Phospholipase D isozymes mediate epigallocatechin gallate-induced cyclooxygenase-2 expression in astrocyte cells

Little is known about the effect of epigallocatechin-3 gallate (EGCG), a major constituent of green tea, on the expression of cyclooxygenase (COX)-2. Here, we studied the role of phospholipase D (PLD) isozymes in EGCG-induced COX-2 expression. Stimulation of human astrocytoma cells (U87) with EGCG induced formation of phosphatidylbutanol, a specific product of PLD activity, and synthesis of COX-2 protein and its product, prostaglandin E(2) (PGE(2)). Pretreatment of cells with 1-butanol, but not 3-butanol, suppressed EGCG-induced COX-2 expression and PGE synthesis. Furthermore, evidence that PLD was involved in EGCG-induced COX-2 expression was provided by the observations that COX-2 expression was stimulated by overexpression of PLD1 or PLD2 isozymes and treatment with phosphatidic acid (PA), and that prevention of PA dephosphorylation by 1-propranolol significantly potentiated COX-2 expression induced by EGCG. EGCG induced activation of p38 mitogen-activated protein kinase (p38 MAPK), and specific inhibition of p38 MAPK dramatically abolished EGCG-induced PLD activation, COX-2 expression, and PGE(2) formation. Moreover, protein kinase C (PKC) inhibition suppressed EGCG-induced p38 MAPK activation, COX-2 expression, and PGE(2) accumulation. The same pathways as those obtained (2)in the astrocytoma cells were active in primary rat astrocytes, suggesting the relevance of the findings. Collectively, our results demonstrate for the first time that PLD isozymes mediate EGCG-induced COX-2 expression through PKC and p38 in immortalized astroglial line and normal astrocyte cells.     

Phospholipase A2

Phospholipase A2 (PLA2) catalyzes the hydrolysis of the sn-2 position of membrane glycerophospholipids to liberate arachidonic acid (AA), a precursor of eicosanoids including prostaglandins (PGs) and leukotrienes (LTs). The same reaction also produces lysophosholipids, which represent another class of lipid mediators. So far, at least 19 enzymes that possess PLA2 activity have been identified in mammals. The secretory PLA2 (sPLA2) family, in which 10 isozymes have been identified, consists of low-molecular-weight, Ca2+-requiring, secretory enzymes that have been implicated in a number of biological processes, such as modification of eicosanoid generation, inflammation, host defense, and atherosclerosis. The cytosolic PLA2 (cPLA2) family consists of 3 enzymes, among which cPLA2alpha plays an essential role in the initiation of AA metabolism. Intracellular activation of cPLA2alpha is tightly regulated by Ca2+ and phosphorylation. The Ca2+-independent PLA2 (iPLA2) family contains 2 enzymes and may play a major role in membrane phospholipid remodeling. The platelet-activating factor (PAF) acetylhydrolase (PAF-AH) family represents a unique group of PLA2 that contains 4 enzymes exhibiting unusual substrate specificity toward PAF and/or oxidized phospholipids. In this review, we will overview current understanding of the properties and functions of each enzyme belonging to the sPLA2, cPLA2, and iPLA2 families, which have been implicated in signal transduction.     

Phospholipase A2 in cnidaria

Phospholipase A2 (PLA2) is an enzyme present in snake and other venoms and body fluids. We measured PLA2 catalytic activity in tissue homogenates of 22 species representing the classes Anthozoa, Hydrozoa, Scyphozoa and Cubozoa of the phylum Cnidaria. High PLA2 levels were found in the hydrozoan fire coral Millepora sp. (median 735 U/g protein) and the stony coral Pocillopora damicornis (693 U/g) that cause skin irritation upon contact. High levels of PLA2 activity were also found in the acontia of the sea anemone Adamsia carciniopados (293 U/g). Acontia are long threads containing nematocysts and are used in defense and aggression by the animal. Tentacles of scyphozoan and cubozoan species had high PLA2 activity levels: those of the multitentacled box jellyfish Chironex fleckeri contained 184 U/g PLA2 activity. The functions of cnidarian PLA2 may include roles in the capture and digestion of prey and defense of the animal. The current observations support the idea that cnidarian PLA2 may participate in the sting site irritation and systemic envenomation syndrome resulting from contact with cnidarians.     

Phospholipase A2 in porifera

Phospholipase A2 (PLA2) catalytic activity was measured in aqueous extracts of 83 freeze-dried specimens representing 55 marine sponge species collected from the east coast of Australia including the Great Barrier Reef. High levels (>500 u/l) of PLA2 activity (defined as the amount of activity that releases 1 micromol of fatty acid per min) were found in four out of 55 species (7%), moderate activities (100-499 u/l) in 6/55 (11%), low activities (1-99 u/l) in 11/55 (20%) and no PLA2 activity in 34/55 (62%). Species with high PLA2 activity levels included Cymbastela coralliophila (2118 u/l, specific activity 10,590 u/g of protein), Acanthella cavernosa (1318 u/l, specific activity 2470 u/g), Spirastrella vagabunda (1036 u/l, specific activity 1727 u/g and Theonella swinhoei (567 u/l, specific activity 354 u/g). It was postulated that poriferan PLA2 may be involved in eicosanoid metabolism and antimicrobial and toxic defence of the animal.     

