Selective release of phospholipase A2 and lysophosphatidylserine-specific lysophospholipase from rat platelets

Rat platelets released phospholipase A2 and lysophospholipase upon activation with thrombin or ADP. The release of phospholipases was energy-dependent and was not in parallel with that of a known lysosomal marker enzyme, N-acetyl-beta-D-glucosaminidase. The phospholipases are derived from other granules (dense granules or alpha-granules) rather than lysosomal granules of the cells. All of the activities of both phospholipases in the cell free fraction obtained from the activated platelet reaction mixture was recovered in the supernatant after centrifugation at 105,000 X g. The degree of hydrolysis of phospholipids by the phospholipase A2 followed the order: phosphatidylethanolamine (PE) greater than phosphatidylserine (PS) greater than phosphatidylcholine (PC). Phospholipase A2 shows a broad pH optimum (greater than pH 7.0) and absolutely requires Ca2+. Lysophospholipase was specific to lysophosphatidylserine (lysoPS), and neither lysophosphatidylethanolamine (lysoPE) nor lysophosphatidylcholine (lysoPC) was hydrolyzed appreciably. Both 1-acyl- and 2-acyl-lysophosphatidylserine were equally hydrolyzed. Lysophospholipase activity shows similar pH optimum to phospholipase A2. The lysophospholipase activity was lost easily at 60 degrees C. The activity was reduced by the presence of EDTA, though low but distinct activity was observed even in the presence of EDTA. Addition of Ca2+ to the mixtures restores the full activity.     

Arachidonate release and eicosanoid generation by group IIE phospholipase A(2)

The heparin-binding group II subfamily of secretory phospholipase A(2)s (sPLA(2)s), such as sPLA(2)-IIA and -IID, augments stimulus-induced arachidonic acid (AA) release through the cellular heparan sulfate proteoglycan (HSPG)-dependent pathway when transfected into HEK293 cells. Here we show that the closest homolog, sPLA(2)-IIE, also promotes stimulus-induced AA release and prostaglandin (PG) production similar to those elicited by HSPG-dependent sPLA(2)s. Confocal laser microscopic analysis demonstrates the location of sPLA(2)-IIE in cytoplasmic punctate compartments. sPLA(2)-IIE also enhances leukotriene (LT) production and granule exocytosis by RBL-2H3 mastocytoma cells. Expression of sPLA(2)-IIE was highly upregulated in mice injected with lipopolysaccharide (LPS) and in mice with experimental atopic dermatitis. These observations suggest that this enzyme plays a role in the inflammatory process, as proposed for other group II subfamily sPLA(2)s.     

Serotonin 5-HT(2A) receptor activation induces 2-arachidonoylglycerol release through a phospholipase c-dependent mechanism

To date, several studies have demonstrated that phospholipase C-coupled receptors stimulate the production of endocannabinoids, particularly 2-arachidonoylglycerol. There is now evidence that endocannabinoids are involved in phospholipase C-coupled serotonin 5-HT(2A) receptor-mediated behavioral effects in both rats and mice. The main objective of this study was to determine whether activation of the 5-HT(2A) receptor leads to the production and release of the endocannabinoid 2-arachidonoylglycerol. NIH3T3 cells stably expressing the rat 5-HT(2A) receptor were first incubated with [(3)H]-arachidonic acid for 24 h. Following stimulation with 10 mum serotonin, lipids were extracted from the assay medium, separated by thin layer chromatography, and analyzed by liquid scintillation counting. Our results indicate that 5-HT(2A) receptor activation stimulates the formation and release of 2-arachidonoylglycerol. The 5-HT(2A) receptor-dependent release of 2-arachidonoylglycerol was partially dependent on phosphatidylinositol-specific phospholipase C activation. Diacylglycerol produced downstream of 5-HT(2A) receptor-mediated phospholipase D or phosphatidylcholine-specific phospholipase C activation did not appear to contribute to 2-arachidonoylglycerol formation in NIH3T3-5HT(2A) cells. In conclusion, our results support a functional model where neuromodulatory neurotransmitters such as serotonin may act as regulators of endocannabinoid tone at excitatory synapses through the activation of phospholipase C-coupled G-protein coupled receptors.     

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

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

Identification of group X secretory phospholipase A(2) as a natural ligand for mouse phospholipase A(2) receptor

Phospholipase A(2) receptor (PLA(2)R) mediates various biological responses elicited by group IB secretory phospholipase A(2) (sPLA(2)-IB). The recently cloned group X sPLA(2) (sPLA(2)-X) possesses several structural features characteristic of sPLA(2)-IB. Here, we detected a specific binding site of sPLA(2)-X in mouse osteoblastic MC3T3-E(1) cells. Cross-linking experiments demonstrated its molecular weight (180 kDa) to be similar to that of PLA(2)R. In fact, sPLA(2)-X was found to bind the recombinant PLA(2)R expressed in COS-7 cells, and its specific binding detected in mouse lung membranes was abolished by the deficiency of PLA(2)R. These findings demonstrate sPLA(2)-X to be one of the high-affinity ligands for mouse PLA(2)R.     

