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PubMed1976

Gonadotropin receptors in plasma membranes of bovine corpus luteum. I. Effect of phospholipases on the binding of 125I-choriogonadotropin by membrane-associated and solubilized receptors.

The ability of bovine corpus luteum plasma membranes to bind 125I-choriogonadotropin has been examined after prior treatment of the membranes with phospholipases A, C, and D. Treatment of the purified membranes with low concentrations of phospholipases A and C resulted in the inhibition of the binding of 125I-choriogonadotropin to its receptors, whereas phospholipase D had no effect. Receptor activity was decreased by low concentrations of phospholipase A from either bee venom, Vipera russelli or Crotalus terrificus terrificus. Similarly, low concentrations of phospholipase C from Clostridium perfringens and Clostridium welchii also inhibited the binding activity while comparatively higher concentrations of phospholipase C from Bacillus cereus were required to achieve comparable inhibition. The time required to produce 50% inhibition of in vitro binding by phospholipases A and C was found to be 6 and 23 min, respectively. Upon either removal or chelation of calcium ions by ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) both enzymes were completely inhibited as evidenced by the complete retention of the membrane binding activity. The decrease in the specific binding of choriogonadotropin to membranes after phospholipase digestion resulted in a decrease in the number of binding sites and was not accompanied by a change in the affinity of the hormone-receptor complex. The rates of association and dissociation of the 125I-choriogonadotropin-receptor complex and the equilibrium dissociation constant (Kd) were nearly identical in untreated and phospholipase-treated membranes. Phospholipases did not have any effect on the preformed hormone-receptor complex or on solubilized receptor. Filtration through Sepharose 6B of solubilized 125I-choriogonadotropin-receptor complex from untreated membranes or membranes which had been pretreated with phospholipase C prior to carrying out hormone binding did not alter the profile (Kav 0.38). Gel filtration of membranes treated with phospholipase A showed two peaks of bound radioactivity with distribution coefficients (Kav) of 0.08 and 0.35, respectively.

Animals↗

PubMed1978

Release of phospholipase A and triglyceride lipase from rat liver.

A new, rapid, and sensitive assay for phospholipase A, utilizing commercially available [14C]phosphatidylethanolamine with 14C label in both palmitic acid moieties, was used to study phospholipase A release from perfused liver, hepatocytes, and intestinal cells from rats. Heparin triggered a prompt release of phospholipase A from perfused liver. Phospholipase A and triglyceride lipase were released from hepatocytes at a linear rate for 1 h and 30 min, respectively. Heparin (20 u/ml) doubled the release of phospholipase A and triglyceride lipase from hepatocytes. Colchicine (0.1 mM), but not puromycin (0.2 mM), inhibited basal and heparin-stimulated phospholipase A release by 40%. Since the amount of phospholipase A and triglyceride lipase released into the medium greatly exceeded intracellular activities, it is possible that secretion is coupled with intracellular conversion from inactive to active forms of the enzymes. Dibutyryl cyclic AMP (1 mM) inhibited phospholipase A (48%) and triglyceride lipase (82%) release from hepatocytes. Epinephrine, dexamethasone, and clofibrate inhibited release of triglyceride lipase but not phospholipase A. Phospholipase A activity of intestinal cells was greater than in hepatocytes, but neither heparin nor dibutyryl cyclic AMP affected phospholipase A release from intestinal cells. These results suggest that the liver is a major source of phospholipase A of postheparin plasma. The fact that dibutyryl cyclic AMP affects the release of these enzymes suggests an additional mechanism for hormonal regulation of lipid and lipoprotein metabolism.

Animals↗

PubMed1987

Different susceptibilities of platelet phospholipids to various phospholipases and modifications induced by thrombin. Possible evidence of rearrangement of lipid domains.

