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PubMed1989

Pertussis toxin-sensitive GTP-binding proteins may regulate phospholipase A2 in response to thrombin in rabbit platelets.

Incubation of rabbit platelets with thrombin resulted in rapid accumulations of inositol trisphosphate (IP3) in [3H]inositol-labeled platelets, increases of [3H]arachidonic acid [( 3H]AA) release, and [3H]serotonin secretion from the platelets prelabeled with these labeled compounds. The experiments using phospholipase A2 or C inhibitor suggested that not only phospholipase C but also phospholipase A2 activity plays an important role in serotonin secretion. We then studied the regulatory mechanisms of phospholipase A2 activity. Guanosine 5'-(3-O-thio)triphosphate (GTP gamma S), guanyl-5'-(beta,gamma-iminio)triphosphate), or AlF4- caused a significant liberation of AA in digitonin-permeabilized platelets but not in intact platelets. Thrombin-stimulated AA release was not observed in permeabilized platelets, whereas thrombin acted synergistically with GTP or GTP analogs to stimulate AA release. GTP analog-stimulated AA release was inhibited by guanosine 5'-(2-O-thio)diphosphate) and was also inhibited by decreased Mg2+ concentrations. Thrombin-induced, GTP-dependent AA release, but not IP3 formation, was diminished by 100 ng/ml of pertussis toxin, associated with ADP-ribosylation of membrane 41-kDa protein(s). Thrombin-stimulated AA release from intact platelets and GTP gamma S-stimulated release from permeabilized platelets were both markedly dependent on Ca2+. However, Ca2+ addition could not enhance AA release without GTP gamma S even when Ca2+ was increased up to 10(-4) M in permeabilized platelets. The results show that thrombin-stimulated AA release from rabbit platelets is mainly mediated by phospholipase A2 activity, not by phospholipase C activity, and that Ca2+ is an important factor to the activation of phospholipase A2 but is not the sole factor to the regulation. GTP-binding protein(s) is involved in receptor-mediated activation of phospholipase A2.

Animals↗

PubMed1988

Phospholipase A2 hydrolysis of membrane phospholipids causes structural alteration of the nicotinic acetylcholine receptor.

Thermal perturbation techniques have been used to probe structural alteration of the nicotinic acetylcholine receptor as a function of perturbations of its native membrane environment. Differential scanning calorimetry and a technique involving heat inactivation of the alpha-bungarotoxin-binding sites on the receptor protein reveal that there is a profound destabilization of the acetylcholine receptor structure when receptor-containing membranes are exposed to phospholipase A2. The characteristic calorimetric transition assigned to irreversible denaturation of the receptor protein and the heat inactivation profile of alpha-bungarotoxin-binding sites are shifted to lower temperatures by approx. 7 and 5 C degrees, respectively, upon exposure to phospholipase A2 at a phospholipase/neurotoxin binding site molar ratio of about 1:100. The effects of phospholipase A2 on receptor structure can be (i) reversed by using bovine serum albumin as a scavenger of phospholipase hydrolysis products of membrane phospholipids, and (ii) stimulated by incorporation into the membranes of free, polyunsaturated fatty acids. In particular, linolenic acid (18:3(n-3] causes detectable destabilization of the alpha-bungarotoxin binding sites on the receptor at free fatty acid/receptor molar ratios as low as 10:1. Furthermore, alteration of receptor structure by added phospholipase occurs very rapidly, which is consistent with the observation of rapid in situ phospholipase A2 hydrolysis of membrane phospholipids, particularly highly unsaturated phosphatidylethanolamine and phosphatidylserine. Based on previously published data on the inhibition of acetylcholine receptor cation-gating activity caused by the presence of either phospholipase A2 or free fatty acids (Andreasen T.J. and McNamee M.G. (1980) Biochemistry 19, 4719), we interpret our data as indicative of a correlation between structural and functional alterations of the membrane-bound acetylcholine receptor induced by phospholipase A2 hydrolysis products.

Animals↗

PubMed1986

Evidence for a catalytic role of phospholipase A in phorbol diester- and zymosan-induced mobilization of arachidonic acid in mouse peritoneal macrophages.

