Regulation of lung surfactant secretion by phospholipase A2

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

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

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

Essential role of Ca2+ -dependent phospholipase A2 in estradiol-induced lysosome activation

The mechanism of lysosome activation by 17beta-estradiol has been studied in mussel blood cells. Cell treatment with estradiol induced a sustained increase of cytosolic free Ca2+ that was completely prevented by preincubating the cells with the Ca2+ chelator BAPTA-AM. Estradiol treatment was also followed by destabilization of the lysosomal membranes, as detected in terms of the lysosomes' increased permeability to neutral red. The effect of estradiol on lysosomes was almost completely prevented by preincubation with the inhibitor of cytosolic Ca2+ -dependent PLA2 (cPLA2), arachidonyl trifluoromethyl ketone (AACOCF3), and was significantly reduced by preincubation with BAPTA-AM. In contrast, it was virtually unaffected by preincubation with the inhibitor of Ca2+ -independent PLA2, (E)-6-(bromomethylene)tetrahydro-3-(1-naphtalenyl)-2H-pyran-2-one (BEL). The Ca2+ ionophore A-23187 yielded similar effects on [Ca2+](i) and lysosomes. Exposure to estradiol also resulted in cPLA2 translocation from cytosol to membranes, lysosome enlargement, and increased protein degradation. These results suggest that the destabilization of lysosomal membranes following cell exposure to estradiol occurs mainly through a Ca2+ -dependent mechanism involving activation of Ca2+ -dependent PLA2. This mechanism promotes lysosome fusion and catabolic activities and may mediate short-term estradiol effects.     

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.     

Cytosolic phospholipase A2 in rat decidual cells: evidence for its role in decidualization

We investigated the existence and possible role of cytosolic phospholipase A2 (cPLA2) in rat decidualized uteri. PLA2 activity in the cytosol of a decidualized uterine horn, induced by intraluminal oil infusion, was significantly higher than that in contralateral intact horn. The activity was almost completely depressed by cPLA2 inhibitors including arachidonyl trifluoromethyl ketone (ATK). The immunoreactive signals for cPLA2 were intense in decidua and glandular epithelial cells. In vivo administration of ATK (0.1-100 microg) caused a dose-dependent inhibition of decidualization. These results show the presence of cPLA2 and its probable implication in decidualization in rat uterus.     

The Ca2+-sensing receptor activates cytosolic phospholipase A2 via a Gqalpha -dependent ERK-independent pathway

The Ca(2+)-sensing receptor (CaR) stimulates a number of phospholipase activities, but the specific phospholipases and the mechanisms by which the CaR activates them are not defined. We investigated regulation of phospholipase A(2) (PLA(2)) by the Ca(2+)-sensing receptor (CaR) in human embryonic kidney 293 cells that express either the wild-type receptor or a nonfunctional mutant (R796W) CaR. The PLA(2) activity was attributable to cytosolic PLA(2) (cPLA(2)) based on its inhibition by arachidonyl trifluoromethyl ketone, lack of inhibition by bromoenol lactone, and enhancement of the CaR-stimulated phospholipase activity by coexpression of a cDNA encoding the 85-kDa human cPLA(2). No CaR-stimulated cPLA(2) activity was found in the cells that expressed the mutant CaR. Pertussis toxin treatment had a minimal effect on CaR-stimulated arachidonic acid release and the CaR-stimulated rise in intracellular Ca(2+) (Ca(2+)(i)), whereas inhibition of phospholipase C (PLC) with completely inhibited CaR-stimulated PLC and cPLA(2) activities. CaR-stimulated PLC activity was inhibited by expression of RGS4, an RGS (Regulator of G protein Signaling) protein that inhibits Galpha(q) activity. CaR-stimulated cPLA(2) activity was inhibited 80% by chelation of extracellular Ca(2+) and depletion of intracellular Ca(2+) with EGTA and inhibited 90% by treatment with W7, a calmodulin inhibitor, or with KN-93, an inhibitor of Ca(2+), calmodulin-dependent protein kinases. Chemical inhibitors of the ERK activator, MEK, and a dominant negative MEK, MEK(K97R), had no effect on CaR-stimulated cPLA(2) activity but inhibited CaR-stimulated ERK activity. These results demonstrate that the CaR activates cPLA(2) via a Galpha(q), PLC, Ca(2+)-CaM, and calmodulin-dependent protein kinase-dependent pathway that is independent the ERK pathway.     

