Purification Of Phospholipase A2 Research Articles

New Mechanistic and Therapeutic Insights into Purified Phospholipase A2 from Pseudechis Australis Snake Venom - a Novel Anticancer Agent for the Treatment of Precursor B-Cell Acute Lymphoblastic Leukaemia

Introduction Precursor B-cell acute lymphoblastic leukaemia (pre-B ALL) is the most common childhood malignancy and the leading cause of cancer-related death in children and young adults. Standard treatment options for pre-B ALL typically involve multiple regimens of DNA-damaging chemotherapy agents, which despite their high curative rates, are often tolerated poorly by patients. Previously our group has purified, and characterised a novel anti-leukaemia compound, termed PA-PLA2 from Pseudechis australis snake venom. Comprised of three phospholipase A2 (PLA2) enzymes, PA-PLA2 was shown to induce necrotic cell death in pre-B ALL cells, with RNA-sequencing analysis revealing enrichment of PI3K/Akt signalling as well as inflammation and immune response genes. Importantly, PA-PLA2 has also been shown to be non-cytotoxic towards normal stem cells from donor peripheral blood and bone marrow, raising the prospect of its use as a novel and selective treatment. In this study, we further explore the mechanism of action and therapeutic potential of PA-PLA2 in relevant models of pre-B ALL. Methods PA-PLA2 was purified and characterised previously using, reverse-phase high-performance liquid chromatography and mass spectrometry (Boncan et al., 2021). Reh and SD-1 pre-B ALL in vitro cell lines, were used to study PA-PLA2's anti-leukaemia action. Protein immunoblotting was used to validate previously performed RNA-sequencing, proteomics and phosphoproteomics analysis. Densitometry analysis of immunoblots was completed using ImageJ software. To assess the therapeutic potential of PA-PLA2 in combination with standard of care therapies, we performed a drug screen, comprised of a custom panel of 48 standard of care and clinical agent compounds. CellTox™ Green cytotoxicity assay was used as a readout of cell death, with effective drug combinations validated using CellTiter® Glo viability assay, and combination indices (CI) calculated using CompuSyn software. Results Time course protein immunoblotting analyses of PA-PLA2-sensitive cell lines, Reh and SD-1 treated with IC50 doses of PA-PLA2 (Reh; 1µg/mL, SD-1; 25µg/mL) revealed activation of pRIPK1Ser166/RIPK1 in both cell lines after one hour treatment (p<0.0001 and p<0.01 respectively). Activation was accompanied by cleavage of RIPK1 after 24 hours in Reh cells only (p<0.05). One hour treatment with PA-PLA2 was also found to activate pAktSer473/Akt(pan) and pAktThr308/Akt(pan) signalling in Reh cells (p<0.001 and p<0.05 respectively). In contrast, a transient and latent response of Akt activation was observed in SD-1 cells, with pAktSer473/Akt(pan) levels initially decreasing after one hour (p<0.05), before increasing again and resulting in activation at 24 hours (p<0.01). Activation of pAktThr308/Akt(pan) was only observed after 24 hours treatment also (p<0.05). Examining the signalling axes driving PA-PLA2's mechanism of action, phosphoproteomics showed a reduction in pc-MycSer348 expression in both cell lines (p<0.05). Immunoblotting analysis confirmed the c-Myc modifying role of PA-PLA2, with c-Myc total expression found to be elevated in SD-1 cells after 8 hours treatment (p<0.001). To evaluate the therapeutic benefit of including PA-PLA2 in standard of care regimens, we performed a drug screen that identified combinations of PA-PLA2 with Cytarabine, Idarubicin-HCl or Vorinostat (SAHA) as being synergistic in Reh cells. SAHA displayed the best synergism profile, with CI values of < 1 being obtained for all combinations of PA-PLA2 (0.1, 0.5, 1, 2.5 and 5µg/mL) with SAHA (1,10 and 20µM). Importantly, proteomics analysis revealed increased expression of HDAC2 in Reh cells (p<0.05), implicating HDAC2 as a possible effector of synergism. Conclusion This study has identified RIPK1 and Akt as effectors of the mechanism of action of PA-PLA2. PA-PLA2 treatment had a modifying role on the expression of the multifunctional transcription factor c-Myc. Importantly PA-PLA2 was shown to act synergistically with Cytarabine as well as SAHA and Idarubicin-HCl, with HDAC2 implicated as a possible effector of synergism with SAHA. Overall, this study has further elucidated the mechanism of action of PA-PLA2, with the identified synergy of PA-PLA2 with standard of care agents possibly yielding a novel, treatment option for patients with pre-B ALL.