Inositol phospholipid degradation and release of phospholipid-bound arachidonic acid was induced in intact peritoneal macrophages by exposure to phorbol myristate acetate (PMA) or zymosan particles. PMA, known to activate protein kinase C, selectively enhanced the deacylation of phosphatidylinositol (i.e., degradation by phospholipase A), while zymosan particles enhanced degradation via both phospholipase A and inositol lipid phosphodiesterase (phospholipase C). The release of arachidonic acid was found to correlate with the degradation of phosphatidylinositol by the phospholipase A pathway and could be dissociated from the phospholipase C-catalyzed cleavage of inositol phospholipids in several experimental situations: (i) when PMA was the stimulus, (ii) by the difference in Ca2+ dependence between the two enzymatic processes when zymosan was the stimulus and (iii) by the parallel inhibition by chlorpromazine of the phospholipase A pathway and arachidonic acid release, but not inositol phospholipid phosphodiesterase. In addition, phloretin, a reported inhibitor of protein kinase C, was found to inhibit arachidonic acid release and the deacylation of phosphatidylinositol. The results are consistent with a model in which arachidonic acid release is mediated by phospholipase(s) A and in which PMA or the phosphodiesterase-catalyzed degradation of phosphoinositides causes activation of the phospholipase A pathway via protein kinase C.

Animals↗

PubMed1986

Endogenous suppression of neutral-active and calcium-dependent phospholipase A2 in human polymorphonuclear leukocytes.

Phospholipase A2 activity was measured in homogenized and acid-extracted human polymorphonuclear leukocytes using [1-14C]oleate-labelled autoclaved Escherichia coli as substrate. In whole homogenate and in the supernatant and particular fractions separated by centrifugation at 150,000 X g, phospholipase activity was barely detectable (1-4 pmol/h per 10(6) cell equivalents). By contrast, acid extracts of these fractions contained over 10-times as much phospholipase activity in the dialyzed supernatants (20-300 pmol/h per 10(6) cell equivalents), whereas phospholipase inhibitor(s) were found in the sediment. The acid-solubilized phospholipase A2 activity was absolutely Ca2+-dependent and optimal at pH 7.0-7.5 with 1.0 mM added Ca2+. Addition of the resuspended sediment of the acid extract dose-dependently suppressed phospholipase activity in the supernatant; less than equivalent amounts were sufficient to inhibit 95%. Suppressor activity was lipid-extractable. After thin layer chromatography of lipid extracts, the bulk of inhibitory activity was recovered from the free fatty acid region. Analysis of the fatty acids by gas liquid chromatography showed that 63% were unsaturated. All unsaturated fatty acids tested were potent inhibitors of phospholipase A2 activity (IC50 3-10 microM). Oleoyl-CoA, hydroxyeicosatetraenoic acids and leukotriene D4 were also inhibitory, while methyl oleate, saturated fatty acids and the prostaglandins E2 and F2 alpha had no effect. These in vitro data indicate that neutral-active and calcium-dependent phospholipase A2 in human polymorphonuclear leukocytes is largely suppressed by endogenous inhibitors and suggest that unsaturated fatty acids and some of their metabolites may partly account for this suppressor activity.

Calcium↗

PubMed1988

Properties and purification of an arachidonoyl-hydrolyzing phospholipase A2 from a macrophage cell line, RAW 264.7.