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.     

Chronic cocaine administration reduces phospholipase A(2) activity in rat brain striatum

BACKGROUND: Phospholipase A(2) (PLA(2)) catalyses the release of free fatty acids used for eicosanoid biosynthesis. We previously reported that calcium-stimulated PLA(2) activity is reduced in the brain of cocaine users and patients with schizophrenia, and have speculated that this is due to dopaminergic hyperactivity in both conditions. METHODS: To investigate these observations under controlled conditions, PLA(2) activity was measured in brain of rats exposed to cocaine and the dopamine receptor antagonist haloperidol. RESULTS: As compared with saline-treated controls, calcium-stimulated PLA(2) activity was reduced (-30%; P<0.01) in the dopamine-rich striatum of animals sacrificed 1 h after chronic (20 mg/kg/day) injection of cocaine, but was normal in haloperidol- (2 mg/kg/day) treated animals, and in the dopamine-poor cortex and cerebellum of animals treated with either drug. CONCLUSION: This confirms and extends our observations in human brain, and further suggests a link between the brain dopaminergic and phospholipid catabolic systems.     

Clearance of group X secretory phospholipase A(2) via mouse phospholipase A(2) receptor.

Given the potent hydrolyzing activity toward phosphatidylcholine, group X secretory phospholipase A(2) (sPLA(2)-X) elicits a marked release of arachidonic acid linked to the potent production of lipid mediators in various cell types. We have recently shown that sPLA(2)-X can also act as a ligand for mouse phospholipase A(2) receptor (PLA(2)R). Here, we found that sPLA(2)-X was internalized and degraded via binding to PLA(2)R associated with the diminished prostaglandin E(2) (PGE(2)) formation in PLA(2)R-expressing Chinese hamster ovary (CHO) cells compared to CHO cells. Indirect immunocytochemical analysis revealed that internalized sPLA(2)-X was co-localized with PLA(2)R in the punctate structures in PLA(2)R-expressing CHO cells. Moreover, in mouse osteoblastic MC3T3-E(1) cells that endogenously express the PLA(2)R, the internalized sPLA(2)-X was localized in lysosomes. These findings demonstrate that PLA(2)R acts as a clearance receptor for sPLA(2)-X to suppress its strong enzymatic activity.     

Toxoplasma gondii secretes a calcium-independent phospholipase A(2)

Phospholipases A(2) (PLA(2)) play an important role in Toxoplasma gondii host cell penetration. They are also key enzymes in the host cell response to the parasite invasion. PLA(2) hydrolyse cellular phospholipids, releasing multiple inflammatory lipidic mediators. We have investigated the biochemical characterisation of T. gondii PLA(2) activity in a mouse-cultured tachyzoite homogenate and in the peritoneal exudate from infected mice, using the hydrolysis of a fluorescent phosphatidylglycerol labelled at the sn-2 position. Spectrofluorimetry and thin-layer chromatography showed a PLA(2) activity (about 0.5-2 nmol/min per mg), calcium-independent, secreted into infected mice peritoneal exudate, with a broad pH activity ranging between 6.5 and 9.5 and resistant to a great number of potential PLA(2) inhibitors except dithio-nitrobenzoic acid (1 mM). An associated phospholipase A(1) activity was also displayed. These results suggest that Toxoplasma gondii displays specific phospholipases different from host enzymes and probably involved at critical steps of infectious cycle.     

Sequential appearance of IL-1 and IL-6 activities in rat carrageenin-induced pleurisy.

IL-1 and IL-6 activities were measured in the pleural exudate of rats during carrageenin-induced pleurisy to examine the relationship of the local production of cytokines to the inflammatory reaction. Time courses of appearance of the cytokines and inflammatory parameters in the exudate were compared. IL-1 activity and exudate volume started to increase at 1 h after the carrageenin injection, and then slightly later IL-6 activity and leukocyte number began to increase. IL-1 showed peak activity of approximately 700 U at 3 h and IL-6, of 6000 U at 5 h in the exudate, whereas exudate volume and number of polymorphonuclear leukocytes continued to increase thereafter. Furthermore, IL-6 level in the plasma of the carrageenin-injected rats showed a peak at 4 h (30 U/ml), and when rhIL-1 alpha (100,000 U) was intrapleurally injected, the more rapid increase in plasma IL-6 level was demonstrated at 1 h (30 U/ml). This latter rise was neutralized with simultaneous injection of anti-rhIL-1 alpha antibody. These facts indicate the possibility that IL-1 produced in the exudate or injected could rapidly propagate a signal to induce IL-6 production in the circulation. It took several hours to transmit an inflammatory signal that stimulated the liver to synthesize the acute-phase protein, T-kininogen. The time lag from the peak induction of IL-1 to the T-kininogen-increase in the pleurisy corresponded well to the interval for T-kininogen-increase by exogenous rhIL-1 alpha injection. These results strongly suggest that the initial inflammatory stimulus induces sequentially IL-1, IL-6, and T-kininogen production in this carrageenin inflammation.     