Functional crosstalk between phospholipase D(2) and signaling phospholipase A(2)/cyclooxygenase-2-mediated prostaglandin biosynthetic pathways

We performed reconstitution analyses of functional interaction between phospholipase A(2) (PLA(2)) and phospholipase D (PLD) enzymes. Cotransfection of HEK293 cells with cytosolic (cPLA(2)) or type IIA secretory (sPLA(2)-IIA) PLA(2) and PLD(2), but not PLD(1), led to marked augmentation of stimulus-induced arachidonate release. Interleukin-1-stimulated arachidonate release was accompanied by prostaglandin E(2) production via cyclooxygenase-2, the expression of which was augmented by PLD(2). Conversely, activation of PLD(2), not PLD(1), was facilitated by cPLA(2) or sPLA(2)-IIA. Thus, our results revealed functional crosstalk between signaling PLA(2)s and PLD(2) in the regulation of various cellular responses in which these enzymes have been implicated.     

Involvement of cytosolic phospholipase A2 in the ovulatory process in gonadotropin-primed immature rats

The preovulatory LH surge induces a remarkable increase in ovarian prostaglandins (PGs) which help to mediate the ovulatory process. We investigated whether cytosolic phospholipase A2 (cPLA2) has a role in this PG production in PMSG/hCG-primed immature rats. The immunoreactive signal for cPLA2 was localized in both thecal and granulosa layers of mature follicles and became evident in response to gonadotropins. The PLA2 activity in the whole ovarian cytosol rose slightly after PMSG stimulation, persisted relatively constant until 24 h after hCG injection and thereafter increased gradually. Intra-ovarian bursal injection of arachidonyl trifluoromethyl ketone, a specific inhibitor for cPLA2 ( 1.0-3.0 mg/ovary), significantly reduced ovarian PGE2 content and the ovulation rate. These results suggest that cPLA2 exists in periovulatory follicles and functions in PG production related to the ovulation process.     

Independent functioning of cytosolic phospholipase A2 and phospholipase D1 in Trp-Lys-Tyr-Met-Val-D-Met-induced superoxide generation in human monocytes

Recently, a novel peptide (Trp-Lys-Tyr-Met-Val-D-Met, WKYMVm) has been shown to induce superoxide generation in human monocytes. The peptide stimulated phospholipase A2 (PLA2) activity in a concentration- and time-dependent manner. Superoxide generation as well as arachidonic acid (AA) release evoked by treatment with WKYMVm could be almost completely blocked by pretreatment of the cells with cytosolic PLA2 (cPLA2)-specific inhibitors. The involvement of cPLA2 in the peptide-induced AA release was further supported by translocation of cPLA2 to the nuclear membrane of monocytes incubated with WKYMVm. WKYMVm-induced phosphatidylbutanol formation was completely abolished by pretreatment with PKC inhibitors. Immunoblot showed that monocytes express phospholipase D1 (PLD1), but not PLD2. GF109203X as well as butan-1-ol inhibited peptide-induced superoxide generation in monocytes. Furthermore, the interrelationship between the two phospholipases, cPLA2 and PLD1, and upstream signaling molecules involved in WKYMVm-dependent activation was investigated. The inhibition of cPLA2 did not blunt peptide-stimulated PLD1 activation or vice versa. Intracellular Ca2+ mobilization was indispensable for the activation of PLD1 as well as cPLA2. The WKYMVm-dependent stimulation of cPLA2 activity was partially dependent on the activation of PKC and mitogen-activated protein kinase, while PKC activation, but not mitogen-activated protein kinase activation, was an essential prerequisite for stimulation of PLD1. Taken together, activation of the two phospholipases, which are absolutely required for superoxide generation, takes place through independent signaling pathways that diverge from a common pathway at a point downstream of Ca2+.     

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

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

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.     

Function, activity, and membrane targeting of cytosolic phospholipase A(2)zeta in mouse lung fibroblasts

Group IVA cytosolic phospholipase A(2) (cPLA(2)alpha) initiates eicosanoid production; however, this pathway is not completely ablated in cPLA(2)alpha(-/-) lung fibroblasts stimulated with A23187 or serum. cPLA(2)alpha(+/+) fibroblasts preferentially released arachidonic acid, but A23187-stimulated cPLA(2)alpha(-/-) fibroblasts nonspecifically released multiple fatty acids. Arachidonic acid release from cPLA(2) alpha(-/-) fibroblasts was inhibited by the cPLA(2)alpha inhibitors pyrrolidine-2 (IC(50), 0.03 microM) and Wyeth-1 (IC(50), 0.1 microM), implicating another C2 domain-containing group IV PLA(2). cPLA(2) alpha(-/-) fibroblasts contain cPLA(2)beta and cPLA(2)zeta but not cPLA(2)epsilon or cPLA(2)delta. Purified cPLA(2)zeta exhibited much higher lysophospholipase and PLA(2) activity than cPLA(2)beta and was potently inhibited by pyrrolidine-2 and Wyeth-1, which did not inhibit cPLA(2)beta. In contrast to cPLA(2)beta, cPLA(2)zeta expressed in Sf9 cells mediated A23187-induced arachidonic acid release, which was inhibited by pyrrolidine-2 and Wyeth-1. cPLA(2)zeta exhibits specific activity, inhibitor sensitivity, and low micromolar calcium dependence similar to cPLA(2)alpha and has been identified as the PLA(2) responsible for calcium-induced fatty acid release and prostaglandin E(2) production from cPLA(2) alpha(-/-) lung fibroblasts. In response to ionomycin, EGFP-cPLA(2)zeta translocated to ruffles and dynamic vesicular structures, whereas EGFP-cPLA(2)alpha translocated to the Golgi and endoplasmic reticulum, suggesting distinct mechanisms of regulation for the two enzymes.     