Phospholipase A2 in astrocytes

Astrocytes comprise the major cell type in the central nervous system (CNS) and they are essential for support of neuronal functions by providing nutrients and regulating cell-to-cell communication. Astrocytes also are immune-like cells that become reactive in response to neuronal injury. Phospholipases A₂ (PLA ₂) are a family of ubiquitous enzymes that degrade membrane phospholipids and produce lipid mediators for regulating cellular functions. Three major classes of PLA ₂ are expressed in astrocytes: group IV calcium-dependent cytosolic PLA ₂ (cPLA₂), group VI calcium-independent PLA ₂ (iPLA₂), and group II secretory PLA ₂ (sPLA₂). Upregulation of PLA ₂ in reactive astrocytes has been shown to occur in a number of neurodegenerative diseases, including stroke and Alzheimer’s disease. This review focuses on describing the effects of oxidative stress, inflammation, and activation of G protein-coupled receptors on PLA ₂ activation, arachidonic acid (AA) release, and production of prostanoids in astrocytes.     

Phospholipase A2 activity in T-lymphocytes

Phospholipase A2 activity was shown indirectly in T-lymphocytes from rat thymus and a permanent T-cell line by the liberation of arachidonic acid from phospholipids. In addition, phospholipase A2 activity was measured directly with two different substrates, phosphatidylcholine and labelled E. coli.     

Phospholipase A(2)

Considerable progress has been made in characterizing the individual participant enzymes and their relative contributions in the generation of eicosanoids, lipid mediators derived from arachidonic acid, such as prostaglandins and leukotrienes. However, the role of individual phospholipase (PL) A(2) enzymes in providing arachidonic acid to the downstream enzymes for eicosanoid generation in biologic processes has not been fully elucidated. In this review, we will provide an overview of the classification of the families of PLA(2) enzymes, their putative mechanisms of action, and their role(s) in eicosanoid generation and inflammation.     

磷脂酶A2的应用

磷脂酶Ａ2 (ｐｈｏｓｐｈｏｌｉｐａｓｅＡ2 ,ＰＬＡ2 ,ＥＣ 3 .1 .1 .4)即磷脂 2 酰基水解酶 ,是专一催化 3 Ｓｎ 磷酸甘油脂Ｃ 2位酯键的水解反应的酶 ,酶解产物为溶血磷脂和脂肪酸。ＰＬＡ2 不仅在生物体内具有很重要的生理功能 ,而且具有很高的应用价值 ,可广泛地应用在科学研究、磷脂改性、油脂精练、饲料添加剂、医疗等诸多方面。1 .用ＰＬＡ2 研究酶学、脂代谢和生物膜结构与功能ＰＬＡ2 (尤其是外分泌型的ＰＬＡ2 )的分子量较小 ,一般在 1 0～ 2 0ｋＤ之间 ,相对而言 ,结构较为简单。在蛇毒中 ,存在许多ＰＬＡ2 的同工酶 ,它们之…     

Action of Phospholipase A2 and Phospholipase C on Bacillus subtilis Protoplasts

Protoplasts prepared from Bacillus subtilis by lysozyme digestion lysed in the presence of pure pancreatic phospholipase A(2). The phospholipids cardiolipin, phosphatidylethanolamine, phosphatidylglycerol and lysylphosphatidylglycerol, which are present in the membrane, are degraded by phospholipase A(2) only after removal of the cell wall, giving free fatty acids and lyso derivatives. The four phospholipids are hydrolyzed equally well at a given enzyme concentration. Differences in the phospholipid composition of the protoplasts were obtained by variations in the growth medium, time of harvesting, and preincubation time with lysozyme. The extent of hydrolysis appeared to depend on the initial phospholipid composition. A relative increase in acidic phospholipids in the membrane facilitated the action of phospholipase A(2), whereas the rate of hydrolysis was diminished when protoplasts were tested which contained a relatively high amount of positively charged phospholipid. Pure phospholipase C from B. cereus preferentially hydrolyzed phosphatidyl-ethanolamine in the B. subtilis membrane. More than 80% of this phospholipid was converted into diglyceride, whereas only 30% of the cardiolipin was hydrolyzed. Such a loss of phospholipids, however, was not followed by lysis of the protoplasts. Liposomes were prepared from the lipid extracts of B. subtilis and incubated with both phospholipases. The hydrolysis pattern of the phospholipids in these model membrane systems was identical to the hydrolysis pattern of the phospholipids in the protoplast membrane. Phospholipase A(2) hydrolyzed all the phospholipids in the liposomes equally well, whereas phospholipase C preferentially degraded phosphatidylethanolamine.     