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

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

Inhibition of phospholipase A(2) as a therapeutic target

The hydrolysis of cell membrane phospholipids by phospholipase A(2) (PLA(2)) leads to the production of numerous lipid mediators of diverse pathological conditions, mainly inflammatory diseases. These include lysophospholipids and their derivatives, and arachidonic acid and its derivatives (the eicosanoids). Both these groups of mediators are produced predominantly by the secretory PLA(2)s (sPLA(2)s) which hydrolyze the phospholipids of the cell surface membrane. Protection of cell membrane from these 'inflammatory enzymes' can therefore be used for the treatment of inflammatory processes. A prototype of cell-impermeable PLA(2) inhibitors, which protect the cell membrane from different sPLA(2)s without affecting vital phospholipid metabolism, is presented and discussed in the present review.     

A major role for phospholipase A2 in antigen-induced arachidonic acid release in rat mast cells.

Cross-linking of IgE receptors by antigen stimulation leads to histamine release and arachidonic acid release in rat peritoneal mast cells. Investigators have reported a diverse distribution of [3H]arachidonate that is dependent on labelling conditions. Mast cells from rat peritoneal cavity were labelled with [3H]arachidonic acid for different periods of time at either 30 or 37 degrees C. Optimum labelling was found to be after 4 h incubation with [3H]arachidonate at 30 degrees C, as judged by cell viability (Trypan Blue uptake), responsiveness (histamine release) and distribution of radioactivity. Alterations in 3H-radioactivity distribution in mast cells labelled to equilibrium were examined on stimulation with antigen (2,4-dinitrophenyl-conjugated Ascaris suum extract). The results indicated that [3H]arachidonic acid was lost mainly from phosphatidylcholine and, to a lesser extent, from phosphatidylinositol. A transient appearance of radiolabelled phosphatidic acid and diacylglycerol indicated phosphatidylinositol hydrolysis by phospholipase C. Pretreatment with a phospholipase A2 inhibitor, mepacrine, substantially prevented the antigen-induced liberation of [3H]arachidonic acid from phosphatidylcholine. It can be thus concluded that, in the release of arachidonic acid by antigen-stimulated mast cells, the phospholipase A2 pathway, in which phosphatidylcholine is hydrolysed, serves as the major one, the phospholipase C/diacylglycerol lipase pathway playing only a minor role.     

A phospholipase A2 in the supernatant fraction of rat spleen. Its similarity to rat pancreatic phospholipase A2

Rat spleen supernatant contained two forms of calcium-dependent cellular phospholipase A2 which could be separated from each other by TEAE-cellulose chromatography. The phospholipase A2, named PLA2 S-1, present in the major flow-through fraction was purified to homogeneity. The structural and catalytic properties of splenic PLA2 S-1 were systematically compared with those of rat pancreatic phospholipase A2. Structural evidence, including the sequence of the N-terminal 32 residues, peptide maps obtained on Achromobacter protease I digestion and cyanogen bromide cleavage, and the amino acid composition, showed the close similarity of the two enzymes. Their catalytic and immunochemical properties were also similar. These results demonstrated the existence of a pancreatic type phospholipase A2 in a non-pancreatic organ as a member of the cellular phospholipases A2 and suggest the potential functional involvement of pancreatic type phospholipase A2 in cellular phospholipid metabolism.     

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

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

Expression and function of phospholipase A(2) in brain

Phospholipase A(2) (PLA(2)) appears to play a fundamental role in cell injury in the central nervous system. We have investigated PLA(2) expression in the astrocytoma cell line 1231N1, and found that GIVA, GIVB, GIVC and GVI PLA(2) messages are expressed. PLA(2) activity is increased by inflammatory/injury stimuli such as interleukin-1beta and lipopolysaccharide in these cells but with very different time courses. The arachidonic acid liberated is converted to prostaglandin E(2), possibly by cyclooxygenase-2, which is induced by inflammatory stimuli. This cell system emerges as a model to study injury/inflammation-related activation of the new PLA(2) forms GIVB and GIVC.     

