Levels Of Phospholipase A2 Activity Research Articles

Stinging caterpillars from the genera Podalia, Leucanella and Lonomia in Misiones, Argentina: A preliminary comparative approach to understand their toxicity

Dermal contact with Lepidoptera specimens at their larval stage (caterpillar) may cause systemic and/or local envenomation. There are multiple venomous species of them in Argentina, but their overall venom composition is poorly known. Lately, several cases of envenomation have been reported in the Misiones province, Northeastern Argentina. Thus, this work aimed to compare the protein composition, and the enzymatic properties of bristle extracts from caterpillars belonging to the families Megalopygidae (Podalia ca. fuscescens) and Saturniidae (Leucanella memusae and Lonomia obliqua) - the most common causative agents of accidents in Misiones -, and additionally to test their cross-reactivity with the L. obliqua antivenom produced in Brazil. Saturniidae venoms exhibited striking similarity in both their electrophoretic protein profile, and antigenic cross-reactivity. All venoms degraded azocasein – with the highest proteolytic activity observed in the P. ca. fuscescens bristle extract –, and hyaluronic acid, but the latter at low levels. Lonomia obliqua venom exhibited the highest level of phospholipase A2 activity. Bristle extracts from P. ca. fuscescens and L. obliqua both degraded human fibrin(ogen) and shortened the clotting time triggered by calcium, while L. memusae venom inhibited plasma coagulation. Proteins related to the coagulation disturbance were identified by mass spectrometry in all samples. Altogether, our findings show for the first time a comparative biotoxinological analysis of three genera of caterpillars with medical relevance. Moreover, this study provides relevant information about the pathophysiological mechanisms whereby these caterpillar bristle extracts can induce toxicity on human beings, and gives insight into future directions for research on them.

Differential effects of RU486 reveal distinct mechanisms for glucocorticoid repression of prostaglandin E release.

In A549 pulmonary cells, the dexamethasone- and budesonide-dependent repression of interleukin-1beta-induced prostaglandin E2 release was mimicked by the steroid antagonist, RU486. Conversely, whereas dexamethasone and budesonide were highly effective inhibitors of interleukin-1beta-induced cyclooxygenase (COX)/prostaglandin E synthase (PGES) activity and COX-2 expression, RU486 (<1 microm) was a poor inhibitor, but was able to efficiently antagonize the effects of dexamethasone and budesonide. In addition, both dexamethasone and RU486 repressed [3H]arachidonate release, which is consistent with an effect at the level of phospholipase A2 activity. By contrast, glucocorticoid response element-dependent transcription was unaffected by RU486 but induced by dexamethasone and budesonide, whilst dexamethasone- and budesonide-dependent repression of nuclear factor-kappaB-dependent transcription was maximally 30-40% and RU486 (<1 microm) was without significant effect. Thus, two pharmacologically distinct mechanisms of glucocorticoid-dependent repression of prostaglandin E2 release are revealed. First, glucocorticoid-dependent repression of arachidonic acid is mimicked by RU486 and, second, repression of COX/PGES is antagonized by RU486. Finally, whilst all compounds induced glucocorticoid receptor translocation, no role for glucocorticoid response element-dependent transcription is supported in these inhibitory processes and only a limited role for glucocorticoid-dependent inhibition of nuclear factor-kappaB in the repression of COX-2 is indicated.

Primitive Myeloid Cells Express High Levels of Phospholipase A2 Activity in the Absence of Leukotriene Release: Selective Regulation by Stem Cell Factor Involving the MAP Kinase Pathway

The activation of phospholipase A2 (PLA2) with release of eicosanoids and prostanoids in mature myeloid cells and the augmentation (priming) of this activity by cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) are central to the inflammatory process. Yet, there are few data concerning PLA2 activity and its regulation by growth factors in primary hematopoietic cells. We therefore analyzed the PLA2activity of mobilized human CD34 antigen-positive (CD34+) stem cells by quantitation of the extracellular release of3H-arachidonate. The PLA2 activity of CD34+ cells stimulated with calcium ionophore (A23187) was of similar magnitude to that of mature neutrophils and monocytes. Preincubation of CD34+ cells with stem cell factor (SCF) before A23187-stimulation resulted in primed PLA2 activity, whereas interleukin-3 (IL-3), GM-CSF, and tumor necrosis factor  had no significant effect. When CD34+ cells were induced to differentiate, PLA2 activity remained responsive to SCF for several days, but after 8 days, at which stage morphological and functional evidence of maturation was occurring, priming of PLA2 by SCF could no longer be elicited, whereas responses to GM-CSF and IL-3 had developed. The further metabolism of arachidonic acid to eicosanoids by CD34+ cells was not detected by either thin-layer chromatography, enzyme immunoassay, or differential spectroscopy. SCF stimulated the rapid but transient activation of ERK2 (p42 MAP kinase) in CD34+ cells, and we used the MAP kinase kinase inhibitor, PD 098059, which at 30 μmol/L blocks ERK2 activation in CD34+ cells, to investigate whether SCF-mediated priming of arachidonate release was mediated by this kinase. PD 098059 only partially inhibited A23187-stimulated PLA2 activity primed by SCF, suggesting the involvement of ERK2 and possibly a further signal transduction pathway. Methyl arachidonyl fluorophosphonate (5 μmol/L), a dual inhibitor of i and cPLA2 isoforms, completely inhibited arachidonate release without affecting ERK2 activation, demonstrating the lack of cellular toxicity. These data provide the first evidence that primitive myeloid cells have the capacity to release arachidonate, which is regulated by an early acting hematopoietic growth factor important for the growth and survival of these cells.

