Enhanced Phospholipase A2 Activity In Cholesterol-Enriched Platelets From Rabbits Fed A Cholesterol-Enriched Diet

1981 ◽  
Author(s):  
A J McLeod ◽  
M Johnson ◽  
K E Sucklino ◽  
P Walton
Keyword(s):  
Arachidonic Acid ◽  
Phospholipase A2 ◽  
Serum Cholesterol ◽  
Control Diet ◽  
Molar Ratio ◽  
Control Group ◽  
Pla2 Activity ◽  
Rate Limiting ◽  
Phospholipase A2 Activity

Phospholipase A2(PLA2) could be the rate-limiting enzyme in the metabolism of arachidonic acid (AA) derived from membrane phospholipid to thromboxane A2 (TXA2). Mal- ondialdehyde (MDA) production, which is considered to be an index of TXA2 synthesis, is increased in platelets which have been enriched in cholesterol by incubation with cholesterol-rich phospholipid dispersions in vitro.Rabbits were fed a diet supplemented with 0.5% w/w cholesterol for 4 weeks after which serum cholesterol was determined and the platelets examined and compared with rabbits fed a control diet. The cholesterol:phospholipid molar ratio (c/p) in the platelets, MDA production (stimulated by AA (imM) and basal) and PLA2 activity were estimated. PLA2 activity was estimated by measuring the % inversion by resuspended washed platelets of 1-acyl-2-(l- 14C)arachidonyl phosphatidylcholine to (1- 14C)arachidonic acid on stimulation with collagen (2μg/ml). Serum cholesterol in the cholesterol-fed group (n=9) was 488 ± 104 mg/ 100ml compared with the control group (n=3) which was 34 ± 4.7 mg/l00ml. Platelets from the cholesterol-fed rabbits showed a 20% increase in C/P (p<0.05); basal and AA stimulated MDA production was increased by 40% and 27% respectively compared with platelets from the control group. PLA2 activity was 1.26% conversion to products in the cholesterol-enriched platelets compared with 0.10% in the control platelets. This increase in activity was significant (p < 0.05).These results suggest that increased AA metabolism in cholesterol-enriched platelets may in part be due to increased PLA2 activity. This may reflect a physical effect of cholesterol on the platelet membrane predisposing arachidonyl phosphatidylcholine to PLA2 catalysed hydrolysis.

Interleukin-1beta stimulates phospholipase A2 activity in adult rat ventricular myocytes

1997 ◽  
Vol 272 (2) ◽  
pp. C450-C456 ◽  
Author(s):  
J. McHowat ◽  
S. Liu
Keyword(s):  
Arachidonic Acid ◽  
Phospholipase A2 ◽  
Ventricular Myocytes ◽  
Arachidonic Acid Release ◽  
Pla2 Activity ◽  
Rat Ventricular Myocytes ◽  
Adult Rat ◽  
Acid Release ◽  
Cytosolic Pla2 ◽  
Phospholipase A2 Activity

We have examined whether interleukin (IL)-1beta modulates phospholipase A2 (PLA2) activity in ventricular myocytes. PLA2 activity was measured in isolated membrane and cytosol fractions with (16:0,[3H]18:1) plasmenylcholine and (16:0,[3H]18:1) phosphatidylcholine in the absence and presence of Ca2+. When measured in the absence of Ca2+ with plasmenylcholine, exposure to 5 ng/ml IL-1beta caused an increase in membrane-associated PLA2 activity for 10 min that returned to basal levels by 20 min. In the presence of Ca2+ with phosphatidylcholine, IL-1beta had no effect on membrane-associated PLA2 but decreased cytosolic PLA2 activity. Additionally, IL-1beta caused an increase in arachidonic acid release in 20 min. Pretreatment with E-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one, a selective Ca2+-independent PLA2 inhibitor, blocked IL-1beta-induced increases in both PLA2 activity and arachidonic acid release. Exposure to IL-1 receptor antagonist (IL-1RA) alone had no effect on membrane-associated PLA2 activity. When incubated with IL-1beta, IL-1RA inhibited the IL-1beta-enhanced PLA2 activity. These results show that, via activation of its receptors, IL-1beta stimulates specifically membrane-associated Ca2+-independent plasmalogen-selective PLA2 in rat ventricular myocytes.