On the membrane surface of the human platelet, phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were hydrolyzed to different extents by the snake venom phospholipases A2 of varying pI values. The susceptibility of platelet phospholipids to basic phospholipase A2 of Naja nigricollis (pI 10.6) has been reported (Wang et al. (1986) Biochim. Biophys. Acta 856, 244-258). The susceptibilities of platelet phospholipids to acidic phospholipase A2 of Naja naja atra (pI 5.2) and to neutral phospholipase A2 of Hemachatus haemachatus (pI 7.3) were investigated in this study. In gel-filtered platelets, acidic phospholipase A2 hydrolyzed 35% PC and 10% PE, while neutral phospholipase A2 hydrolyzed 18% PC and 3% PE. In thrombin-induced shape-changed platelets, acidic phospholipase A2 hydrolyzed 20% PC and 10% PE, while neutral phospholipase A2 hydrolyzed 15% PC and 6% PE. In thrombin-activated platelets, acidic phospholipase A2 hydrolyzed 25% PC and 7% PE, while neutral phospholipase A2 hydrolyzed 25% PC and 10% PE. Sequential lipid hydrolysis experiments showed that basic phospholipase A2 of Naja nigricollis could hydrolyze the remaining PC and PE in the membrane previously treated with the neutral enzyme. The results may mean that: the PC and the PE domains exist on the platelet membrane surface; and the lipid domains on the membrane surface of resting platelets are rearranged by thrombin.

Blood Platelets↗

PubMed1997

Group II phospholipase A2 as an autocrine growth factor mediating interleukin-1 action on mesangial cells.

The proliferation of mesangial cells plays a central role in the progression of glomerulonephritis. We studied the role of group II phospholipase A2 in interleukin-1-stimulated proliferation of mesangial cells. Cultured rat mesangial cells secreted 5.3 units group II phospholipase A2/24 h per 10(5) cells in response to stimulation of 200 U/ml of interleukin-1. Northern hybridization analysis showed that mRNA for group II phospholipase A2 was induced by exogenously added group II phospholipase A2 (15 U/ml) as well as interleukin-1. The pretreatment of quiescent mesangial cells with interleukin-1 augmented [3H]thymidine incorporation caused by platelet derived growth factor. Exogenous group II phospholipase A2 (5-36 U/ml) purified homogeneously from rat spleen also increased [3H]thymidine incorporation by platelet derived growth factor-stimulated mesangial cells in a dose dependent manner (36 U/ml phospholipase A2; 1.9-fold). The stimulatory effect of interleukin-1 on DNA synthesis of mesangial cells was specifically blunted by immunoglobulin raised against group II phospholipase A2. Group II phospholipase A2 (16 U/ml) amplified a platelet derived growth factor-stimulated increase in the mesangial cell number by 1.5-fold. Among the products of the phospholipase A2-catalyzed reaction, lysophospholipids including lysophosphatidylcholine, lysophosphatidylethanolamine and lysophosphatidic acid, but not fatty acids, mimicked the stimulatory effect of interleukin-1 and phospholipase A2. These results suggest that group II phospholipase A2 acts as a signaling molecule that mediates interleukin-1-induced growth of rat mesangial cells through yielding lysophospholipids.

Animals↗

PubMed1999

Cloning, expression and biochemical characterization of a basic-acidic hybrid phospholipase A2-II from Agkistrodon halys pallas.

A cDNA encoding a basic-acidic hybrid phospholipase A2-II from Agkistrodon halys Pallas with an N-terminus highly homologous to that of BPLA2 and a C-terminus sequence almost the same as that of APLA2 was inserted into a bacterial expression vector and effectively expressed in Escherichia coli RR1. The protein was produced as insoluble inclusion bodies. After partial purification by washing, the inclusion bodies with Triton X-100, denaturing and refolding, the renatured recombinant protein was purified by FPLC column superose 12. The purified recombinant enzyme with an isoelectric point of pH 6.8 could cross-react with antiserum prepared against acidic phospholipase A2. The enzymatic activity of the expressed basic-acidic hybrid phospholipase A2-II is close to that of denatured-refolded native basic phospholipase A2, and has the same inhibiting effect on platelet aggregation as denatured-refolded acidic phospholipase A2, but lacks the hemolytic activity of denatured-refolded basic phospholipase A2. To study the structural relationships among basic phospholipase A2, acidic phospholipase A2 and basic-acidic hybrid phospholipase A2-II, molecular modeling of basic-acidic hybrid phospholipase A2-II was done. The roles of various amino acid residues in the enzymatic activity and pharmacological activities of phospholipase A2 are discussed.

Agkistrodon↗

PubMed1987

Stimulation of phospholipase A2 activity in bovine rod outer segments by the beta gamma subunits of transducin and its inhibition by the alpha subunit.