Roe-derived phospholipid administration enhances lymphatic docosahexaenoic acid-containing phospholipid absorption in unanesthetized rats

Plasma n-3 fatty acids are important as the supplying pool of n-3 fatty acids to various tissues including the brain, although the relationship between dietary n-3 fatty acids and their molecular species in the plasma are not fully clarified. We investigated the intestinal absorption of docosahexaenoic acid (DHA) derived from fish roe phospholipid (Roe-PL) and compared it with fish oil triacylglycerol and free DHA using unanesthetized lymph-cannulated rats. The DHA absorption from intraduodenally administered three samples were not significantly different, whereas Roe-PL administration resulted in a significantly higher level of DHA in the phospholipid fraction than the other two samples administrations. DHA in Roe-PL at the sn–2 position was less hydrolyzed by pancreatin than by purified phospholipase A2 in vitro and simultaneous administration of free DHA and lysophosphatidylcholine did not produce the same results as the Roe-PL administration. Our results indicate that dietary DHA-containing phospholipid is effective to increase the systemic DHA incorporated into phospholipids via intestinal absorption and biosynthesis.

Inhibition of the toxic effects of Bothrops asper venom by pinostrobin, a flavanone isolated from Renealmia alpinia (Rottb.) MAAS

Ethnopharmacological relevanceRenealmia alpinia has been traditionally used to treat snakebites by indigenous Embera-Katíos tribes belonging to the regions of Antioquia and Chocó, Colombia, and it has been shown to inhibit the enzymatic and biological activities of Bothrops venoms and their purified phospholipase A2 (PLA2) toxins. In addition to its common local usage against snakebites, Renealmia alpinia is commonly used to treat pain. To evaluate the inhibitory ability of pinostrobin, the main compound in the dichloromethane extract of Renealmia alpinia, on the toxic effects of Bothrops asper venom through in vitro and in vivo models and to evaluate its activity against pain and edema. Materials and methodsPinostrobin was isolated from the dichloromethane extract of Renealmia alpinia leaves. The protective properties of the extract and of pinostrobin against the indirect hemolytic, coagulant and proteolytic effects of Bothrops asper venom were evaluated in vitro, and the anti-hemorrhagic and anti-inflammatory activity were evaluated in vivo. ResultsRenealmia alpinia extract significantly inhibited the proteolytic activity and indirect hemolytic activity of Bothrops asper venom at a venom:extract ratio of 1:20. Moreover, the present data demonstrate that pinostrobin may mitigate some venom-induced local tissue damage due to hemorrhagic effects, and the compound is also responsible for the analgesic and anti-inflammatory activity of the extract from Renealmia alpinia. This is the first report to describe pinostrobin in the species Renealmia alpinia and its properties in vitro against Bothrops asper venom. ConclusionOur studies of the activity of Renealmia alpinia against the venom of Bothrops asper have confirmed that this species possesses inhibitory effects against Bothrops asper venom in both in vitro and in vivo models and that these effects may be due to pinostrobin, supporting the traditional usage of the plant. Additionally, pinostrobin may be responsible for the anti-hemorrhagic and analgesic activity (peripheral analgesic activity) of Renealmia alpinia.

Effect of Methanol Extract of Mangifera indica Linn. Kernel on Partially Purified Phospholipase A2 (PLA2) from Venom of Naja nigricollis: An in vitro Study

Effect of Methanol Extract of Mangifera indica Linn. Kernel on Partially Purified Phospholipase A2 (PLA2) from Venom of Naja nigricollis: An in vitro Study

Acute toxicity of vipoxin and its components: is the acidic component an “inhibitor” of PLA2 toxicity?