Phospholipase A2 and reacylation of phospholipids

Transacetylation of labeled CoA-oleate and oleate into liposomes from dipalmitoyl phosphatidylcholine catalyzed by phospholipase A2 from rat liver mitochondria was studied. It was shown that the efficiency of CoA-oleate incorporation slightly exceeded that of oleic acid both in the phosphatidylcholine and lysophosphatidylcholine fractions. It was found also that some amount of the labeled substrate remains bound to the enzyme; the type of oleate and CoA-oleate binding differs, depending on their concentrations. The autonomy of lipid component formation in mitochondrial membranes is discussed.     

Comparative hemostatic protein alterations accompanying pregnancy and parturition

Pregnancy and parturition represent adaptive, physiological stress situations that elicit both blood coagulation protein changes and endocrine alterations. This paper briefly reviews the interrelationships of these responses by comparing the available data in several mammalian species including man, dog, cow, and pig. Hemostasis is an enzymatically controlled cascade process involving platelets and specific coagulation proteins that results in the formation of insoluble strands of fibrin. Although qualitatively similar in most mammals, quantitative differences exist in the circulating levels of individual coagulation proteins which may contribute towards the different rates of clot formation. In the late gestation stage of human pregnancy, significant increases are observed in the circulating biological activities of the coagulation factors VII, VIII:C, IX, and X. In the dog, the activities of factors VII and IX reach peak values in midpregnancy, while in the cow little or no changes are observed except for factor VII values which show a significant increase only around the time of parturition. These coagulation changes appear to be associated with fluctuations in circulating hormone values, particularly plasma estrogen. In human pregnancy, the circulating levels of fibrinogen, the precursor of fibrin, increase during the gestation period reaching maximum values during and immediately following parturition. In the dog and the cow two distinct increases in fibrinogen values are observed, the first in the midgestation period and the second in the immediate postparturition period. In the pig, fibrinogen values also rise significantly at parturition. Oral contraceptive studies in the human female have indicated that changes in circulating activities of coagulation proteins are hormonally influenced.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phospholipase A2 and phospholipase B activities in fungi

As saprophytes or disease causing microorganisms, fungi acquire nutrients from dead organic material or living host organisms. Lipids as structural components of cell membranes and storage compartments play an important role as energy-rich food source. In recent years, it also has become clear that lipids have a wide range of bioactive properties including signal transduction and cell to cell communication. Thus, it is not surprising that fungi possess a broad range of hydrolytic enzymes that attack neutral lipids and phospholipids. Especially during infection of a mammalian host, phospholipase A(2) (PLA(2)) enzymes released by fungi could play important roles not only for nutrient acquisition and tissue invasion, but for intricate modulation of the host's immune response. Sequencing of fungal genomes has revealed a wide range of genes encoding PLA(2) activities in fungi. We are just beginning to become aware of the significance these enzymes could have for the fungal cells and their interaction with the host.     

Phospholipase A2 activity in rat and human lymphocytes

Partial characterization of phospholipase A from rat and human lymphocytes showed that it was much less active in man than in rat. The use of phosphatidylethanolamine labelled in the 2 position as substrate established that phospholipase A activity was 2 acyl-specific. It was maximal at pH 7.0 to 8.0, totally Ca2+ dependent and inhibited by detergents and Indomethacin.     

Phospholipase A2-like catalytic antibody

The phospholipase A2-like catalytic antibody 13C2-1F6 was elicited against the hapten 1 as the transition state analog for the hydrolysis of the C2 ester in the phospholipid. The Michaelis-Menten kinetics for the hydrolysis of the phospholipid 2 by 13C2-1F6 afforded a kcat of 1.0 x 10(-2) min(-1) and aKm of 71 microM. This antibody hydrolyzes the C2 ester in (R)-2, regio- and enantioselectively.     

Studies on Plasmalogen-Selective Phospholipase A2 in Brain

Plasmalogen-selective phospholipase A₂ (PlsEtn-PLA₂) has been purified from pig brain using multiple column chromatographic procedure. The purified enzyme migrates as a single band on polyacrylamide. It is stimulated by Triton X-100 and inhibited by sodium deoxycholate. Purified PlsEtn-PLA₂ is inhibited by iodoacetate, and this inhibition can be prevented by β-meracaptoethanol. Treatment of neuronal cell cultures with kainic acid stimulates PlsEtn-PLA₂ activity in a dose-dependent manner, and this stimulation can be blocked by Ly294486, a selective kainic acid receptor antagonist. Activities of PlsEtn-PLA₂ are markedly increased in plasma membrane and synaptosomal plasma membrane fraction prepared from nucleus basalis and hippocampal region of brains from Alzheimer disease patients compared to age-matched controls. It is proposed that accumulation of ceramide and increased expression of cytokines may be responsible for the stimulation of PlsEtn-PLA₂ in Alzheimer disease.     

Phospholipase A2 activity in rat platelets]

Rat blood platelets show phospholipase A2 activities twenty times higher than human platelets. The breakdown of PE at pH 7.2 is linear for 15 minutes. There is no degradation at pH less than 5; the maximal activity is in the pH range 5.5-7.5. The presence of Ca2+ increases the phospholipase activity; an excess is not inhibitory. The optimal activity is obtained with 16 microM substrate concentration. Substrate inhibition is observed when the concentration exceeds 25 microM.