In the rod outer segments (ROS) of bovine retina, light activation of phospholipase A2 has been shown to occur by a transducin-dependent mechanism. In this report, the transducin-mediated stimulation of phospholipase A2 is shown to require dissociation of the alpha beta gamma heterotrimer. Addition of transducin to dark-adapted transducin-poor ROS stimulated phospholipase A2 activity only with coincident exposure to white light or, in the dark, with addition of the hydrolysis-resistant GTP analog, guanosine 5'-[gamma-thio]triphosphate (GTP[gamma-S]). Both light and GTP[gamma-S] induced dissociation of the transducin subunits and led to severalfold increases in the phospholipase A2 activity of transducin-rich, but not transducin-poor, ROS. In contrast, pertussis toxin treatment of transducin, which stabilizes the associated state of this G protein, prevented the stimulation of phospholipase A2 by exogenous transducin in the presence of light. Addition of purified transducin subunits to dark-adapted transducin-poor ROS revealed that phospholipase A2 stimulation occurred by action of the beta gamma subunits. This is in contrast to the transducin-mediated increase in cGMP phosphodiesterase activity, where activation occurs by action of the alpha subunit. The alpha subunit, which itself slightly stimulated phospholipase A2 activity, inhibited the beta gamma-induced stimulation of phospholipase A2. This inhibition appears to be the result of subunit reassociation since addition of GTP[gamma-S] abolished the inhibitory effect of the alpha subunit on the beta gamma-induced increase in phospholipase A2, while pertussis toxin treatment of the subunits further inhibited phospholipase A2 activity. Modulation of phospholipase A2 activity by the transducin subunit is, therefore, a mode of action for these subunits in signal transduction.

Animals↗

PubMed1989

Biochemical characterization of phospholipase D activity from human neutrophils.

We have found a phospholipase D activity in the postnuclear fraction of human neutrophils, employing phosphatidylinositol as exogenous substrate. This phospholipase D activity was assessed by both phosphatidate formation and by free inositol release in the presence of 15 mM LiCl in the reaction mixture and in the absence of Mg2+ ions to prevent inositol-1-phosphate phosphatase activity. To assess further the phospholipase D activity, we studied its capacity to catalyze a transphosphatidylation reaction, as a unique feature of the enzyme. It was detected as [14C]phosphatidylethanol formation when the postnuclear fraction was incubated with [14C]phosphatidylinositol in the presence of ethanol. The phospholipase D showed a major optimum pH at 7.5 and a minor one at pH 5.0. Neutral and acid phospholipase D activities were differentially located in subcellular fractionation studies of resting neutrophils, namely in the cytosol and in the azurophilic granules, respectively. Neutral phospholipase D required Ca2+ ions to the active, whereas the acid enzyme activity was Ca2(+)-independent. The neutral phospholipase D activity showed a certain specificity for phosphatidylinositol, as it was able to hydrolyze phosphatidylinositol at a much higher rate than phosphatidylcholine, in the absence and in the presence of different detergents. This neutral phospholipase D activity behaved as a protein of high molecular mass (350-400 kDa) by gel filtration chromatography. Moreover, neutral phospholipase D activity was detected in the postnuclear fraction of human monocytes, by measuring free inositol release from phosphatidylinositol as exogenous substrate, under the same experimental conditions as those used with neutrophils. The enzyme displayed similar specific activities in both cell types as well as the same degree of activation after cell stimulation with the calcium ionophore A23187. These results demonstrate the existence of two phospholipase D activities with different pH optima and intracellular location in human neutrophils. Furthermore, these results suggest that this phospholipase D can play a role in signal-transducing processes during cell stimulation in human phagocytes.

Cell Fractionation↗

PubMed1980

The action of human and rabbit serum phospholipase A2 on Escherichia coli phospholipids.