Vipoxin is a heterodimeric neurotoxin isolated from the venom of the Bulgarian long-nosed viper Vipera ammodytes meridionalis. Vipoxin represents a noncovalent association of two subunits – a basic and toxic phospholipase A2 enzyme, and an acidic non-enzymatic component (vipoxin's acidic component). It was postulated that the phospholipase A2 subunit was more toxic than the whole vipoxin complex and the function of the acidic component was to reduce the enzymatic and toxic activities of the basic phospholipase A2. In the present study, we report new data on the acute toxicity (LD50) of vipoxin and its individual separated components. Vipoxin LD50 (mice, i.p. and i.v.) values were found to be 0.7–1.2 mg/kg b.w. (i.p.) and 0.9–1.3 mg/kg b.w. (i.v.). The established LD50 values for the separated pure phospholipase A2 subunit are higher – 10.0–13.0 mg/kg b.w (i.p.) and 2.2–3.0 mg/kg b.w. (i.v.), i.e. the individual phospholipase A2 subunit displays less toxic activity than vipoxin, contrary to the data published in the literature. The reconstituted vipoxin complex (obtained after preliminary incubation of pure separated phospholipase A2 and acidic component showed enzyme activity and toxicity comparable to that of the native vipoxin complex. Addition of acidic component to the phospholipase A2 subunit showed a positive effect on the enzymatic activity, reaching maximal enzyme reaction rate of acidic component to phospholipase A2 molar ratio of 0.8:1 on using 4-nitro-3-octanoyloxy-benzoic acid as substrate. For the first time we showed that the acidic subunit was absolutely required for the toxic activity of vipoxin. Based on the obtained results, we assume that the function of the acidic component is to stabilize the neurotoxin's quaternary structure, required for its toxic and enzymatic activities, similarly to the role of the acidic component of crotoxin.

Anti‐inflammatory Effects of Cocoa Procyanidins

Inflammation, as evaluated by increased inflammatory cytokines and other markers, represents a link between obesity and its comorbidities. Cocoa (Theobroma cacao) is a procyanidin‐rich food with putative anti‐inflammatory effects; however, the mechanism of action remains to be fully elucidated. The objective of the present study is to determine the effects of cocoa procyanidins (CPs) with varying degree of polymerization (DP) on inflammatory markers related to eicosanoid metabolism and cytokine production using purified enzymes and cultured RAW 264.7 murine macrophage cells. CPs with DP ≥7 dose‐dependently inhibited purified phospholipase A2 (IC50 =3.2 μg/mL) and cyclooxygenase‐2 (IC50= 57.7 μg/mL) in vitro, and significantly decreased prostaglandin E2 production in lipopolysaccharide (LPS)‐stimulated macrophages. Pre‐treatment of macrophages with CP octamer (50 μM) significantly suppressed secretion of the proinflammatory cytokines tumor necrosis factor‐α (70%), interleukin‐6 (100%), monocyte chemoattractant protein‐1 (50%) compared to LPS‐stimulated controls. These results demonstrate the anti‐inflammatory effects of CPs and provide potential mechanistic biomarkers to be further tested in obese subjects under inflammatory conditions. This study was supported in part by a grant from Hershey Foods and by NIH Grant AT004678.Grant Funding Source: NIH Grant AT004678 and Hershey Foods

Anti-snake venom effect of secodolastane diterpenes isolated from Brazilian marine brown alga Canistrocarpus cervicornis against Lachesis muta venom

The effect of a Brazilian algae extract and also a mixture of two secodolastane diterpenes (linearol/isolinearol) that were isolated from the marine brown alga Canistrocarpus cervicornis were evaluated against biological activities of Lachesis muta snake venom. In vitro assays showed that the crude extract and the diterpenes were able to inhibit the clotting and proteolytic activity induced by L. muta crude venom, but not the hemolytic activity. However, only the diterpenes inhibited the hemolysis caused by a purified phospholipase A2 previously isolated from L. muta venom, denoted LM-PLA2-I. Interestingly, the crude algal extract and the diterpenes were able to protect mice from hemorrhage induced by L. muta venom. Thus, we may conclude that marine algae are rich and powerful sources of molecules that may be used against L. muta accidents in order to improve treatment of envenomation by this snake.