Primitive Myeloid Cells Express High Levels of Phospholipase A2 Activity in the Absence of Leukotriene Release: Selective Regulation by Stem Cell Factor Involving the MAP Kinase Pathway

AbstractThe activation of phospholipase A2 (PLA2) with release of eicosanoids and prostanoids in mature myeloid cells and the augmentation (priming) of this activity by cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) are central to the inflammatory process. Yet, there are few data concerning PLA2 activity and its regulation by growth factors in primary hematopoietic cells. We therefore analyzed the PLA2activity of mobilized human CD34 antigen-positive (CD34+) stem cells by quantitation of the extracellular release of3H-arachidonate. The PLA2 activity of CD34+ cells stimulated with calcium ionophore (A23187) was of similar magnitude to that of mature neutrophils and monocytes. Preincubation of CD34+ cells with stem cell factor (SCF) before A23187-stimulation resulted in primed PLA2 activity, whereas interleukin-3 (IL-3), GM-CSF, and tumor necrosis factor α had no significant effect. When CD34+ cells were induced to differentiate, PLA2 activity remained responsive to SCF for several days, but after 8 days, at which stage morphological and functional evidence of maturation was occurring, priming of PLA2 by SCF could no longer be elicited, whereas responses to GM-CSF and IL-3 had developed. The further metabolism of arachidonic acid to eicosanoids by CD34+ cells was not detected by either thin-layer chromatography, enzyme immunoassay, or differential spectroscopy. SCF stimulated the rapid but transient activation of ERK2 (p42 MAP kinase) in CD34+ cells, and we used the MAP kinase kinase inhibitor, PD 098059, which at 30 μmol/L blocks ERK2 activation in CD34+ cells, to investigate whether SCF-mediated priming of arachidonate release was mediated by this kinase. PD 098059 only partially inhibited A23187-stimulated PLA2 activity primed by SCF, suggesting the involvement of ERK2 and possibly a further signal transduction pathway. Methyl arachidonyl fluorophosphonate (5 μmol/L), a dual inhibitor of i and cPLA2 isoforms, completely inhibited arachidonate release without affecting ERK2 activation, demonstrating the lack of cellular toxicity. These data provide the first evidence that primitive myeloid cells have the capacity to release arachidonate, which is regulated by an early acting hematopoietic growth factor important for the growth and survival of these cells.

Characterization of a Mammalian Peroxiredoxin That Contains One Conserved Cysteine

A new type of peroxidase enzyme, named thioredoxin peroxidase (TPx), that reduces H2O2 with the use of electrons from thioredoxin and contains two essential cysteines was recently identified. TPx homologs, termed peroxiredoxin (Prx), have also been identified and include several proteins, designated 1-Cys Prx, that contain only one conserved cysteine. Recombinant human 1-Cys Prx expressed in and purified from Escherichia coli has now been shown to reduce H2O2 with electrons provided by dithiothreitol. Furthermore, human 1-Cys Prx transiently expressed in NIH 3T3 cells was able to remove intracellular H2O2 generated in response either to the addition of exogenous H2O2 or to treatment with platelet-derived growth factor. The conserved Cys47-SH group was shown to be the site of oxidation by H2O2. Thus, mutation of Cys47 to serine abolished peroxidase activity. Moreover, the oxidized intermediate appears to be Cys-SOH. In contrast to TPx, in which one of the two conserved cysteines is oxidized to Cys-SOH and then immediately reacts with the second conserved cysteine of the second subunit of the enzyme homodimer to form an intermolecular disulfide, the Cys-SOH of 1-Cys Prx does not form a disulfide. Neither thioredoxin, which reduces the disulfide of TPx, nor glutathione, which reduces the Cys-SeOH of oxidized glutathione peroxidase, was able to reduce the Cys-SOH of 1-Cys Prx and consequently could not support peroxidase activity. Human 1-Cys Prx was previously shown to exhibit a low level of phospholipase A2 activity at an acidic pH; the enzyme was thus proposed to be lysosomal, and Ser32 was proposed to be critical for lipase function. However, the mutation of Ser32 or Cys47 has now been shown to have no effect on the lipase activity of 1-Cys Prx, which was also shown to be a cytosolic protein. Thus, the primary cellular function of 1-Cys Prx appears to be to reduce peroxides with the use of electrons provided by an as yet unidentified source; the enzyme therefore represents a new type of peroxidase.