The lipid mediators, platelet activating factor (PAF) and the eicosanoids, can be coordinately produced from the common phospholipid precursor, 1-O-alkyl-2-arachidonoylglycerophosphocholine (1-O-alkyl-2-arachidonoyl-GPC), through the initial action of a phospholipase A2 that cleaves arachidonic acid from the sn-2 position. The mouse macrophage cell line RAW 264.7, which was used as a model macrophage system to study the arachidonoyl-hydrolyzing phospholipase A2 enzyme(s), could be induced to release arachidonic acid in response to inflammatory stimuli. A phospholipase A2 that hydrolyzed 1-O-hexadecyl-2-[3H]arachidonoyl-GPC was identified in the cytosolic fraction of these macrophages. This phospholipase activity was optimal at pH 8 and dependent on calcium. Enzyme activity could be stimulated 3-fold by heparin, suggesting the presence of phospholipase inhibitory proteins in the macrophage cytosol. Compared to 1-alkyl-2-arachidonoyl-GPC, the enzyme hydrolyzed 1-acyl-2-arachidonoylglycerophosphoethanolamine (1-acyl-2-arachidonoyl-GPE) with similar activity but showed slightly greater activity against 1-acyl-2-arachidonoyl-GPC, suggesting no specificity for the sn-1 linkage or the phospholipid base group. Although comparable activity against 1-acyl-2-arachidonoylglycerophosphoinositol (1-acyl-2-arachidonoyl-GPI) could be achieved, the enzyme exhibited much lower affinity for the inositol-containing substrate. The enzyme did, however, show apparent specificity for arachidonic acid at the sn-2 position, since much lower activity was observed against choline-containing substrates with either linoleic or oleic acids at the sn-2 position. The cytosolic phospholipase A2 was purified by first precipitating the enzyme with ammonium sulfate followed by chromatography over Sephadex G150, where the phospholipase A2 eluted between molecular weight markers of 67,000 and 150,000. The active peak was then chromatographed over DEAE-cellulose, phenyl-Sepharose, Q-Sepharose, Sephadex G150 and finally hydroxylapatite. The purification scheme has resulted in over a 1000-fold increase in specific activity (2 mumol/min per mg protein). Under non-reducing conditions, a major band on SDS-polyacrylamide gels at 70 kDa was observed, which shifted to a lower molecular weight, 60,000, under reducing conditions. The properties of the purified enzyme including the specificity for sn-2-arachidonoyl-containing phospholipids was similar to that observed for the crude enzyme. The results demonstrate the presence of a phospholipase A2 in the macrophage cell line. RAW 264.7, that preferentially hydrolyzes arachidonoyl-containing phospholipid substrates.

Acetophenones↗

PubMed1989

In vivo release and clearance of rat platelet phospholipase A2.

Intravenous injection of ADP into rats produced a rapid increase of plasma phospholipase A2 activity with a concomitant decrease in platelet count. Phospholipase A2 activity in plasma reached a peak within 1 min and thereafter declined sharply. This phospholipase A2 activity was reactive with a monoclonal antibody specific for rat platelet-derived phospholipase A2. These findings, together with the fact that ADP is a stimulant specific for platelets, suggest that the phospholipase A2 observed may be released into plasma from activated platelets in vivo. When platelet-derived phospholipase A2 was purified, labeled with 125I, and injected intravenously into rats, the radioactivity in the plasma decreased rapidly: the half-life of the enzyme in the blood stream was less than 30 s. Addition of either heparin or anti-phospholipase A2 monoclonal antibody directed against the domain of the enzyme responsible for binding to heparin retarded the clearance of the enzyme, suggesting that phospholipase A2 might be adsorbed onto heparan sulfate proteoglycans on the endothelial cell surface. Examination of the distribution of radioactivity in vivo revealed that most of the enzyme was rapidly taken up by the liver and degraded to acid-soluble materials.

Adenosine Diphosphate↗

PubMed1985

Inhibition of purified lysosomal phospholipase A1 by beta-adrenoceptor blockers.

Inhibition of rat liver lysosomal phospholipases is one of the main events that leads to accumulation of tissue phospholipids during drug-induced phospholipidosis. Drug inhibition of lysosomal phospholipase A may occur by direct effects of drugs on the enzyme (or substrate) or by drug-induced increases in intralysosomal pH. Although beta-adrenoceptor blockers have not been reported to cause lipid storage, they do inhibit lysosomal phospholipase A. To investigate the structural requirements for drug inhibition, we studied the effects of six beta-adrenoceptor blockers on purified rat liver lysosomal phospholipase A1. The agents studied include: propranolol, timolol, metoprolol, practolol, atenolol and the combined alpha and beta adrenoceptor blocking agent, labetalol. The drugs varied by two logs in their abilities to inhibit phospholipase A1 activity. The relative inhibitory potencies were propranolol greater than labetalol much greater than timolol greater than metoprolol much greater than practolol greater than atenolol. Our studies identify drug hydrophobicity as a key determinant for phospholipase A1 inhibition. A strong negative correlation was noted between the octanol/water partition coefficients and IC50 for phospholipase inhibition (r = -0.91). The ability of propranolol to inhibit phospholipase A1 was identical for the d, l and the d and l stereoisomers.