Synthesis and Evaluation of Benzophenone Oximes Derivatized with Sydnone as Inhibitors of Secretory Phospholipase A2 with Anti-inflammatory Activity

A series of benzophenone oximes appended with sydnone (3a--h) bearing different substituents on aroyl moiety were synthesized to evaluate in vivo and in vitro for their inhibitory activity against purified phospholipase A2 (PLA2) enzymes from snake venom and human inflammatory pleural and ascites fluid. In vivo and in vitro inhibition studies were carried out against PLA2 with respect to the modification of the pharmacophore (substituent) to analyze the specificity for PLA2. The substituent at the aroyl ring was responsible for enhancing the inhibition towards PLA2 enzymes. Most of the newly synthesized compounds inhibit the purified PLA2 enzyme, and the inhibition was more in hydrophobic and aromatic substituents and less when no such substituents were present. The inhibitory effect of the compounds appeared to be due to the direct interaction of compounds with the enzyme. Inhibition is substrate dependent, and the inhibition competes with the substrate for the same binding site of the enzyme. The most active interacting compound 3h from in vitro inhibition of PLA2 activity showed similar potency in the in vivo neutralization of PLA2 induced mouse paw edema and hemolytic activity. Thus, the in vitro inhibition correlated well with the in vivo inhibition and hence the reported derivatives are therapeutically important anti-inflammatory drugs.

Toxicity of phospholipases A2 D49 (6-1 and 6-2) and K49 (Bj-VII) from Bothrops jararacussu venom

Purified phospholipase A2 (PLA2) enzymes from Bothrops jararacussu snake venom were examined to evaluate NIH 3T3 and COS7 fibroblast cytotoxicity, as well as muscle myotoxic and inflammatory activities. Separation of fractions Bj-VII (from BthTX-I; a Lys49 PLA2 homolog) and 6-1 and 6-2 (from BthTX-II; an Asp49 PLA2) from B. jararacussu snake venom by SDS-PAGE in tricine buffer in the absence and presence of dithiothreitol revealed a homodimer with an estimated molecular mass of approximately 30 kDa (monomer mass approximately 15 kDa). This finding indicates that these toxins form dimeric complexes-a previously reported tendency among PLA2s. These toxins were assayed for viability with the MTT assay, which is used to examine the effects of phospholipases on the mitochondrial viability of cells. The toxins were also assayed for cytolysis of the fibroblast cell lines NIH 3T3 and COS7 by quantification of lactate dehydrogenase released into the medium. The results indicate that the PLA2s 6-1, 6-2 and the Bj-VII PLA2 homolog studied here induce moderate footpad edema and local myotoxicity. Moreover, exposure to these phospholipases led to a reduction in fibroblast viability; at the 1 muM dose of PLA2 tested, a reduction of 50% in cell viability was observed. The present findings indicate that the inflammatory activity observed in envenomation may be correlated with the cytotoxicity observed in fibroblasts.

P.1.c.011 Chronic treatment with paroxetine decreases defensive reactions elicited by golden hamsters in confront with venomous coral snakes