A controlled biochemical and immunohistochemical study of human synovial-type (group II) phospholipase A2 and inflammatory cells in macroscopically normal, degenerated, and herniated human lumbar disc tissues.

Group II phospholipase A2 enzyme activity was studied biochemically and immunohistochemically in tissue samples from disc prolapses, degenerated discs, and macroscopically normal discs. In parallel, phospholipase A2 and inflammatory cells were studied by indirect immunocytochemistry. To compare phospholipase A2 activity in normal discs and abnormal discs by an identical assay for phospholipases A2, and to compare the occurrence of inflammatory cells with phospholipase A2 activity and immunoreactivity. It has been suggested that a high phospholipase A2 enzyme activity in herniated disc tissue could be significant in abnormal states such as sciatica and discogenic pain. No comparison between healthy disc tissue and samples of abnormal discs (degenerated or herniated) has been carried out. In particular, an identical assay for phospholipase A2 for such tissue samples, supported by immunohistochemical staining data, has never been applied in parallel to normal and abnormal disc tissue, and neither have such results been compared with the demonstration of inflammatory cells. Group II phospholipase A2 enzyme activity was determined, in parallel, using an identical assay for tissue samples from 11 macroscopically normal discs, 33 disc herniations, and six discs showing degeneration by discography. For determination of phospholipase A2 enzyme activity, a radioassay using 1-palmitoyl-2-(1-14C)linoleoyl-L-3-phosphatidylethanolamine as the phospholipid substrate was used. Total tissue DNA as an estimate of total tissue cell number was measured in parallel with phospholipase A2 activity. All tissue samples also were studied by indirect immunocytochemistry, locating phospholipase A2 and T and B lymphocytes. Neither degenerated nor herniated disc tissue samples demonstrated a higher phospholipase A2 activity than control disc tissue samples. Average phospholipase A2 activity was actually higher in the control samples than in herniated disc samples (Mann-Whitney test, P < 0.001), possibly a result of a higher total DNA (P < 0.005). The observed level of phospholipase A2 activity was lower than that of inflammatory human synovial fluid. Neither was there marked immunoreactivity for phospholipase A2, which was observed in chondrocytes in areas of cartilage and occasional disc cells, supporting the biochemical results. Lymphocytes were more numerous only in herniated disc samples (15%), and their presence showed little overlap with phospholipase A2 immunoreactivity. Synovial-type (Group II) phospholipase A2 enzyme activity is not particularly high in disc tissue and does not appear to be higher in herniated or degenerated discs than control disc tissue. Immunoreactivity to phospholipase A2 is seen only occasionally and is strong only when cartilage tissue is present. Neither are inflammatory lymphocytes commonly observed.

Prostaglandin production after experimental discectomy.

This study ascertained the effects of discectomy on prostaglandin synthesis. The purpose of these novel experiments was to measure the levels of two prostaglandins in lumbar epidural fluid obtained from an area subjected to discectomy. For comparison, lumbar epidural fluid from a site not disturbed by discectomy and fluid from a subcutaneous site were analyzed for the prostaglandins. Previous studies have shown that nuclear material obtained from degenerative discs manifests an extraordinarily high level of phospholipase A2 activity. Others have hypothesized that the known inflammatory effects of phospholipase A2 are due to the release of arachidonic acid, which is converted to various eicosanoids, including several algesic prostaglandins (PGI2 and PGE2). No previous study has continuously measured prostaglandin levels in epidural fluid or assessed the effect of discectomy on prostaglandin production. An ultrafiltrate of lumbar epidural fluid of dogs was obtained from indwelling catheters located adjacent to spinal areas that were and were not subjected to discectomy as well as from subcutaneous tissue. The fluid was collected daily for 14 days and analyzed for PGE2 and 6-keto PGF1(alpha) (the stable metabolite of PGI2) by radioimmunoassay. The concentration of 6-keto PGF1(alpha) and PGE2 in fluid collected during the first 24 hours was significantly higher in the area of discectomy than in the epidural region that was not subjected to discectomy and significantly higher than in fluid obtained from the subcutaneous site. The high level of these prostaglandins at the discectomy site fell rapidly, so that by the end of 48 hours the differences in values between spinal fluid from the discectomy and nondiscectomy regions were not statistically significant. The concentration of the prostaglandins in epidural fluid decreased with time and became minimal within the second week. The removal of normal discs is accompanied for 24 hours by a marked rise in the synthesis of two prostaglandins known to produce pain. Because the concentration of prostaglandins in epidural fluid decreased rapidly thereafter, the initial surge obtained appears to be associated more with chemical factors such as phospholipase A2 than with wound healing.