We have compared the properties of phospholipase A (E.C. 3.1.1.4) activity in whole human and rabbit serum toward the phospholipids of Escherichia coli. Using as substrate E. coli labeled during growth with either [1-(14)C]-palmitic acid or [1-(14)C]oleic acid, and then autoclaved to inactivate E. coli phospholipases and to render the labeled phospholipids accessible to exogenous phospholipases, we show that the deacylating activity in both human and rabbit serum is almost exclusively of the A(2) type. Rabbit serum is at least 20-fold more active than human serum. Activity in both sera is maximal at physiological Ca(2+) concentrations (2 mM) and is abolished by ethylenediaminetetraacetic acid. To examine hydrolysis of intact (unautoclaved) E. coli treated with 25% serum, use was made of a phospholipase A-deficient E. coli strain (E. coli S17), thereby eliminating the possible contribution of bacterial phospholipases to degradation. Human and rabbit serum are about equally bactericidal toward E. coli and cause comparable structural damage. However, only rabbit serum produces substantial hydrolysis of the phospholipids of intact E. coli S17. Heated (56 degrees C, 30 min) rabbit serum is non-bactericidal and retains phospholipase A(2) activity toward autoclaved, but not intact E. coli. The ability of heated serum to degrade phospholipids of intact E. coli S17 is restored, however, by adding 25% normal human serum, which is bactericidal. In this combination, doses of heated rabbit serum containing as much phospholipase A(2) activity (toward autoclaved E. coli) as is present in 25% unheated rabbit serum, produce roughly the same extent of hydrolysis of intact E. coli as does normal rabbit serum alone. Low doses with a phospholipase A(2) activity comparable to that of normal human serum elicit little or no hydrolysis. These findings indicate that hydrolysis of the phospholipids of intact E. coli S17 by serum occurs when: 1) the serum is bactericidal, and 2) when sufficient phospholipase A(2) is present. The difference in phospholipid hydrolysis that accompanies killing of E. coli by human or rabbit serum appears to reflect, therefore, the different amounts of phospholipase A(2) activity in the two sera. Phospholipid degradation is not required for the bactericidal action of serum. Bacterial phospholipid breakdown may be important, however, in the overall destruction and digestion of invading bacteria by the host.-Kaplan-Harris, L., J. Weiss, C. Mooney, S. Beckerdite-Quagliata, and P. Elsbach. The action of human and rabbit serum phospholipase A(2) on Escherichia coli phospholipids.

Animals↗

PubMed1986

Properties of acid phospholipases in lysosome and extracellular medium of Tetrahymena pyriformis.

Phospholipase activity in the lysosomes of the protozoan Tetrahymena pyriformis strain NT-1 was studied using phospholipids radioactively labeled in the fatty acid moieties. Lysosomal homogenates showed high phospholipase activity with an acidic pH optimum. Unlike the phospholipases in rat liver lysosomes, almost all activity was recovered from the membranous fraction of the lysosomes. The activity was partially solubilized by treatment of the membranes with a detergent or trypsin. Using specifically labeled phospholipids revealed that phospholipase. A1 and C are predominant in Tetrahymena lysosomes, no appreciable phospholipase A2 or lysophospholipase activity was detected in the fraction. There are two catabolic pathways of the hydrolysis of phospholipid: Hydrolysis is initiated by deacylation at the 1-position by phospholipase A1 and the 2-acyllysophospholipid thus formed is successively attacked by (lyso)phospholipase C; hydrolysis is initiated by cleavage of phosphodiester by phospholipase C and the diacylglycerol thus formed is attacked by lipase. Both pathways give the same end products, free fatty acid and 2-monoacylglycerol. The former pathway might be predominant in Tetrahymena lysosomes under physiological conditions since the pathway is independent of detergent. Phospholipases A1 and C activities were partially released into the medium. At least two different phospholipases C are present in the medium as judged by chromatographic behavior and their substrate specificities.

Animals↗

PubMed1995

Plasma type II phospholipase A2 levels are elevated in severe preeclampsia.