Some differences between the reaction of antiserum to reconstituted crotoxin complex, to ‘native’ crotoxin and to whole Crotalus durissus terrificus (South American rattlesnake) venom were detected by enzyme-linked immunosorbent assay (ELISA) and by immunodiffusion. ELISA showed the presence of antibodies to the components of the crotoxin complex, phospholipase A2 and crotapotin. When the antiserum was tested against North American rattlesnake venoms some species gave a positive reaction (C. horridus atricaudatus and C. basiliscus), whilst the venoms of other species were virtually negative (C. durissus totonacus, C. horridus horridus, C. viridis viridis and C. atrox). Confirmation of antigenic similarities between crotoxin and Mojave toxin (from the venom of C. scutulatus scutulatus) was obtained with ELISA and some implications of these species differences in antigen-antibody reaction are discussed. No paraspecificity was observed with heterologous snake venoms from other Crotalidae, Elapidae, Hydrophiidae or Viperidae. ELISA also showed a lack of cross-reactivity of the antiserum to heterologous purified phospholipases A2 from Enhydrina schistosa, Naja nigricollis or Apis mellifera venoms or from porcine pancreas. The antiserum reacted to the homologous phospholipase A2 inactivated with p-bromophenacyl bromide as well as to the detoxified crotoxin complex reconstituted with this modified phospholipase A2. This may be useful in the raising of high titres of antibody to crotoxin.

Selective Inhibition of Phospholipases by Atiprimod, a Macrophage Targeting Antiarthritic Compound

Azaspiranes are cationic amphiphilic compounds that are active in a number of models of autoimmune disease and transplantation. Repeated administration of cationic amphiphiles induces phospholipid accumulation in a variety of species. The present study was conducted to explore the mechanism of phospholipid accumulation in rats caused by treatment with the novel azaspirane, SK&F 106615 (atiprimod). Atiprimod inhibited the activities of partially purified phospholipases A2 and C, but not D, in a noncompetitive manner in vitro. Treatment of rats for 28 days with 10 mg/kg/day of atiprimod increased the contents of arachidonate-containing molecular species within plasmalogen subclasses of hepatic phosphatidylcholine and phosphatidylethanolamine. In contrast, diacyl-linked species were not affected, indicating a selective effect upon an hepatic plasmalogen-selective phospholipase A2. Taken together, the data suggest that the beneficial effects of atiprimod in autoimmune diseases may involve inhibition of phospholipase A2 and C activities. Further, the data suggest that atiprimod is a selective inhibitor of plasmalogen-selective phospholipase A2in vivo.

Tight binding inhibitors of 85-kDa phospholipase A2 but not 14-kDa phospholipase A2 inhibit release of free arachidonate in thrombin-stimulated human platelets.

An analogue of arachidonic acid in which the COOH group is replaced by a trifluoromethyl ketone group (COCF3) has recently been shown to be a tight binding inhibitor of the 85-kDa cytosolic phospholipase A2 that is found in platelets and other cells (Street, I. P., Lin, H.-K., Laliberté, F., Ghomashchi, F. G., Wang, Z., Perrier, H., Tremblay, N. M., Huang, Z., Weech, P. K., and Gelb, M. H. (1993) Biochemistry 32, 5935-5940). This trifluoromethyl ketone inhibits most of the arachidonate release from the phospholipid pool in thrombin-stimulated human platelets at concentrations of 0-40 microM with 4 x 10(8) platelets/ml. A structure-function analysis of related compounds reveals a good correlation between the inhibition of the purified phospholipase A2 and the blockage of arachidonate release in platelets. A number of recently described potent inhibitors of the 14-kDa phospholipase A2 that is secreted from activated platelets have no effect on the level of free arachidonate production. Furthermore, the addition of a large amount of recombinant 14-kDa phospholipase A2 to platelets does not produce free arachidonate, and it does not alter the amount of arachidonate released following platelet activation with thrombin. These studies provide strong pharmacological evidence for the role of the cytosolic phospholipase A2 in producing most, if not all, of the liberated arachidonate in thrombin-stimulated human platelets, and they show that tight binding membrane-residing inhibitors of the cytosolic phospholipase A2 can block the eicosanoid cascade in living cells.

Suppression of platelet-derived growth factor receptor tyrosine kinase activity by unsaturated fatty acids.