Adrenergic beta-Antagonists↗

PubMed1985

Inhibition of [3H]nitrendipine binding by phospholipase A2.

Phospholipase A2 from several sources inhibited [3H]nitrendipine binding to membranes from brain, heart and ileal longitudinal muscle. The enzymes from bee venom and Russell's viper venom were most potent, having IC50 values of approximately 5 and 14 ng/ml, respectively, in all three membrane preparations. Inhibition of binding by bee venom phospholipase A2 was time- and dose-dependent. Mastoparan, a known facilitator of phospholipase A2 enzymatic activity, shifted the bee venom phospholipase A2 dose-response curve to the left. Pretreatment of brain membranes with bee venom phospholipase A2 (10 ng/ml) for 15 min caused a 2-fold increase in the Kd without changing the Bmax compared with untreated membranes. Extension of the preincubation period to 30 min caused no further increase in the Kd but significantly decreased the Bmax to 71% the value for untreated membranes. [3H]Nitrendipine, preincubated with bee venom phospholipase A2, was recovered and found to be fully active, indicating that the phospholipase A2 did not modify the ligand. It is concluded that phospholipase A2 acts on the membrane at or near the [3H]nitrendipine binding site and that phospholipids play a key role in the interactions of 1,4 dihydropyridine calcium channel antagonists with the dihydropyridine binding site.

Animals↗

PubMed1987

Dantrolene and mepacrine antagonize the hemolysis of human red blood cells by halothane and bee venom phospholipase A2.

Dantrolene is an effective antagonist of anesthesia-induced malignant hyperthermia due to a poorly understood action on skeletal muscle. The present study examines whether the red blood cell can be used as a model to investigate the mechanism of dantrolene action. Halothane (4.7 mM) caused 9% hemolysis of red blood cells. Phospholipase A2 (1 microM) alone caused less than 2% hemolysis, despite high levels (54%) of phosphatidylcholine hydrolysis. Incubation of red blood cells with halothane and phospholipase A2 caused 72% hemolysis. Halothane addition caused 100% hydrolysis of all diacylphosphoglycerides by phospholipase A2, suggesting a mutual potentiation. The major products of phospholipase A2 activity, arachidonic acid and lysophosphatidylcholine, when exogenously added, also greatly increased hemolysis induced by halothane, with arachidonic acid most closely resembling the synergism observed with phospholipase A2. Dantrolene (10 microM) and mepacrine (10 microM) significantly antagonized hemolysis induced by halothane and phospholipase A2 or halothane and exogenously added arachidonic acid and lysophosphatidylcholine. Dantrolene and mepacrine did not antagonize phospholipid hydrolysis or free fatty acid levels. Dantrolene and mepacrine antagonized the synergism between halothane and phospholipase A2 most likely by reducing the lytic action of halothane in the presence of arachidonic acid. The red blood cell is a useful model for studying the antagonism of halothane and phospholipase A2 toxicity by dantrolene and mepacrine.

Bee Venoms↗

PubMed1989

Immunological relationships of phospholipase A2 neurotoxins from snake venoms.

Polyclonal rabbit antisera were raised against ten snake phospholipase A2 neurotoxins and one snake phospholipase A2 cytotoxin. Immunological cross-reactivities between these toxins, two other snake phospholipase A2 enzymes and pancreatic phospholipase A2 were studied using ELISA technology. All snake phospholipase A2 neurotoxins fell into two main antigenic classes. One antigenic class was composed of all the elapid toxins tested (textilotoxin, taipoxin, notexin, pseudexin and beta-bungarotoxin), the cytotoxic phospholipase A2 from Naja naja atra and pancreatic phospholipase A2. beta-Bungarotoxin seemed to be in an immunological subclass of its own compared to the rest of the elapid toxins. The second antigenic class was comprised of crotalid and viperid phospholipase A2 neurotoxins (crotoxin, concolor toxin, Mojave toxin, vegrandis toxin, ammodytoxin and caudoxin). Our data indicated that the viperid toxins, caudoxin and ammodytoxin, were an immunological subclass apart from the crotalid toxins.