Phenytoin potentiates interleukin-1-induced prostaglandin biosynthesis in human gingival fibroblasts.

1. The effect of phenytoin (PHT) on prostaglandin E2 (PGE2) biosynthesis in human gingival fibroblasts stimulated by interleukin-1 (IL-1 alpha, IL-1 beta) or by tumour necrosis factor alpha (TNF alpha) was studied. 2. IL-1 alpha (1.5-6.0 ng ml-1) and IL-1 beta (30-300 pg ml-1), dose-dependently, stimulated PGE2 formation, in 24 h cultures, with IL-beta being the most potent agonist. 3. PHT (2.5-20 micrograms ml-1) did not induce PGE2 formation itself but potentiated IL-1 alpha- and IL-1 beta-induced PGE2 formation in the gingival fibroblasts in a manner dependent on the concentrations of both IL-1 and PHT. 4. IL-1 beta (0.1-1.0 ng ml-1) induced release of [3H]-arachidonic acid ([3H]-AA) from prelabelled fibroblasts that was potentiated by PHT (20 micrograms ml-1). 5. TNF-alpha (greater than or equal to 0.01 micrograms ml-1) significantly stimulated the biosynthesis of PGE2 by a process that was potentiated by PHT. 6. Addition of exogenous arachidonic acid (AA) (greater than or equal to 1 microM) caused an increase of PGE2 formation in the fibroblasts that was not potentiated by PHT (20 micrograms ml-1). 7. The results indicate that treatment with PHT results in upregulation of prostaglandin biosynthesis in gingival fibroblasts challenged with IL-1 or TNF alpha, at least partly due to enhanced level of phospholipase A2 activity.

Phospholipase A2 is a differentiation-dependent enzymatic activity for adipogenic cell line and adipocyte precursors in primary culture.

Phospholipase A2 enzymatic activity was measured in the teratoma-derived adipogenic cell line 1246 and in adipocyte precursors in primary cultures. It was shown that enzymatic activity was low while the cells were undifferentiated and increased by 20-24-fold after the cells had undergone adipocyte differentiation. The increase of phospholipase A2 activity follows the same time course as that observed for glycerol-3-phosphate dehydrogenase activity used as a marker of differentiation. In contrast, the differentiation-deficient, insulin-independent cell line 1246-3A always contained very low levels of phospholipase A2 activity. Phospholipase A2 activity measured in the 1246 cells was inhibited in a dose-dependent fashion by incubation with ONO-RS-082 and quinacrine which are inhibitors of phospholipase A2 activity. Measurements of arachidonate metabolites in 1246 cells showed that production of prostaglandin F2 alpha by the 1246 cells followed the same time course as the increase of phospholipase A2 activity during differentiation. Similar results were obtained with primary cultures of adipocyte precursors. These results indicate that phospholipase A2 is a differentiation-dependent enzymatic activity for the adipogenic cell line 1246 and for adipocyte precursors in primary culture. These data suggest that metabolic pathways controlled by phospholipase A2 activity could play an important physiological role in adipose tissue differentiation.

Purification of rabbit platelet secretory phospholipase A2 and its characteristics.

