamma in cellular homeostasis and signaling, a functional link between peroxisomal AA release and eicosanoid generation, and a potential contribution of iPLA2gamma to tumorigenesis.     

Role of group VIA calcium-independent phospholipase A2 in arachidonic acid release, phospholipid fatty acid incorporation, and apoptosis in U937 cells responding to hydrogen peroxide

Group VIA calcium-independent phospholipase A2 (iPLA2) has been shown to play a major role in regulating basal phospholipid deacylation reactions in certain cell types. More recently, roles for this enzyme have also been suggested in the destruction of membrane phospholipid during apoptosis and after oxidant injury. Proposed iPLA2 roles have rested heavily on the use of bromoenol lactone as an iPLA2-specific inhibitor, but this compound actually inhibits other enzymes and lipid pathways unrelated to PLA2, which makes it difficult to define the contribution of iPLA2 to specific functions. In previous work, we pioneered the use of antisense technology to decrease cellular iPLA2 activity as an alternative approach to study iPLA2 functions. In the present study, we followed the opposite strategy and prepared U937 cells that exhibited enhanced iPLA activity by stably expressing a plasmid containing iPLA2 cDNA. Compared with control cells, the iPLA2 -overexpressing U937 cells showed elevated responses to hydrogen peroxide with regard to both arachidonic acid mobilization and incorporation of the fatty acid into phospholipids, thus providing additional evidence for the key role that iPLA2 plays in these events. Long-term exposure of the cells to hydrogen peroxide resulted in cell death by apoptosis, and this process was accelerated in the iPLA2-overexpressing cells. Increased phospholipid hydrolysis and fatty acid release also occurred in these cells. Unexpectedly, however, abrogation of U937 cell iPLA2 activity by either methyl arachidonyl fluorophosphonate or an antisense oligonucleotide did not delay or decrease the extent of apoptosis induced by hydrogen peroxide. These results indicate that, although iPLA2-mediated phospholipid hydrolysis occurs during apoptosis, iPLA2 may actually be dispensable for the apoptotic process to occur. Thus, beyond a mere destructive role, iPLA2 may play other roles during apoptosis.     

Role of Ca2+-independent phospholipase A2 on arachidonic acid release induced by reactive oxygen species

Previous studies have shown that reactive oxygen species (ROS) enhance arachidonic acid (AA) release and the subsequent AA metabolism in macrophages. The purpose of this study was determined the implication of phospholipases A2 (PLA2s) in these events. Our results show that oxidative stress induced by exogenous adding of hydrogen peroxide or superoxide anion in macrophage RAW 264.7 and mouse peritoneal macrophage cultures caused a marked enhancement of calcium-independent PLA2 (iPLA2) activity,whereas the increment of secreted PLA2 (sPLA2) and calcium-dependent cytosolic PLA2 (cPLA2) activities were slight. This increase of iPLA2 activity by ROS was rapid and dose-dependent. ROS also induced a significant [3H] arachidonic acid (AA) release. The iPLA2 selective inhibitor, bromoenol lactone, almost completely suppressed the mobilization of [3H]AA induced by ROS whereas antisense oligonucleotide against cPLA2 did not have any appreciable effect. Thus, our data show that iPLA2 activity is involved in the mechanism by which ROS increases the availability of free AA in macrophages RAW 264.7. Moreover, the protein kinase C (PKC) inhibitor, calphostin C, and calcium chelators had no effect on the [3H]AA release induced by ROS, suggesting this is a regulatory role of iPLA2.     

Bromoenol lactone promotes cell death by a mechanism involving phosphatidate phosphohydrolase-1 rather than calcium-independent phospholipase A2