Animals↗

PubMed1982

Phospholipase A2 activity in pancreatic islets is calcium-dependent and stimulated by glucose.

Phospholipase A2 activity in islet cell homogenates and dispersed islet cells of the rat was determined using an exogenous radiolabeled phospholipid substrate from E. coli membranes. Phospholipase A2 activity in islet homogenates was found to have two pH optima in acid or neutral/alkaline pH ranges. The enzyme activity at pH 7.5 was calcium dependent and responded to increasing calcium concentrations with graded increases in phospholipid hydrolysis. Preincubation of islets with a concentration of glucose known to elicit maximum rates of insulin secretion resulted in a stable activation of phospholipase A2 activity which was assayable in islet homogenates. Glucose stimulated phospholipase A2 in these preparations by as much as 220% above control. 2-Deoxy-D-glucose, a nonsecretory analogue of glucose, did not elicit a significant increase in islet phospholipase A2 activity. The glucose sensitive enzyme was associated with a membrane-enriched subcellular fraction in which the glucose-stimulated activity was greater than 2-fold higher than control activity. Glucose stimulation potentiated the phospholipase A2 activity measured in the presence of high calcium concentrations. Phospholipase A2 activity was also found in dispersed islet cell preparations where glucose stimulation of what may be a partly externalized membrane enzyme was most apparent at low calcium concentrations. These data indicate that islet cells possess phospholipase A2 activity which may be in part localized to the plasma membrane as well as other membrane systems, and which exhibits the characteristic properties of pH and calcium dependency, and sensitivity to secretagogue stimulation reported for the enzyme in other secretory systems.

Animals↗

PubMed1990

Characterization of distinct phospholipases mediating bradykinin and noradrenaline hyperalgesia.

Specific prostaglandins have been identified that mediate the sympathetic postganglionic neuron-terminal dependent hyperalgesia induced by bradykinin and norepinephrine, prostaglandin E2 and prostacyclin, respectively. In this study we evaluated the hypothesis that bradykinin and norepinephrine stimulate prostaglandin production in the rat, via distinct phospholipases. We found that, in normal skin, bradykinin hyperalgesia is inhibited by the phospholipase A2 inhibitor, mepacrine, but not by the phospholipase C inhibitor, neomycin and is mimicked by phospholipase A2. In chloroform-treated skin or when co-injected with A23187, bradykinin-induced hyperalgesia was found to consist of two components, one resulting from prostaglandin E2 synthesis (phospholipase A2-dependent) and one resulting from prostacyclin synthesis (phospholipase C-dependent). This latter component is blocked by Quin 2 and verapamil and also inhibited by yohimbine, an alpha 2 receptor antagonist. Arachidonic acid induces a dose-dependent hyperalgesia that was found to be like bradykinin-hyperalgesia in untreated skin (prostaglandin E2-mediated and phospholipase A2-dependent). In chloroform-treated skin or in the presence of A23187, arachidonic acid like bradykinin led to the production of prostacyclin as well as prostaglandin E2. Norepinephrine does not produce hyperalgesia in untreated skin, but in chloroform pretreated skin or in the presence of the calcium ionophore A23187, norepinephrine produces a potent dose-dependent hyperalgesia. This hyperalgesia is prevented by sympathectomy and suppressed by the calcium antagonists Quin 2 and verapamil. It is also suppressed by indomethacin and neomycin but not by SC19220 and mepacrine and is mimicked by phospholipase C.(ABSTRACT TRUNCATED AT 250 WORDS)

Aminoquinolines↗

PubMed1999

Lysosomal phospholipase activity is decreased in mucolipidosis II and III fibroblasts.