OBJECTIVE: Our purpose was to compare plasma concentrations of type II phospholipase A2 in normal and preeclamptic pregnancies and to determine whether plasma from preeclamptic patients increases endothelial cell secretion of type II phospholipase A2. STUDY DESIGN: We compared the plasma levels of type II phospholipase A2 in 23 patients with normal pregnancies and 25 patients with strictly defined preeclampsia. Plasma samples were collected throughout pregnancy and stored at -80 degrees C. Patients with preeclampsia were matched closely as possible with controls for maternal age, race, gestational age at blood sampling, and parity. Plasma levels of type II phospholipase A2 were measured by enzyme immunoassay. To investigate endothelial cell type II phospholipase A2 production, we measured levels of prostacyclin and type II phospholipase A2 in conditioned media from cultured human umbilical vein endothelial cells exposed to plasma from preeclamptic and normal pregnant patients. RESULTS: The mean plasma type II phospholipase A2 level in the mild preeclampsia group (6.1 +/- 3.0 ng/ml, n = 6) was not different from that in control patients (6.0 +/- 3.2 ng/ml, n = 23). However, the mean type II phospholipase A2 level in patients with severe preeclampsia (19.9 +/- 12.3 ng/ml, n = 19) was significantly higher when compared with the other two groups (p < 0.001). Prostacyclin concentrations in conditioned media of endothelial cells exposed to plasma from patients with preeclampsia were elevated compared with cells incubated with control plasma (8.6 +/- 1.7 vs 4.0 +/- 5.0 ng/ml, p < 0.05). However, the concentration of type II phospholipase A2 in conditioned medium was not significantly different (1.6 +/- 0.3 vs. 1.8 +/- 1.0 ng/ml, p = 0.40). CONCLUSIONS: Plasma concentrations of type II phospholipase A2 are elevated in preeclampsia and correlate well with the severity of disease. Plasma from preeclamptic women increased secretion of prostacyclin, but not type II phospholipase A2 by endothelial cells in culture.

Case-Control Studies↗

PubMed1984

Biphasic effect on platelet aggregation by phospholipase a purified from Vipera russellii snake venom.

A basic phospholipase A was isolated from Vipera russellii snake venom. It induced a biphasic effect on washed rabbit platelets suspended in Tyrode's solution. The first phase was a reversible aggregation which was dependent on stirring and extracellular calcium. The second phase was an inhibitory effect on platelet aggregation, occurring 5 min after the addition of the venom phospholipase A without stirring or after a recovery from the reversible aggregation. The aggregating phase could be inhibited by indomethacin, tetracaine, papaverine, creatine phosphate/creatine phosphokinase, mepacrine, verapamil, sodium nitroprusside, prostaglandin E1 or bovine serum albumin. The venom phospholipase A released free fatty acids from synthetic phosphatidylcholine and intact platelets. p-Bromophenacyl bromide-modified venom phospholipase A lost its phospholipase A enzymatic and platelet-aggregating activities, but protected platelets from the aggregation induced by the native enzyme. The second phase of the venom phospholipase A action showed a different degree of inhibition on platelet aggregation induced by some activators in following order: arachidonic acid greater than collagen greater than thrombin greater than ionophore A23187. The longer the incubation time or the higher the concentration of the venom phospholipase A, the more pronounced was the inhibitory effect. The venom phospholipase A did not affect the thrombin-induced release reaction which was caused by intracellular Ca2+ mobilization in the presence of EDTA, but inhibited collagen-induced release reaction which was caused by Ca2+ influx from extracellular medium. The inhibitory effect of the venom phospholipase A and also lysophosphatidylcholine or arachidonic acid could be antagonized or reversed by bovine serum albumin. It was concluded that the first stimulatory phase of the venom phospholipase A action might be due to arachidonate liberation from platelet membrane. The second phase of inhibition of platelet aggregation and the release of ATP might be due to the inhibitory action of the split products produced by this venom phospholipase A.

Adenosine Triphosphate↗

PubMed1983

The effects of polyamines and aminoglycosides on phosphatidylinositol-specific phospholipase C from human amnion.