It was reported previously that rat platelets release phospholipase A2 upon in vitro stimulation by thrombin, ADP, or A23187 (Horigome, K., Hayakawa, M., Inoue, K., & Nojima, S. (1987) J. Biochem. 101, 53-61). Secretion of phospholipase A2 was also observed with rabbit platelets. Rabbit platelets seem to release phospholipase A2 upon stimulation in vivo, because the rabbit plasma taken immediately after intravenous injection of PAF contained an appreciable level of phospholipase A2 activity and fewer platelets. Rabbit platelet phospholipase A2 released in vitro was purified by column chromatography using Sepharose CL-4B conjugated with anti-rat platelet derived phospholipase A2 monoclonal antibody, followed by reversed-phase HPLC. The purified enzyme was subjected to structural analysis by HPLC peptide mapping and primary sequence determination of the separated peptides. Based on the homology with rat platelet secretory phospholipase A2 (Hayakawa, M., Kudo, I., Tomita, M., Nojima, S., & Inoue, K. (1988) J. Biochem. 104, 767-772), a partial primary structure (62 amino acid residues) of the rabbit enzyme was tentatively determined; the two sequences were highly homologous (72%). The rabbit sequence was also nearly identical to that of rabbit ascitic fluid phospholipase A2, which was determined by Forst et al. (Forst, S., Weiss, J., Elsbach, P., Maraganore, J.M., Reardon, I., & Heinrikson, R.L. (1986) Biochemistry 25, 8381-8385). Phospholipase A2 from the membrane fraction of rabbit platelets was also purified; it had the same characteristics and th same amino-terminal sequence as the purified secretory enzyme. Secretory and membrane-bound phospholipase A2 of rabbit platelets may in fact be identical.(ABSTRACT TRUNCATED AT 250 WORDS)

The level of phospholipase A2 activity is raised in the uninvolved epidermis of psoriasis.

SUMMARY The activity of phospholipase A2 was measured in uninvolved psoriatic epidermis and normal epidermis. The mean phospholipase A2 activity in uninvolved psoriatic epidermis, expressed as nmol fatty acid released per mg protein per h, was raised compared with normal epidermis (7.88±s.e. 1·20 vs 2.18±s.e. 0·38; P<0·01). This observation is, we believe, the first demonstration of an abnormality in the uninvolved untreated psoriatic epidermis which could account for the pathology of the disease.

Rat mast cell phospholipase A2: activity toward exogenous phosphatidylserine and inhibiton by N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylserine.

The presence of phospholipase A2 in intact rat peritoneal mast cells was investigated by using two synthetic radiolabeled phosphatidylserine (PS) substrates. Incubation of intact cells with 1-oleoyl-2-[3H]oleoyl-PS resulted in the release of a considerable quantity of [3H]oleic acid from the substrate. To establish that [3H]oleic acid release was mediated via direct enzymatic attack at the sn-2 position, we measured release of the [3H]serine moiety from the glycerol backbone of 1,2-dimyristoylphosphatidyl[3H]serine. This activity, which represents the combined actions of phospholipases C and D, was 10-fold lower than [3H]oleic acid release, indicating that neither of these enzymes is required for the release of the preponderance of [3H]oleic acid. These results establish the existence in intact rat mast cells of a phospholipase A2 active toward exogenous PS. Over the concentration range at which exogenous PS activates mast cell secretion, intact mast cells and broken cells possessed nearly equal levels of phospholipase A2 activity, and enzyme activity was 3--4-fold higher toward PS than phosphatidylcholine. Several agents were tested for their ability to inhibit phospholipase A2 in intact mast cells. Of the agents tested, an N-substituted derivative of PS previously identified as an inhibitor of mast cell secretion was shown to be a particularly potent and efficacious inhibitor of mast cell phospholipase A2. The concentration dependence of enzyme inhibition paralleled inhibition of histamine secretion, providing a strong positive correlation between the level of phospholipase A2 in mast cells and the capacity for secretion.

Mode of action of staphylococcal leukocidin: effects of the S and F components on the activities of membrane-associated enzymes of rabbit polymorphonuclear leukocytes.

The cytotoxic action of the S component of leukocidin from Staphylococcus aureus on rabbit polymorphonuclear leukocytes was supported by the following observations, (i) Leukocytes displayed a large chemotactic response to the S component (10(-10) M) as well as to the chemotactic factor N-formylmethionylleucylphenylalanine (10(-11) M). (ii) The S component stimulated high levels of phospholipase A2 activity in the cell membranes, with concomitant synthesis and release of prostaglandins. (iii) Uptake of 45Ca into leukocytes exposed to the S component was about double the rate of uptake into untreated cells. The increased 45Ca uptake into the cells was not inhibited by trifluoperazine and ruthenium red. (iv) Indomethacin and alloxazine, which had no effects on the binding of the S component to the cells, attenuated markedly the stimulation of phospholipase A2 activity, the syntheses of prostaglandins, and the increased uptake of 45Ca caused by the S component. The F component of leukocidin, bound to rabbit leukocytes with the aid of the S component, rapidly induced complete release of 86Rb from preloaded leukocytes. This release resulted from stimulation of ouabain-insensitive (Na+ + K+)-adenosine triphosphatase activity and inhibition of cyclic AMP-dependent protein kinase.