scholarly journals Intracellular Regulation of the Metabolism of Arachidonic Acid in Human Platelets

1977 ◽  
Author(s):  
Thomas K. Bills ◽  
J. Bryan Smith ◽  
Melvin J. Silver
Keyword(s):  
Fatty Acids ◽  
Arachidonic Acid ◽  
Fatty Acid ◽  
Phospholipase A2 ◽  
Unsaturated Fatty Acids ◽  
Human Platelets ◽  
Intracellular Regulation ◽  
Rate Limiting ◽  
Phospholipase A2 Activity ◽  
Oleic And Linoleic Acids

The synthesis of prostaglandins and thromboxanes by human platelets is limited by the availability of the fatty acid precursor, arachidonic acid. Although large amounts of arachidonic acid are esterified to platelet phospholipids, only the free acid can be utilized by the oxygenation pathways of platelets. Since there are only trace amounts of free arachidonic acid in platelets, the enzymatic liberation of this fatty acid from platelet phospholipids can be considered the initial and rate limiting step of these oxygenation pathways. This process is catalyzed by a phospholipase A2 whose role as the rate limiting enzyme makes it a prime target for the intracellular regulation of prostaglandin and thromboxane synthesis.A second mechanism for the regulation of prostaglandin synthesis has been hypothesized. Several commonly occurring unsaturated fatty acids, e.g., oleic and linoleic acids, are capable of inhibiting prostaglandin cyclooxygenase. If these fatty acids are liberated from phospholipids along with arachidonic acid, they could limit the production of prostaglandins and thromboxanes. Thus, the types and amounts of fatty acids released from platelet phospholipids could regulate the amounts of prostaglandins and thromboxanes produced.We have investigated these two types of intracellular regulation and have found that 1) intracellular cyclic nucleotides and divalent cations are involved in the regulation of the platelet phospholipase A2 activity stimulated by thrombin and 2) this phospholipase A2 activity catalyzes the specific release of arachidonic acid from phospholipids, thereby obviating the regulatory role of liberated oleic and linoleic acids.

scholarly journals Demonstration of calcium-dependent phospholipase A2 activity in membrane preparation of rabbit neutrophils. Absence of activation by fMet-Leu-Phe, phorbol 12-myristate 13-acetate and A-kinase

1988 ◽  
Vol 250 (2) ◽  
pp. 343-348 ◽  
Author(s):  
T Matsumoto ◽  
W Tao ◽  
R I Sha'afi
Keyword(s):  
Arachidonic Acid ◽  
Cyclic Amp ◽  
Phospholipase A2 ◽  
Kinase Inhibitor ◽  
Calcium Ionophore ◽  
Membrane Preparation ◽  
Free Calcium ◽  
Ionophore A23187 ◽  
Pla2 Activity ◽  
Intact Cells

The presence of a phospholipase A2 (PLA2) activity in rabbit neutrophil membrane preparation that is able to release [1-14C]oleic acid from labelled Escherichia coli has been demonstrated. The activity is critically dependent on the free calcium concentration and marginally stimulated by GTP gamma S. More than 80% of maximal activity is reached at 10 microM-Ca2+. The chemotactic factor, fMet-Leu-Phe, does not stimulate the PLA2 activity in this membrane preparation. Pretreatment of the membrane preparation, under various experimental conditions, or intact cells, before isolation of the membrane with phorbol 12-myristate 13-acetate (PMA), does not affect PLA2 activity. Addition of the catalytic unit of cyclic AMP-dependent kinase to membrane preparation has no effect on PLA2 activity. Pretreatment of the intact neutrophil with dibutyryl-cAMP before isolation of the membrane produces a small but consistent increase in PLA2 activity. The activity of PLA2 in membrane isolated from cells treated with the protein kinase inhibitor 1-(5-isoquinolinesulphonyl)-2-methyl piperazine dihydrochloride (H-7) is significantly decreased. Furthermore, although the addition of PMA to intact rabbit neutrophils has no effect on the release of [3H]arachidonic acid from prelabelled cells, it potentiates significantly the release produced by the calcium ionophore A23187. This potentiation is not due to an inhibition of the acyltransferase activity. H-7 inhibits the basal release of arachidonic acid but does not inhibit the potentiation by PMA. These results suggest several points. (1) fMet-Leu-Phe does not stimulate PLA2 directly, and its ability to release arachidonic acid in intact neutrophils is mediated through its action on phospholipase C. (2) The potentiating effect of PMA on A23187-induced arachidonic acid release is most likely due to PMA affecting either the environment of PLA2 and/or altering the organization of membrane phospholipids in such a way as to increase their susceptibility to hydrolysis. (3) The intracellular level of cyclic AMP probably does not directly affect the activity of PLA2.