Originally described as a serine protease inhibitor, bromoenol lactone (BEL) has recently been found to potently inhibit Group VI calcium-independent phospholipase A2 (iPLA2). Thus, BEL is widely used to define biological roles of iPLA2 in cells. However, BEL is also known to inhibit another key enzyme of phospholipid metabolism, namely the magnesium-dependent phosphatidate phosphohydrolase-1 (PAP-1). In this work we report that BEL is able to promote apoptosis in a variety of cell lines, including U937, THP-1, and MonoMac (human phagocyte), RAW264.7 (murine macrophage), Jurkat (human T lymphocyte), and GH3 (human pituitary). In these cells, long term treatment with BEL (up to 24 h) results in increased annexin-V binding to the cell surface and nuclear DNA damage, as detected by staining with both DAPI and propidium iodide. At earlier times (2 h), BEL induces the proteolysis of procaspase-9 and procaspase-3 and increases cleavage of poly(ADP-ribose) polymerase. These changes are preceded by variations in the mitochondrial membrane potential. All these effects of BEL are not mimicked by the iPLA2 inhibitor methylarachidonyl fluorophosphonate or by treating the cells with a specific iPLA2 antisense oligonucleotide. However, propranolol, a PAP-1 inhibitor, is able to reproduce these effects, suggesting that it is the inhibition of PAP-1 and not of iPLA2 that is involved in BEL-induced cell death. In support of this view, BEL-induced apoptosis is accompanied by a very strong inhibition of PAP-1-regulated events, such as incorporation of [3H]choline into phospholipids and de novo incorporation of [3H]arachidonic acid into triacylglycerol. Collectively, these results stress the role of PAP-1 as a key enzyme for cell integrity and survival and in turn caution against the use of BEL in studies involving long incubation times, due to the capacity of this drug to induce apoptosis in a variety of cells.     

Studies of insulin secretory responses and of arachidonic acid incorporation into phospholipids of stably transfected insulinoma cells that overexpress group VIA phospholipase A2 (iPLA2beta ) indicate a signaling rather than a housekeeping role for iPLA2beta

A cytosolic 84-kDa group VIA phospholipase A(2) (iPLA(2)beta) that does not require Ca(2+) for catalysis has been cloned from several sources, including rat and human pancreatic islet beta-cells and murine P388D1 cells. Many potential iPLA(2)beta functions have been proposed, including a signaling role in beta-cell insulin secretion and a role in generating lysophosphatidylcholine acceptors for arachidonic acid incorporation into P388D1 cell phosphatidylcholine (PC). Proposals for iPLA(2)beta function rest in part on effects of inhibiting iPLA(2)beta activity with a bromoenol lactone (BEL) suicide substrate, but BEL also inhibits phosphatidate phosphohydrolase-1 and a group VIB phospholipase A(2). Manipulation of iPLA(2)beta expression by molecular biologic means is an alternative approach to study iPLA(2)beta functions, and we have used a retroviral construct containing iPLA(2)beta cDNA to prepare two INS-1 insulinoma cell clonal lines that stably overexpress iPLA(2)beta. Compared with parental INS-1 cells or cells transfected with empty vector, both iPLA(2)beta-overexpressing lines exhibit amplified insulin secretory responses to glucose and cAMP-elevating agents, and BEL substantially attenuates stimulated secretion. Electrospray ionization mass spectrometric analyses of arachidonic acid incorporation into INS-1 cell PC indicate that neither overexpression nor inhibition of iPLA(2)beta affects the rate or extent of this process in INS-1 cells. Immunocytofluorescence studies with antibodies directed against iPLA(2)beta indicate that cAMP-elevating agents increase perinuclear fluorescence in INS-1 cells, suggesting that iPLA(2)beta associates with nuclei. These studies are more consistent with a signaling than with a housekeeping role for iPLA(2)beta in insulin-secreting beta-cells.     

Electrospray ionization/mass spectrometric analyses of human promonocytic U937 cell glycerolipids and evidence that differentiation is associated with membrane lipid composition changes that facilitate phospholipase A2 activation