The effects of polyvalent cations (polyamines and aminoglycoside antibiotics) on Ca2+-dependent phosphatidylinositol-specific phospholipase C activity of human amnion tissue were examined. In the presence of 1 mM Ca2+, the hydrolysis of phosphatidylinositol (2 mM) by phospholipase C was increased greatly (240-560% of control) by spermine (0.4 mM), spermidine (1 mM), neomycin (0.1 mM), gentamicin (0.2 mM), kanamycin (0.4 mM) and streptomycin (0.8 mM). Putrescine and cadaverine (0.1-2.0 mM), however, stimulated phospholipase C activity only slightly. The effects of spermidine, spermine and gentamicin on phospholipase C activity were characterized and found to be dependent upon the concentrations of phosphatidylinositol, Ca2+ and the particular polyvalent cation. At low concentrations of phosphatidylinositol and Ca2+ the predominant effect of polyamines and aminoglycosides was to inhibit phospholipase C activity. When the concentrations of phosphatidylinositol and Ca2+ were increased, spermidine, spermine and gentamicin stimulated phospholipase C activity. In the presence of 16 mM Ca2+, however, phospholipase C activity was maximal and was unaffected by either polyamines or aminoglycosides. At all concentrations of Ca2+ examined, the maximal stimulation of phospholipase C activity by a given polyvalent cation occurred at a fixed molar ratio of the particular polyvalent cation to phosphatidylinositol. Polyamines and aminoglycosides appeared to modulate the Ca2+ requirement for phospholipase C activity, but could not substitute completely for Ca2+. The activities of phospholipase A2, diacylglycerol lipase, monoacylglycerol lipase and diacylglycerol kinase in amnion tissue were unaffected by any of the polyvalent cations examined. It is proposed that any in vivo influences (stimulatory or inhibitory) of polyamines and aminoglycosides on amnion phospholipase C activity would depend upon the effective concentrations of Ca2+ and phosphatidylinositol.

Aminoglycosides↗

PubMed1994

Human recombinant phospholipase A2 inhibits platelet aggregation in vitro and in vivo in rat and guinea pig.

Platelets contain a phospholipase A2 in their granules which can be released in response to various activating stimuli in vitro as well as in vivo. Human recombinant phospholipase A2 (1-10 micrograms/ml) had no direct effect on platelet aggregation in vitro using rabbit platelet rich plasma. In contrast human recombinant phospholipase A2 (1-20 micrograms/ml) was able to inhibit aggregation of washed rabbit platelet in vitro induced by collagen (0.250-2.0 micrograms/ml). When rabbit platelet rich plasma was recalcified with CaCl2 1 M in the presence of the thrombin inhibitor hirulog (10 micrograms/ml), human recombinant phospholipase A2 (10-40 micrograms/ml) was able to inhibit platelet aggregation. The anti-aggregatory effect was removed by incubation of platelet rich plasma with a monoclonal anti-human recombinant phospholipase A2 antibody. Human recombinant phospholipase A2 (1-10 micrograms) inhibited 111In-labelled platelet accumulation within the thoracic region of rats and guinea pigs induced by i.v. administration of submaximal doses of collagen or adenosine diphosphate. Phospholipase A2 (1-20 micrograms/ml) from Naja mocambique mocambique snake venom had no direct effect on platelet aggregation in vitro. However, Naja phospholipase A2 administered i.v. to rats or guinea pigs was able to induce a dose related accumulation of 111In-labelled platelet within the thoracic region. The inhibitory effect of exogenously added human recombinant phospholipase A2 on platelet aggregation in vivo suggests a possible pathophysiological role for the extracellular form of phospholipase A2 but this property is not a feature of all phospholipase A2 preparations.

6-Ketoprostaglandin F1 alpha↗

PubMedDate not supplied

Role of cytosolic phospholipase A2 in cytokine-stimulated prostaglandin release by human gallbladder cells.

Eicosanoids are involved in gallbladder inflammation, epithelial water transport, and mucous secretion. Phospholipase Asubscript2 enzymes liberate arachidonic acid from membrane phospholipids for the synthesis of eicosanoids. The purpose of this study was to determine the effect of selective cytoplasmic and secretory phospholipase A2 inhibitors on basal and stimulated arachidonic acid and prostaglandin E2 release in gallbladder cells. Western immunoblotting was employed to evaluate both cytosolic and secretory phospholipase A2 enzymes in human gallbladder cells. Cells were incubated for 22 hours with (3)H-labeled arachidonic acid. Arachidonic acid and prostaglandin E2 release was then measured in the supernate after 2 hours of exposure to human interleukin-1beta, alone or after pretreatment for 1 hour with the inhibitors. Unstimulated gallbladder cells express both 85 kDa cytosolic and 14 kDa secretory phospholipase A2++. The 85 kDa phospholipase A2 was induced by interleukin-1beta, whereas there was no apparent change in secretory phospholipase A2 enzyme concentrations. Both the secretory phospholipase A2 inhibitor p-bromophenylacyl bromide and the cytosolic phospholipase A2 inhibitor arachidonyl trifluoromethyl ketone decreased basal and interleukin-1beta-stimulated arachidonic acid release. In contrast, only inhibition of cytosolic phospholipase A2 led to a decrease in interleukin-1beta-stimulated prostaglandin E2 release. Basal and interleukin-1beta-stimulated arachidonic acid release appears to be the result of the activity of both cytosolic and secretory phospholipase A2. Interleukin-1beta-stimulated prostaglandin E2 release appears to be dependent on the activity of cytosolic phospholipase A2.