scholarly journals Cerebrospinal Fluid Secretory Ca2+-Dependent Phospholipase A2 Activity Is Increased in Alzheimer Disease

2009 ◽  
Vol 55 (12) ◽  
pp. 2171-2179 ◽  
Author(s):  
Sonia Chalbot ◽  
Henrik Zetterberg ◽  
Kaj Blennow ◽  
Tormod Fladby ◽  
Inge Grundke-Iqbal ◽  
...  
Keyword(s):  
Cerebrospinal Fluid ◽  
Alzheimer Disease ◽  
Phospholipase A2 ◽  
Microtiter Plate ◽  
Pla2 Activity ◽  
Activity Assay ◽  
Proinflammatory Mediators ◽  
Spla2 Activity ◽  
Phospholipase A2 Activity ◽  
Microtiter Plate Format

Abstract Background: The phospholipase A2 (PLA2) family comprises multiple isoenzymes that vary in their physicochemical properties, cellular localizations, calcium sensitivities, and substrate specificities. Despite these differences, PLA2s share the ability to catalyze the synthesis of the precursors of the proinflammatory mediators. To investigate the potential of PLA2 as a biomarker in screening neuroinflammatory disorders in both clinical and research settings, we developed a PLA2 assay and determined the predominant types of PLA2 activity in cerebrospinal fluid (CSF). Methods: We used liposomes composed of a fluorescent probe (bis-Bodipy® FL C11-PC [1,2-bis-(4,4- difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-undecanoyl)-sn-glycero-3-phosphocholine]) and 1,2-dioleoyl-l-α-phosphatidylcholine as a substrate to measure CSF PLA2 activity in a 96-well microtiter plate format. We established the type of CSF PLA2 activity using type-specific inhibitors of PLA2. Results: Using 5 μL CSF per assay, our PLA2 activity assay was reproducible with CVs &lt;15% in 2 CSF samples and for recombinant secretory Ca2+-dependent PLA2 (sPLA2) in concentrations ranging from 0.25 to 1 μmol/L. This PLA2 assay allowed identification of sPLA2 activity in lumbar CSF from healthy individuals 20–77 years old that did not depend on either sex or age. Additionally, CSF sPLA2 activity was found to be increased (P = 0.0008) in patients with Alzheimer disease. Conclusions: Adult human CSF has sPLA2 activity that can be measured reliably with the assay described. This enzyme activity in the CSF is independent of both sex and age and might serve as a valuable biomarker of neuroinflammation, as we demonstrated in Alzheimer disease.

scholarly journals Ruthenium, Not Carbon Monoxide, Inhibits the Procoagulant Activity of Atheris, Echis, and Pseudonaja Venoms

2020 ◽  
Vol 21 (8) ◽  
pp. 2970 ◽  
Author(s):  
Vance G. Nielsen
Keyword(s):  
Carbon Monoxide ◽  
Phospholipase A2 ◽  
Procoagulant Activity ◽  
Inhibitory Effects ◽  
Snake Venoms ◽  
Radical Intermediate ◽  
Experimental Systems ◽  
Phospholipase A2 Activity