Upon differentiation, U937 promonocytic cells gain the ability to release a large fraction of arachidonate esterified in phospholipids when stimulated, but the mechanism is unclear. U937 cells express group IV phospholipase A(2) (cPLA(2)), but neither its level nor its phosphorylation state increases upon differentiation. A group VI PLA(2) (iPLA(2)) that is sensitive to a bromoenol lactone inhibitor catalyzes arachidonate hydrolysis from phospholipids in some cells and facilitates arachidonate incorporation into glycerophosphocholine (GPC) lipids in others, but it is not known whether U937 cells express iPLA(2). We confirm that ionophore A23187 induces substantial [(3)H]arachidonate release from differentiated but not control U937 cells, and electrospray ionization mass spectrometric (ESI/MS) analyses indicate that differentiated cells contain a higher proportion of arachidonate-containing GPC species than control cells. U937 cells express iPLA(2) mRNA and activity, but iPLA(2) inhibition impairs neither [(3)H]arachidonate incorporation into nor release from U937 cells. Experiments with phosphatidate phosphohydrolase (PAPH) and phospholipase D (PLD) inhibitors coupled with ESI/MS analyses of PLD-PAPH products indicate that differentiated cells gain the ability to produce diacylglycerol (DAG) via PLD-PAPH. DAG promotes arachidonate release by a mechanism that does not require DAG hydrolysis, is largely independent of protein kinase C, and requires cPLA(2) activity. This may reflect DAG effects on cPLA(2) substrate state.     

Complex regulation of store-operated Ca2+ entry pathway by PKC-epsilon in vascular SMCs

The role of PKC in the regulation of store-operated Ca2+ entry (SOCE) is rather controversial. Here, we used Ca2+-imaging, biochemical, pharmacological, and molecular techniques to test if Ca2+-independent PLA2beta (iPLA2beta), one of the transducers of the signal from depleted stores to plasma membrane channels, may be a target for the complex regulation of SOCE by PKC and diacylglycerol (DAG) in rabbit aortic smooth muscle cells (SMCs). We found that the inhibition of PKC with chelerythrine resulted in significant inhibition of thapsigargin (TG)-induced SOCE in proliferating SMCs. Activation of PKC by the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol (OAG) caused a significant depletion of intracellular Ca2+ stores and triggered Ca2+ influx that was similar to TG-induced SOCE. OAG and TG both produced a PKC-dependent activation of iPLA2beta and Ca2+ entry that were absent in SMCs in which iPLA2beta was inhibited by a specific chiral enantiomer of bromoenol lactone (S-BEL). Moreover, we found that PKC regulates TG- and OAG-induced Ca2+ entry only in proliferating SMCs, which correlates with the expression of the specific PKC-epsilon isoform. Molecular downregulation of PKC-epsilon impaired TG- and OAG-induced Ca2+ influx in proliferating SMCs but had no effect in confluent SMCs. Our results demonstrate that DAG (or OAG) can affect SOCE via multiple mechanisms, which may involve the depletion of Ca2+ stores as well as direct PKC-epsilon-dependent activation of iPLA2beta, resulting in a complex regulation of SOCE in proliferating and confluent SMCs.     

Sphingosine 1-phosphate induces arachidonic acid mobilization in A549 human lung adenocarcinoma cells

In the present paper, the effect of sphingosine 1-phosphate (Sph-1-P) on arachidonic acid mobilization in A549 human lung adenocarcinoma cells was investigated. Sph-1-P provoked a rapid and relevant release of arachidonic acid which was similar to that elicited by bradykinin, well-known pro-inflammatory agonist. The Sph-1-P-induced release of arachidonic acid involved Ca(2+)-independent phospholipase A(2) (iPLA2) activity, as suggested by the dose-dependent inhibition exerted by the rather specific inhibitor bromoenol lactone. The Sph-1-P-induced release of arachidonic acid was pertussis toxin-sensitive, pointing at a receptor-mediated mechanism, which involves heterotrimeric Gi proteins. The action of Sph-1-P was totally dependent on protein kinase C (PKC) catalytic activity and seemed to involve agonist-stimulated phospholipase D (PLD) activity. This study represents the first evidence for Sph-1-P-induced release of arachidonic acid which occurs through a specific signaling pathway involving Gi protein-coupled receptor(s), PKC, PLD and iPLA2 activities.     

Involvement of different protein kinases and phospholipases A2 in phorbol ester (TPA)-induced arachidonic acid liberation in bovine platelets

The effect of various phospholipase A2 and protein kinase inhibitors on the arachidonic acid liberation in bovine platelets induced by the protein kinase activator 12-O-tetradecanoylphorbol-13-acetate (TPA) was studied. TPA stimulates arachidonic acid release mainly by activating group IV cytosolic PLA2 (cPLA2), since inhibitors of this enzyme markedly inhibited arachidonic acid formation. However, group VI Ca2+-independent PLA2 (iPLA2) seems to contribute to the arachidonic acid liberation too, since the relatively specific iPLA2 inhibitor bromoenol lactone (BEL) decreased arachidonic acid generation in part. The pronounced inhibition of the TPA-induced arachidonic acid release by the protein kinase C (PKC) inhibitors GF 109203X and Ro 31-82220, respectively, and by the p38 MAP kinase inhibitor SB 202190 suggests that the activation of the PLA2s by TPA is mediated via PKC and p38 MAP kinase.     