Arachidonic Acid↗

PubMed1980

Venom from the snake Bothrops asper Garman. Purification and characterization of three phospholipases A2.

The water-soluble venom of Bothrops asper Garman (San Juan Evangelista, Veracruz, México) showed 15 polypeptide bands on polyacrylamide-gel electrophoresis. This material exhibited phospholipase, hyaluronidase, N-benzoyl-l-arginine ethyl hydrolase, N-benzoyl-l-tyrosine ethyl hydrolase and phosphodiesterase activity, but no alkaline phosphatase or acid phosphatase activity. Fractionation on Sephadex G-75 afforded seven protein fractions, which were apparently less toxic than the whole venom (LD(50)=4.3mug/g mouse wt.). Subsequent separation of the phospholipase-positive fraction (II) on DEAE-cellulose with potassium phosphate buffers (pH7.55) gave several fractions, two being phospholipase-positive (II.6 and II.8). These fractions were further purified on DEAE-cellulose columns with potassium phosphate buffers (pH8.6). Fraction II.8.4 was rechromatographed in the same DEAE-cellulose column, giving a pure protein designated phospholipase 1. The fraction II.6.3 was further separated by gel disc electrophoresis yielding two more pure proteins designated phospholipase 2 and phospholipase 3. Analysis of phospholipids hydrolysed by these enzymes have shown that all three phospholipases belong to type A(2). Amino acid analysis has shown that phospholipase A(2) (type 1) has 97 residues with a calculated mol.wt. of 10978+/-11. Phospholipase A(2) (type 2) has 96 residues with a mol.wt. of 10959+/-11. Phospholipase A(2) (type 3) has 266 residues with 16 half-cystine residues and a calculated mol.wt of 29042+/-31. Automated Edman degradation showed the N-terminal sequence to be: Asx-Leu-Trp-Glx-Phe-Gly-Glx-Met-Met-Ser-Asx-Val- Met-Arg-Lys-Asx-Val-Val-Phe-Lys-Tyr-Leu- for phospholipase A(2) (type 2).

Amino Acids↗

PubMed1991

Cyclic AMP mimics, but does not mediate, interleukin-1- and tumour-necrosis-factor-stimulated phospholipase A2 secretion from rat renal mesangial cells.

We have previously shown that recombinant interleukin 1 (IL-1) and recombinant tumour necrosis factor (TNF) synergistically stimulate phospholipase A2 release from mesangial cells. We now report that treatment of mesangial cells with the beta-agonist salbutamol, prostaglandin E2 (PGE2), cholera toxin or forskolin, which all activate adenylate cyclase, increased release of phospholipase A2 activity. Likewise, addition of a membrane-permeant cyclic AMP (cAMP) analogue or the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine enhanced release of phospholipase A2 activity from mesangial cells. There was a lag period of about 8 h before a significantly enhanced secretion could be detected. Furthermore, actinomycin D or cycloheximide completely suppressed cAMP-stimulated secretion of phospholipase A2. Angiotensin II, the phorbol ester phorbol 12-myristate 13-acetate, the Ca2+ ionophore A23187 and a membrane-permeant cGMP analogue did not stimulate phospholipase A2 release from the cells. Treatment with indomethacin completely inhibited IL-1 beta- and TNF-stimulated PGE2 synthesis, without having any effect on phospholipase A2 secretion, thus excluding cytokine-induced PGE2 synthesis as the mediator of phospholipase A2 release. Neither IL-1 beta nor TNF induced any increase in intracellular cAMP in mesangial cells. Furthermore, incubation of the cells with 2',5'-dideoxyadenosine, an inhibitor of adenylate cyclase, did not block cytokine-stimulated phospholipase A2 secretion. In addition, IL-1 beta and TNF synergistically interacted with forskolin to stimulate phospholipase A2 release from the cells. The protein kinase inhibitors H-8, staurosporine, K252a and amiloride inhibited IL-1 beta- and TNF-stimulated phospholipase A2 secretion. However, high concentrations that inhibit other protein kinases were needed. These observations suggest that IL-1 beta and TNF cause secretion of phospholipase A2 by a mechanism independent of cAMP. The signalling pathways used by IL-1 beta and TNF may involve a protein kinase that is probably different from protein kinase A or protein kinase C.