Mucolipidosis (ML) II and III are rare autosomal recessively inherited diseases characterized by deficiency of multiple lysosomal enzymes and, as a result, a generalized storage of macromolecules in lysosomes of cells of mesenchymal origin. In ML II and ML III fibroblasts, most, but not all, newly synthesized lysosomal enzymes are secreted into the medium instead of being targeted correctly to lysosomes. Defects in the enzyme UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase underlie this effect. It is unknown how lysosomal phospholipases are targeted to the lysosomes of fibroblasts. In the present study lysosomal phospholipase activity was determined in delipidated fibroblast homogenates and plasma from ML II and ML III patients and controls using a [3H]choline-labeled phosphatidylcholine. After incubation, residual phosphatidylcholine and its labeled degradation products (lysophosphatidylcholine, glycerophosphorylcholine and choline phosphate) were quantified. We found that ML II and ML III fibroblasts are deficient in lysosomal phospholipase A and C activity. These enzymes were present in elevated amounts in plasma of ML II and ML III patients. These data indicate that phospholipases, like most other lysosomal enzymes in these diseases, are secreted into the blood instead of being targeted specifically to lysosomes. Thus, the mannose-6-phosphate receptor pathway is needed for proper delivery of lysosomal phospholipases to lysosomes. We also found that production of labeled choline phosphate was mainly due to the activity of acid sphingomyelinase instead of phospholipase C under the assay conditions used. Other active lipolytic enzymes were phospholipase A and lysophospholipase. No evidence for phospholipase D activity was found.

Biological Transport, Active↗

PubMed1988

No direct correlation between Ca2+ mobilization and dissociation of Gi during platelet phospholipase A2 activation.

Stimulation of human platelets with thrombin is accompanied by activation of both phospholipases C and A2. These have been considered to be sequential events, with phospholipase A2 activation resulting from the prior hydrolysis of inositol phospholipids and mobilization of intracellular Ca2+ stores. However, our and other laboratories have recently questioned this proposal, and we now present further evidence that these enzymes may be activated by separate mechanisms during thrombin stimulation. Alpha-thrombin induced the rapid hydrolysis of inositol phospholipids, and formation of inositol trisphosphate and phosphatidic acid. This was paralleled by mobilization of Ca2+ from internal stores. These responses were blocked by about 50% by prostacyclin. In contrast, the liberation of arachidonic acid induced by alpha-thrombin was totally inhibited by prostacyclin. The less-effective agonists, platelet activating factor (PAF) and gamma-thrombin also both stimulated phospholipase C, but whereas PAF evoked a rapid and transient response, that of gamma-thrombin was delayed and more sustained. The abilities of these agonists to induce the release of Ca2+ stores closely paralleled phospholipase C activation. However, the maximal intracellular Ca2+ concentrations achieved by these two agents were the same. Despite this, gamma-thrombin and not PAF, was able to release a small amount of arachidonic acid. When alpha-thrombin stimulation of platelets was preceded by epinephrine, there was a potentiation of phospholipase C activation, Ca2+ mobilization and aggregation. The same was true for gamma-thrombin and PAF. However, unlike alpha-thrombin, the gamma-thrombin-stimulated arachidonic acid release was not potentiated by epinephrine, but rather somewhat reduced. These results suggested that phospholipase C and phospholipase A2 were separable events in activated platelets. The mechanism by which alpha-thrombin stimulated phospholipase A2 did not appear to be through dissociation of the inhibitory GTP-binding protein, Gi, since gamma-thrombin decreased the pertussis toxin-induced ADP-ribosylation of the 41 kDa protein as much as did alpha-thrombin, but was a much less effective agent than alpha-thrombin at inducing arachidonic acid liberation.

Adenosine Diphosphate Ribose↗

PubMed2003

Role of group IIA phospholipase A2 in rat colitis induced by dextran sulfate sodium.

Although group IIA phospholipase A(2) has been suggested to be implicated in inflammatory bowel disease, its pathophysiological role in colitis remains unclear. We investigated whether group IIA phospholipase A(2) had pro-inflammatory roles in dextran sulfate sodium-induced colitis in the rat. Secretory phospholipase A(2) activity was markedly increased in the distal colon with two peaks. Strong immunostaining for group IIA phospholipase A(2) was found in subepithelial tissue and lamina propria at early stage and in deeper tissues of the erosion area at later stage. Treatment with a specific group IIA phospholipase A(2) inhibitor, S-3013/LY333013 (methyl[[3-(aminooxoacetyl)-2-ethyl-1-(phenylmethyl)]-1H-indol-4yl]oxy) acetate), reduced erosion area, shortening of colon and colonic inflammation, and strongly inhibited the increase in secretory phospholipase A(2) activity and moderately reduced myeloperoxidase activity in the colon in vivo, while eicosanoid levels were not affected. Marked group IIA phospholipase A(2) expression in the erosion area and the improvement of colitis by the group IIA phospholipase A(2) inhibitor strongly suggest that this enzyme plays pro-inflammatory roles in this colitis model.