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

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

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.     

Nuclear translocation of cytosolic phospholipase A2 is induced by ATP depletion

Phospholipase A(2) (PLA(2)) enzymes may play a role in cellular injury due to ATP depletion. Renal Madin-Darby canine kidney cells were subjected to ATP depletion to assess the effects of cellular energy metabolism on cytosolic PLA(2) (cPLA(2)) regulation. ATP depletion results in a decrease in soluble cPLA(2) activity and an increase in membrane-associated activity, which is reversed upon restoration of ATP levels by addition of dextrose. In ATP-depleted cells cPLA(2) mass shifts from cytosol to nuclear fractions. GFP-cPLA(2) is localized at the nuclear membrane of stably transfected ATP-depleted LLC-PK(1) cells under conditions where [Ca(2+)](i) is known to increase. cPLA(2) translocation does not occur if the increase in [Ca(2+)](i) increase is inhibited. If [Ca(2+)](i) is allowed to increase when ATP is depleted and the cells are then lysed, cPLA(2) remains associated with nuclear fractions even if the homogenate [Ca(2+)] is markedly reduced. In contrast, cPLA(2), which becomes associated with the nucleus when [Ca(2+)](i) is increased using ionophore, readily dissociates from the nuclear fractions of ATP-replete cells upon reduction of homogenate [Ca(2+)]. Okadaic acid inhibits the ATP depletion-induced association of cPLA(2) with nuclear fractions. Thus energy deprivation results in [Ca(2+)]-induced nuclear translocation, which is partially prevented by a phosphatase inhibitor.     

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

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

D609, an inhibitor of phosphatidylcholine-specific phospholipase C, inhibits group IV cytosolic phospholipase A2

As an inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), D609 has been widely used to explain the role of PC-PLC in various signal transduction pathways. This study shows that D609 inhibits group IV cytosolic phospholipase A2 (cPLA2), but neither secretory PLA2 nor a Ca2+ -dependent PLA2. Dixon plot analysis shows a mixed pattern of noncompetitive and uncompetitive inhibition with Ki = 86.25 microM for the cPLA2 purified from bovine spleen. D609 also time- and dose-dependently reduces the release of arachidonic acid from a Ca2+- ionophore A23187-stimulated MDCK cells. In the AA release experiment, IC50 of D609 was approximately 375 microM, suggesting that this reagent may not enter the cells easily. The present study indicates that the inhibitory effects of D609 on various cellular responses may be partially attributable to the inhibition of cPLA2.     

Cytosolic phospholipase A(2) activity associated with nuclei is not inhibited by arachidonyl trifluoromethyl ketone in macrophages stimulated with receptor-recognized forms of alpha(2)-macroglobulin

We have studied the translocation of cytosolic phospholipase A(2) (cPLA(2)) to nuclei in macrophages stimulated with receptor-recognized forms of alpha(2)-macroglobulin (alpha(2)M*). Translocation of phosphorylated cPLA(2) to nuclei was determined by immunoprecipitation of cPLA(2) in (32)P(i)-labeled cells. The identity of cPLA(2) was established by comparing its mobility on gels with an authentic cPLA(2) standard. cPLA(2) activity was quantified by measuring the release of [(14)C]arachidonic acid from the substrate 1-palmitoyl-2-[1-(14)C]arachidonyl-sn-glycerophosphatidylcholine. alpha(2)M* caused a two- to threefold increase in cPLA(2) phosphorylation and its translocation to nuclei. The p38 MAPK inhibitor SB203580, PKC inhibitor chelerythrin, or depletion of intracellular Ca(2+) profoundly decreased cPLA(2) activity in nuclei isolated from agonist-stimulated cells. The requirement for Ca(2+), PKC, and p38 MAPK activation appears to be of major importance for nuclear cPLA(2) activity. In contrast to cellular cPLA(2) activity, nuclear cPLA(2) activity was not inhibited by arachidonyl trifluoromethyl ketone (AACOCF(3)) in agonist-stimulated cells. It is concluded that the association of cPLA(2) with nuclear membranes in agonist-stimulated cells modifies the activity and the sensitivity of the enzyme to inhibition by AACOCF(3) in this phospholipid environment.