Animals↗

PubMed2001

Inhibition of 1,25-dihydroxyvitamin-D-induced keratinocyte differentiation by blocking the expression of phospholipase C-gamma1.

Keratinocytes produce vitamin D3 and convert it to the most active form, 1,25-dihydroxyvitamin D3, which regulates keratinocyte proliferation and differentiation. Phospholipase C-gamma1 is the most abundant member of the phospholipase C family in keratinocytes and is induced by 1,25-dihydroxyvitamin D3. Therefore, phospholipase C-gamma1 might be important in the signaling pathway mediating 1,25-dihydroxyvitamin-D3-induced keratinocyte differentiation. To test this hypothesis, phospholipase C-gamma1 expression in human keratinocytes was reduced by transfecting the cells with an antisense phospholipase C-gamma1 construct and then evaluating the response of the keratinocyte differentiation markers involucrin and transglutaminase to 1,25-dihydroxyvitamin D3. The results showed that involucrin and transglutaminase protein and mRNA levels were markedly reduced in keratinocytes transfected by the antisense phospholipase C-gamma1 construct. Cotransfection of keratinocytes with the involucrin or transglutaminase promoter construct and the antisense phospholipase C-gamma1 construct showed decreased involucrin or transglutaminase promoter activity in response to 1,25-dihydroxyvitamin D3. To further investigate the mechanism by which phospholipase C-gamma1 regulates keratinocyte differentiation, the calcium and inositol triphosphate levels in keratinocytes transfected by the antisense phospholipase C-gamma1 construct were measured following 1,25-dihydroxyvitamin D3 administration. The increase in keratinocyte intracellular free calcium and inositol triphosphate levels following 1,25-dihydroxyvitamin D3 administration were markedly reduced by the transfection of the antisense phospholipase C-gamma1 construct. These studies indicate that phospholipase C-gamma1 plays a critical role in the signal transduction pathway mediating 1,25-dihydroxyvitamin-D3-induced keratinocyte differentiation at least in part by mediating the increase in inositol triphosphate production and intracellular calcium mobilization following 1,25-dihydroxyvitamin D3 administration.

Calcium↗

PubMed2003

Neuronal lineage-specific induction of phospholipase Cepsilon expression in the developing mouse brain.

Phospholipase C is a key enzyme of intracellular signal transduction in the central nervous system. We and others recently discovered a novel class of phospholipase C, phospholipase Cepsilon, which is regulated by Ras and Rap small GTPases. As a first step toward analysis of its function, we have examined the spatial and temporal expression patterns of phospholipase Cepsilon during mouse development by in situ hybridization and immunohistochemistry. Around embryonic day 10.5, abundant expression of phospholipase Cepsilon is observed specifically in the outermost layer of the neural tube. On embryonic day 12 and later, it is observed mainly in the marginal zone of developing brain and spinal cord as well as in other regions undergoing neuronal differentiation, such as the retina and olfactory epithelium. The phospholipase Cepsilon-expressing cells almost invariably express microtubule-associated protein 2, but hardly express nestin or glial fibrillary acidic protein, indicating that the expression of phospholipase Cepsilon is induced specifically in cells committed to the neuronal lineage. The expression of phospholipase Cepsilon persists in the terminally differentiated neurons and exhibits no regional specificity. Further, an in vitro culture system of neuroepithelial stem cells is employed to show that abundant expression of phospholipase Cepsilon occurs in parallel with the loss of nestin expression as well as with the induction of microtubule-associated protein 2 expression and neuronal morphology. Also, glial fibrillary acidic protein-positive glial lineage cells do not exhibit the high phospholipase Cepsilon expression. These results suggest that the induction of phospholipase Cepsilon expression may be a specific event associated with the commitment of the neural precursor cells to the neuronal lineage.

Animals↗