Localization and regulation of cytosolic phospholipase A(2)

Liberation of arachidonic acid by cytosolic phospholipase A(2) (cPLA(2)) upon cell activation is often the initial and rate-limiting step in leukotriene and prostaglandin biosynthesis. This review discusses the essential features of cPLA(2) isoforms and addresses intriguing insights into the catalytic and regulatory mechanisms. Gene expression, posttranslational modification and subcellular localization can regulate these isoforms. Translocation of cPLA(2)alpha from the cytosol to the perinuclear region in response to calcium transients is critical for the immediate arachidonic acid release. Therefore, particular emphasis is placed on the mechanism of the translocation and the role of the proteins and lipids implicated in this process. The regional distribution and cellular localization of cPLA(2) may help to better understand its function as an arachidonic acid supplier to downstream enzymes and as a regulator of specific cellular processes.     

Calcium-independent phospholipase A2 in rat tissue cytosols

Cytosols (105,000 X g supernatant) from seven rat tissues were assayed for Ca2+-independent phospholipase A2 activity with either 1-acyl-2-[1-14C]linoleoyl-sn-glycero-3-phosphocholine, 1-acyl-2-[1-14C]linoleoyl-sn-glycero-3-phosphoethanolamine or 1-O-hexadecyl-2-[9,10-3H2]oleoyl-sn-glycero-3-phosphocholine as substrate. Low but consistent activities ranging from 10-120 pmol/min per mg protein were found in all tissues. The highest activities were present in liver, lung and brain. Total activities in mU/g wet weight were rather constant, ranging from 0.43 (heart) to 1.36 (liver). The soluble enzyme from rat lung cytosol was further investigated and was found to be capable of hydrolyzing microsomal membrane-associated substrates without exhibiting much selectivity for phosphatidylcholine species. Comparative gel filtration experiments of cytosol prepared from non-perfused and perfused lungs indicated that part of the Ca2+-independent phospholipase A2 originated from blood cells, but most of it was derived from lung cells. Lung cytosol also contained Ca2+-dependent phospholipase A2 activity, a small part of which originated from blood cells, presumably platelets. The major amount of Ca2+-dependent phospholipase A2 activity, however, came from lung cells. Neither this enzyme nor the Ca2+-independent phospholipase A2 from lung tissue showed immunological cross-reactivity with monoclonal antibodies against Ca2+-dependent phospholipase A2 isolated from rat liver mitochondria.     

(S)-tetrahydroisoquinoline alkaloid inhibits LPS-induced arachidonic acid release through downregulation of cPLA2 expression

Sepsis, a systemic inflammatory response syndrome, remains a potentially lethal condition. (S)-1-α-Naphthylmethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (CKD712) is noted as a drug candidate for sepsis. Many studies have demonstrated its significant anti-inflammatory effects. Here we first examined whether CKD712 inhibits lipopolysaccharide (LPS)-induced arachidonic acid (AA) release in the RAW 264.7 mouse monocyte cell line, and subsequently, its inhibitory mechanisms. CKD712 reversed LPS-associated morphological changes in the RAW 264.7 cells, and inhibited LPS-induced release of AA in a concentrationdependent manner. The inhibition was apparently due to the diminished expression of a cytosolic form of phospholipase A₂ (cPLA₂) by CKD712, resulting from reduced NF-κB activation. Furthermore, CKD712 inhibited the activation of ERK1/2 and SAP/JNK, but not of p38 MAPK. CKD712 had no effect on the activity or phosphorylation of cPLA₂ and on calcium influx. Our results collectively suggest that CKD712 inhibits LPS-induced AA release through the inhibition of a MAPKs/NF-κB pathway leading to reduced cPLA₂ expression in RAW 264.7 cells.     

Secreted phospholipase A(2) as a new enzymatic trigger mechanism for localised liposomal drug release and absorption in diseased tissue

Polymer-coated liposomes can act as versatile drug-delivery systems due to long vascular circulation time and passive targeting by leaky blood vessels in diseased tissue. We present an experimental model system illustrating a new principle for improved and programmable drug-delivery, which takes advantage of an elevated activity of secretory phospholipase A(2) (PLA(2)) at the diseased target tissue. The secretory PLA(2) hydrolyses a lipid-based proenhancer in the carrier liposome, producing lyso-phospholipids and free fatty acids, which are shown in a synergistic way to lead to enhanced liposome destabilization and drug release at the same time as the permeability of the target membrane is enhanced. Moreover, the proposed system can be made thermosensitive and offers a rational way for developing smart liposome-based drug delivery systems. This can be achieved by incorporating specific lipid-based proenhancers or prodestabilisers into the liposome carrier, which automatically becomes activated by PLA(2) only at the diseased target sites, such as inflamed or cancerous tissue.