Animals↗

PubMed2002

Roles for cytosolic phospholipase A2alpha as revealed by gene-targeted mice.

Cytosolic phospholipase A2alpha (cPLA2alpha) has unique characteristics among phospholipase A2 (PLA2) family members. Under regulation by intracellular signaling system, cytosolic phospholipase A2alpha liberates arachidonic acid that can be metabolized by downstream enzymes to generate prostaglandins (PGs) and leukotrienes (LTs). Mice deficient in this enzyme have been generated by gene-targeting techniques. Cytosolic phospholipase A2alpha-deficient mice have a normal appearance and grow normally. Close examinations have revealed a renal concentration defect and intestinal ulcerative lesions. There may also be other disadvantages that are not manifested in well-regulated housing conditions. Although female mice are fertile, they become pregnant less frequently and have small litter sizes; moreover, impaired parturition results in few surviving pups. Primary cultured cells prepared from cytosolic phospholipase A2alpha-deficient mice produce significantly smaller amounts of prostaglandins and leukotrienes. Various disease models such as anaphylaxis, acute lung injury, brain injury induced by ischemia/reperfusion and neurotoxin, and polyposis have been investigated. In all these settings, cytosolic phospholipase A2alpha-deficient mice show significantly milder phenotypes. The mechanisms by which deficiencies of this enzyme exert protective effects may differ, but, a cytosolic phospholipase A2alpha inhibitor could have a wide spectrum of clinical targets. Specific functions of cytosolic phospholipase A2alpha have been clearly demonstrated using the gene-targeted mice. Also, comparisons with mice in which related enzymes and receptors have been manipulated using genetic technologies provide further insights into roles of lipid mediators in physiology and pathology.

Anaphylaxis↗

PubMed1982

[Activable phospholipase a2 of rat blood platelets].

Rat blood platelet lysate showed high phospholipase A2 activity and contained much less than activating factor much greater than which increased phospholipase A2 activity. The phospholipase A2 and the much less than activating factor much greater than were eluted together in the same first protein fraction obtained by chromatography on a G 100 Sephadex column washed with tris-HCl buffer (pH 7.2). This mixture of proteins can be further fractionated on a Sepharose Blue CL 6B column in 2 protein fractions. The first fraction eluted by tris-HCl buffer (pH 7.2) showed 10 percent of the initial phospholipase activity; the second fraction eluted by NaCl 1 M-tris HCl buffer (pH 7.2) which showed no phospholipase activity, contains activating factor; if both fractions are mixed the initial phospholipase activity is almost totally restored. In conclusion, in rat blood platelets, phospholipase activity originates in a great part from the association of an much less than activable phospholipase much greater than and an much less than activating factor much greater than.

Animals↗

PubMed2005

Clonidine increases membrane-associated phospholipase A 2.

BACKGROUND AND OBJECTIVE: An anti-inflammatory effect of alpha2-adrenoreceptor agonists has been suggested. Phospholipase A2 is a key enzyme in the production of precursors of inflammatory lipid mediators. The aim of the present study was to investigate the effect of clonidine on phospholipase A2 activity in an established in vitro model. METHODS: Human being platelet membranes containing active phospholipase A2 were exposed to buffer control or to three increasing concentrations of clonidine. Phospholipase A2 was measured by a radioisotope technique. RESULTS: A massive increase in phospholipase A2 activity was measured after clonidine exposure leading to final values of 92.5 +/- 3.1 pmol mg protein(-1) min(-1) (4.5-fold higher than control values; P < or = 0.01 vs. control). After clonidine exposure the maximal reaction velocity increased, while the Michaelis-Menten constant did not change. The Lineweaver-Burk representation suggested an interaction of clonidine with the phospholipase A2-substrate complex as well as the phospholipase A2 molecule. CONCLUSION: We conclude that the putative anti-inflammatory effect of clonidine was not caused by inhibition of phospholipase A2.

Adjuvants, Anesthesia↗