The demonstration that carbon monoxide releasing molecules (CORMs) affect experimental systems by the release of carbon monoxide, and not via the interaction of the inactivated CORM, has been an accepted paradigm for decades. However, it has recently been documented that a radical intermediate formed during carbon monoxide release from ruthenium (Ru)-based CORM (CORM-2) interacts with histidine and can inactivate bee phospholipase A2 activity. Using a thrombelastographic based paradigm to assess procoagulant activity in human plasma, this study tested the hypothesis that a Ru-based radical and not carbon monoxide was responsible for CORM-2 mediated inhibition of Atheris, Echis, and Pseudonaja species snake venoms. Assessment of the inhibitory effects of ruthenium chloride (RuCl3) on snake venom activity was also determined. CORM-2 mediated inhibition of the three venoms was found to be independent of carbon monoxide release, as the presence of histidine-rich albumin abrogated CORM-2 inhibition. Exposure to RuCl3 had little effect on Atheris venom activity, but Echis and Pseudonaja venom had procoagulant activity significantly reduced. In conclusion, a Ru-based radical and ion inhibited procoagulant snake venoms, not carbon monoxide. These data continue to add to our mechanistic understanding of how Ru-based molecules can modulate hemotoxic venoms, and these results can serve as a rationale to focus on perhaps other, complementary compounds containing Ru as antivenom agents in vitro and, ultimately, in vivo.

Arachidonic acid analogues as inhibitors of phospholipase A2 activity

1987 ◽  
Vol 15 (3) ◽  
pp. 418-419 ◽  
Author(s):  
KEITH A FOSTER ◽  
DEREK R. BUCKLE ◽  
KIM L. CRESCENZI ◽  
ASHLEY E. FENWICK ◽  
JOHN E. TAYLOR
Keyword(s):  
Arachidonic Acid ◽  
Phospholipase A2 ◽  
Phospholipase A2 Activity

Platelet inhibition by aspirin is diminished in patients during carotid surgery: a form of transient aspirin resistance?

2004 ◽  
Vol 92 (07) ◽  
pp. 89-96 ◽  
Author(s):  
David Payne ◽  
Chris Jones ◽  
Paul Hayes ◽  
Sally Webster ◽  
A. Naylor ◽  
...  
Keyword(s):  
Arachidonic Acid ◽  
Platelet Aggregation ◽  
Platelet Rich Plasma ◽  
Platelet Inhibition ◽  
Aspirin Resistance ◽  
Arachidonic Acid Metabolism ◽  
Significant Rise ◽  
Control Group

SummaryThe majority of patients who suffer peri-operative thromboembolic complication while undergoing vascular procedures do so despite taking aspirin. This study examined the antiplatelet effect of aspirin during surgery in patients undergoing carotid endarterectomy (CEA). Fifty patients undergoing CEA were standardised to 150 mg aspirin daily for ≥2 weeks. Platelet aggregation in response to arachidonic acid (AA) was measured in platelet rich plasma prepared from blood taken prior to, during, and at the end of surgery. Spontaneous platelet aggregation was also studied, as was the role of physiological agonists (ADP, collagen, thrombin, and epinephrine) in mediating the in vivo and in vitro responses to AA. Eighteen patients undergoing leg angioplasty, also on 150 mg aspirin, without general anaesthesia, served as a control group. In the CEA patients aggregation induced by AA (5 mM) increased significantly from 7.6 ± 5.5% pre-surgery to 50.8 ± 29.5% at the end of surgery (p <0.0001). Aggregation to AA was even greater in samples taken mid-surgery from a sub-set of patients (73.8 ± 7.2%; p = 0.0001), but fell to 45.9 ± 7.4% by the end of surgery. The increased aggregation in response to AA was not due to intra-operative release of physiological platelet agonists since addition of agents that block/neutralise the effects of ADP (apyrase; 4 µg/ml), thrombin (hirudin; 10 units/ml), or epinephrine (yohimbine; 10 µM/l) to the samples taken at the end of surgery did not block the increased aggregation.The patients undergoing angioplasty also showed a significant rise in the response to AA (5 mM), from 5.6 ± 5.5% pre-angioplasty to 32.4 ± 24.9% at the end of the procedure (p <0.0001), which fell significantly to 11.0 ± 8.1% 4 hours later. The antiplatelet activity of aspirin, mediated by blockade of platelet arachidonic acid metabolism, diminished significantly during surgery, but was partially restored by the end of the procedure without additional aspirin treatment.This rapidly inducible and transient effect may explain why some patients undergoing cardiovascular surgery remain at risk of peri-operative stroke and myocardial infarction.

scholarly journals Purification of a 100 kDa phospholipase A2 from spleen, lung and kidney: antiserum raised to pig spleen phospholipase A2 recognizes a similar form in bovine lung, kidney and platelets, and immunoprecipitates phospholipase A2 activity