Ca2(+)-dependence of arachidonic acid redistribution among phospholipids of cultured mouse keratinocytes

Mouse keratinocytes cultured in a medium containing less than 0.1 mM Ca2+ (low Ca2+) incorporated [1-14C]arachidonic acid (AA) into phospholipids by kinetics including; (i) a rapid labelling of phosphatidylinositol (PtdIns), phosphatidylserine (PtdSer) and both acid-stable and alkenylacyl forms of phosphatidylcholine (PtdCho); and (ii) a slow but long-lasting radiolabel incorporation into both acid-stable and alkenylacyl forms of phosphatidylethanolamine (PtdEtn), partly associated with a net radioactivity loss from acid stable-PtdCho. Under low Ca2+ conditions no radioactivity transfer apparently occurred between PtdIns and other phospholipid classes. When cells were prelabelled for 24 h with [1-14C]AA and reincubated in label-free medium containing 1.2 mM Ca2+ (normal Ca2+), an early and extensive loss of radioactivity from PtdIns was observed, reasonably in connection with Ca2+ stimulation of phosphoinositide turnover. Cell shift to normal Ca2+ did not result in an increased synthesis of labelled eicosanoids, but was consistent with an increase of radioactivity incorporation into diacylglycerol (DAG) and with a complex pattern of [1-14C]AA redistribution, eventually leading to a marked radioactivity incorporation into acid stable-PtdEtn (but not into alkenylacyl-PtdEtn) and to a labelling decrease of acid stable-PtdCho. The possible mechanisms driving AA recycling after cell shift to normal Ca2+ are discussed.     

Purification and characterization of a cytosolic phospholipase A2 from rat liver

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

Group VIA PLA2 (iPLA2β) is activated upstream of p38 mitogen-activated protein kinase (MAPK) in pancreatic islet β-cell signaling

Group VIA phospholipase A(2) (iPLA(2)β) in pancreatic islet β-cells participates in glucose-stimulated insulin secretion and sarco(endo)plasmic reticulum ATPase (SERCA) inhibitor-induced apoptosis, and both are attenuated by pharmacologic or genetic reductions in iPLA(2)β activity and amplified by iPLA(2)β overexpression. While exploring signaling events that occur downstream of iPLA(2)β activation, we found that p38 MAPK is activated by phosphorylation in INS-1 insulinoma cells and mouse pancreatic islets, that this increases with iPLA(2)β expression level, and that it is stimulated by the iPLA(2)β reaction product arachidonic acid. The insulin secretagogue D-glucose also stimulates β-cell p38 MAPK phosphorylation, and this is prevented by the iPLA(2)β inhibitor bromoenol lactone. Insulin secretion induced by d-glucose and forskolin is amplified by overexpressing iPLA(2)β in INS-1 cells and in mouse islets, and the p38 MAPK inhibitor PD169316 prevents both responses. The SERCA inhibitor thapsigargin also stimulates phosphorylation of both β-cell MAPK kinase isoforms and p38 MAPK, and bromoenol lactone prevents both events. Others have reported that iPLA(2)β products activate Rho family G-proteins that promote MAPK kinase activation via a mechanism inhibited by Clostridium difficile toxin B, which we find to inhibit thapsigargin-induced β-cell p38 MAPK phosphorylation. Thapsigargin-induced β-cell apoptosis and ceramide generation are also prevented by the p38 MAPK inhibitor PD169316. These observations indicate that p38 MAPK is activated downstream of iPLA(2)β in β-cells incubated with insulin secretagogues or thapsigargin, that this requires prior iPLA(2)β activation, and that p38 MAPK is involved in the β-cell functional responses of insulin secretion and apoptosis in which iPLA(2)β participates.