Treatment of cells with platelet-derived growth factor (PDGF) was previously shown to increase the release of unesterified fatty acids from phospholipids. In view of these observations we examined the effect of various fatty acids on PDGF receptor activity. Unsaturated (oleic, linoleic, linolenic, and arachidonic) but not saturated (stearic and arachidic) fatty acids significantly inhibited the tyrosine kinase activity of the PDGF receptor in intact cells and membrane preparations. Half-maximal inhibition (IC50) was observed between 60 and 100 microM and maximal inhibition between 170 and 200 microM. Lysophospholipids and phospholipids had no effect on receptor activity. Activation of endogenous phospholipase A2 by mellitin in vivo or hydrolysis of phosphatidylcholine by purified phospholipase A2 in vitro inhibited PDGF receptor autophosphorylation similar to that of purified fatty acids. Dimerization of PDGF receptors in vivo was significantly reduced by concentrations of arachidonic acid inhibitory for receptor kinase activity while the binding of PDGF was only marginally affected. These data suggest a possible feedback mechanism resulting in the reduction of PDGF receptor activity by unsaturated fatty acids generated downstream within the PDGF-dependent signal transduction pathway and this effect may be as a direct result of decreased receptor dimerization and/or kinase activity.

Immunohistochemical study of phospholipase A2 in boyine prostate

Approximately 1,100-fold purified phospholipase A2 (PLA2) from bovine prostate was injected into rabbit to prepare polyclonal antibodies. Antibodies produced showed specific immunoprecipitation only with the purified enzyme, as well as with homogenate of bovine prostatic tissue. By Western blot analysis or by immunodiffusion test, no cross-reactivity with PLA2 purified from human seminal plasma, bovine pancreas, Crotalus adamanteus venom, or with partially purified PLA2 from bovine seminal vesicle fluid or Cowper's glands was observed. Using the indirect peroxidase technique, PLA2 was localized in the cytoplasm of bovine prostatic epithelial cells. By immunogold microscopy, this enzyme was directly visualized inside the lysosomes, as well as in the endoplasmic reticulum of the glandular epithelial cells. Enzyme activity was localized in two principal subcellular sites: the mitochondria and lysosome-enriched fraction, and in the microsomal fraction.

Augmentation of Prostaglandin E2 Production by Mammalian Phospholipase A2 Added Exogenously1

Rat group II phospholipase A2 added exogenously to A23187-activated HL-60 granulocytes augmented their production of prostaglandin E2. Human group II phospholipase A2 and porcine group I phospholipase A2 augmented the prostaglandin E2 production in a similar manner. No significant increase in prostaglandin E2 production was observed when cells were treated with purified phospholipase A2 in the absence of A23187. Extracellular phospholipase A2 at inflamed sites may contribute to the generation of pro-inflammatory lipid mediators by hydrolyzing the cellular phospholipids of activated inflammatory cells.

A 32 kDa lipocortin from human mononuclear cells appears to be identical with the placental inhibitor of blood coagulation

A 32 kDa protein isolated from human mononuclear cells is a member of the lipocortin family, a new group of Ca2+-dependent lipid-binding proteins thought to be involved in the regulation of phospholipase A2, in exocytosis and in membrane-cytoskeleton interactions. Purification of this protein was based on its ability to associate with membrane phospholipids in a Ca2+-dependent manner and its capacity to inhibit purified phospholipase A2 from pig pancreas. Using immunological detection, we show that it is present in various cells involved in the inflammatory and coagulation processes. We present extensive amino acid data that strongly suggest that this protein is identical with a recently described inhibitor of blood coagulation, with endonexin II and with lipocortin V. Sequence alignment with other known proteins show a significant degree of homology with lipocortins I and II, the substrates of the epidermal-growth-factor receptor tyrosine kinase and the oncogene pp60src tyrosine kinase respectively, and with protein II. The possible physiological role of this 32 kDa lipocortin is discussed.

Ultracytochemical Demonstration of Phospholipids in the Surface Layer of the Guinea Pig Eustachian Tube

The surface layer, especially the extracellular mucous blanket, in the eustachian tube of adult guinea pigs, was preserved very effectively with an intravascular perfusion of twice diluted Karnovsky's fixative following overnight incubation in the same fixative containing 1% tannic acid. Multilamellar bodies were observed in both mucous lining layers of the tube and the epithelial cells of its lining, but neither reticular nor lattice-like ultrastructures could be detected. Mucous blankets on the pharyngeal portion were composed of an electron-dense surface film and a hypophase. The hypophase was marked heterogenous, and it could hardly be observed on the isthmus. The enzymic digestive method using a purified phospholipase A2 was applied to the middle portion of the tube, and the reaction products, probably phospholipids, could be seen both on the surface film of the lining layers and around some of the dark inclusion bodies in the secretory cells. These findings suggest that such phospholipids may play an important role in reducing surface tension of the eustachian tube.