1993 ◽  
Vol 294 (1) ◽  
pp. 261-270 ◽  
Author(s):  
D K Kim ◽  
J V Bonventre
Keyword(s):  
Arachidonic Acid ◽  
Phospholipase A2 ◽  
Large Scale ◽  
Biochemical Characterization ◽  
Tissue Injury ◽  
Rat Kidney ◽  
Deae Cellulose ◽  
Pla2 Activity ◽  
Purification Scheme ◽  
Pla2 Enzyme

Phospholipase A2 (PLA2) plays a key role in the production of intracellular and extracellular chemical mediators such as arachidonic acid, eicosanoids and platelet-activating factor, which modulate membrane channel activity, signal transduction, are vasoactive and chemotactic, and are implicated in many pathophysiological mechanisms of inflammation and tissue injury. We previously identified, purified and characterized an arachidonic acid-selective cytosolic 100-110 kDa PLA2 from bovine platelets and rat kidney that is activated during cell stimulation. The purification schemes previously published resulted in low yields of enzyme, insufficient for extensive biochemical characterization. We report the purification of a large-molecular-mass (100 kDa) PLA2 from pig spleen, bovine kidney and bovine lung, using a novel large-scale purification scheme. The enzyme was purified to near homogeneity from an acidified extract obtained from 4.8 kg of pig spleen by sequential use of DEAE-cellulose anionic exchange, Butyl-Toyopearl hydrophobic chromatography and DEAE-5PW h.p.l.c., and further purified by non-denaturing PAGE. This purification scheme will permit the preparation of quantities of purified native enzyme sufficient to study its properties and regulation. To generate antiserum against the PLA2 enzyme, the 100 kDa protein was excised and electroeluted from SDS/PAGE gels of the active fractions after DEAE-5PW h.p.l.c., and this was used as antigen. This polyclonal antibody against pig spleen 100 kDa PLA2 protein reacted with 100 kDa bands in preparations partially purified from bovine platelets, kidney and lung as well as pig spleen, and immunoprecipitated PLA2 activity from these sources. The antibody also immunoprecipitated a 100 kDa protein from cytosolic fractions of cultured renal mesangial cells, human erythroleukaemia cells and human monocytic U937 cells. Considerable PLA2 activity was present in the immunoprecipitates. To our knowledge this antibody is unique in its ability to permit measurement of PLA2 activity in the immunoprecipitate itself, and will be a useful tool for the study of the regulation and the activation mechanisms of the native PLA2 enzyme.

scholarly journals Plasma from uninephrectomized rats stimulates phospholipid methylation and arachidonic acid release in renal-cortical slices

1989 ◽  
Vol 260 (2) ◽  
pp. 365-369 ◽  
Author(s):  
H Banfić ◽  
Z Gatalica
Keyword(s):  
Arachidonic Acid ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase A2 ◽  
Arachidonic Acid Release ◽  
Kinase C ◽  
Graded Response ◽  
Acid Release ◽  
Cortical Slices

Phospholipid methylation and arachidonic acid release in renal-cortical slices was investigated in vitro after addition of plasma from uninephrectomized or sham-operated rats. Plasma from uninephrectomized rats (‘uni-plasma’) stimulated phospholipid methylation when obtained within the first 3 h after uninephrectomy. With different amounts of added plasma a graded response in phospholipid methylation was obtained. Addition of 50 nM-12-O-tetradecanoylphorbol 13-acetate for 10 min to intact slices also stimulated phospholipid methylation, whereas incubation of slices before addition of ‘uni-plasma’ with 100 microM-1-(5-isoquinolinylsulphonyl)-2-methylpiperazine prevented it, suggesting that protein kinase C stimulates phospholipid methylation in renal-cortical slices. Plasma from uninephrectomized rats also stimulates [3H]arachidonic acid release from phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn) via activation of phospholipase A2. Two mechanisms of phospholipase A2 activation are proposed: first, in which it is activated by protein kinase C and releases 3H radioactivity from PtdCho, and second, in which phospholipase A2 is stimulated by Ca2+ ions and releases 3H radioactivity from PtdEtn.

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