Quinacrine prevention of intestinal ischaemic mucosal damage is partly mediated through inhibition of intraluminal phospholipase A2.

The influence of total ischaemia and revascularization on luminal phospholipid metabolism in the rat small intestine was investigated. Two hours of total ischaemia followed by five minutes of revascularization caused increases in phospholipase A2 activity, and lysophosphalidylcholine content in the gut lumen. The above treatment also resulted in mucosal damage expressed as an increase in N-acetyl-beta-glucosaminidase activity in the lumen. Pretreatment of animals with the phospholipase A2 inhibitor, quinacrine prevented the increases in luminal phospholipase A2 activity and mucosal damage following ischaemia and revascularization. Intraluminal injection of either phospholipase A2 purified from snake venom or Triton X-100 resulted in increased activity of N-acetyl-beta-glucosaminidase in the luminal content. Again, quinacrine pretreatment of animals prevented the increases in mucosal permeability and activity of N-acetyl-beta-glucosaminidase after intraluminal injection of purified phospholipase A2. On the other hand quinacrine pretreatment had no influence on the observed effects of Triton X-100 treatment. These findings suggest that an increase in luminal phospholipase A2 could be involved in mediating the mucosal injury caused by small intestinal ischaemia.

Enzymatic modification of low density lipoprotein by purified lipoxygenase plus phospholipase A2 mimics cell-mediated oxidative modification

Low density lipoprotein (LDL) can be oxidatively modified by cultured endothelial cells or by cupric ions, resulting in increased macrophage uptake of the lipoprotein. This process could be relevant to the formation of macrophage-derived foam cells in the early atherosclerotic lesion. The mechanism of endothelial cell modification of LDL is unknown. In the present work we show that incubation of LDL with purified soybean lipoxygenase, in the presence of pure phospholipase A2, can mimic endothelial cell-induced oxidative modification. Typically, incubation with lipoxygenase plus phospholipase A2 caused: 1) generation of about 15 nmol of thiobarbituric acid-reactive substances per mg of LDL protein; 2) a 4- to 7-fold increase in the rate of subsequent macrophage degradation of the LDL; 3) a 10-fold decrease in recognition by fibroblasts; 4) a marked increase in electrophoretic mobility in agarose gels; and, 5) disappearance of intact apoprotein B on SDS polyacrylamide gels. Degradation of the enzymatically modified LDL by macrophages was competitively inhibited by endothelial cell-modified LDL and by polyinosinic acid, but only partially suppressed by acetylated LDL. The lipoxygenase plus phospholipase A2-induced modification of LDL is not necessarily identical to endothelial cell modification, but it is a useful model for studying the mechanism of oxidative modification of LDL. This work also represents the first example of oxidative modification of LDL by specific enzymes leading to enhanced recognition by macrophages.

Selective internalization of arachidonic acid by endothelial cells.

The asymmetric distribution of phospholipids in bovine endothelial-cell membranes was probed with 2,4,6-trinitrobenzenesulphonate and purified phospholipase A2. The data suggest that phosphotidylethanolamine is primarily located in the inner lipid bilayer, as reported for other cell types. Stearic acid is taken up by the endothelial cells and is randomly distributed among the membrane phospholipids. In contrast, the polyunsaturated fatty acids (arachidonic, eicosatrienoic and eicosapentaenoic acids) have initial incorporation into the phosphatidylcholine fraction. These fatty acids then undergo a time-dependent transfer from phosphatidylcholine to phosphatidylethanolamine. Thus we propose that endothelial cells possess a mechanism for the selective internalization of polyunsaturated fatty acids.