Forms Of Phospholipase A2 Research Articles

Human Eosinophils Express, Relative to Other Circulating Leukocytes, Large Amounts of Secretory 14-kD Phospholipase A2

Human eosinophils perform several functions dependent on phospholipase A2 (PLA2) activity, most notably the synthesis of platelet-activating factor (PAF) and leukotriene C4 (LTC4). Several forms of PLA2 have been identified in mammalian cells. In the present study, the 14-kD, secretory form of PLA2 was detected in human eosinophils by immunocytochemical staining with the specific monoclonal antibody (MoAb) 4A1. In contrast, preparations of neutrophils, monocytes, lymphocytes, and basophils did not show detectable staining. With two MoAbs in a sandwich enzyme-linked immunosorbent assay (ELISA), large amounts of sPLA2 were detected in lysates of eosinophils, that were 20-fold to 100-fold higher than in the other circulating leukocytes (ie, neutrophils, basophils, monocytes, and lymphocytes). In addition, with a commercially available sPLA2 activity assay kit, we were able to show high activity of sPLA2 in human eosinophils relative to neutrophils. Investigations at the ultrastructural level showed that sPLA2 in eosinophils is mainly located in specific granules. Immunoelectron microscopy also visualized sPLA2 within phagosomes after addition of opsonized particles to the eosinophils. However, sPLA2 was not detected in the cell-free supernatants of activated eosinophils, in contrast to eosinophil-cationic protein (ECP), which colocalizes with sPLA2 in resting eosinophils. These findings warrant further studies into the role of sPLA2 in eosinophil function.

Exogenous phospholipase A2 affects inflammatory gene expression in primary bovine mammary epithelial cells.

This Research Communication addresses the hypothesis that exogenously administered phospholipase A2 (PLA2) affects the inflammatory responses of bovine mammary epithelial cells (bMEC) in vitro with the aim of providing preliminary justification of investigation into the uses of exogenously administered PLA2 to manage or treat bovine mastitis. Primary bMEC lines from 11 lactating Holstein dairy cows were established and the expression of 14 pro-inflammatory genes compared under unchallenged and lipopolysaccharide (LPS)-challenged conditions, with and without concurrent treatment with bovine pancreatic PLA2G1B, a secreted form of PLA2. No differences in the expression of these genes were noted between PLA2-treated and untreated bMEC under unchallenged conditions. Following LPS challenge, untreated bMEC exhibited significant downregulation of CXCL8, IL1B, CCL20, and CXCL1. In contrast, PLA2-treated bMEC exhibited significant downregulation of IL1B and CCL20 only. These findings indicate that exogenous PLA2 affects the expression of some pro-inflammatory factors in immune-stimulated bMEC, but does not influence the constitutive expression of these factors. Further investigation of the influence of exogenous PLA2 in the bovine mammary gland is justified.

Transglutaminase 2 and phospholipase A2 interactions in the inflammatory response in human Thp-1 monocytes

Several experimental approaches have demonstrated that transglutaminase 2 (TG2) increased activity is involved in monocyte activation and inflammatory response. Preliminary results also demonstrate a TG-mediated post-translational modification of phospholipase A2 (PLA2), which catalyzes the release of arachidonic acid from its lipid storage sites. The control of PLA2-mediated production of eicosanoids has been found to be of great benefit for inflammatory disease treatment. However, the identification of the mechanisms of PLA2 activation is a very complex issue, because of the presence of multiple PLA2 forms. The aim of this study was to characterize the interactions between TG2 and sPLA2 in LPS-stimulated THP-1 cells, which were treated with TPA to induce early differentiated macrophage-type model. We demonstrated that increases in TG2 enzyme activity and protein expression may be considered an early event in monocyte/macrophage activation by LPS. Under these conditions, TG2 protein was co-immunoprecipitated with PLA2 by monoclonal antibody directed against the secretory form of the enzyme (sPLA2-V). Concomitantly, the PLA2 enzyme activity increased in TPA-treated cells exposed to LPS; these high levels of enzyme activity were significant reduced by R283, a site-specific inhibitor of TG2. Moreover, confocal laser scanning microscopy analysis of double-immunostained cytochemical specimens confirmed a co-localization of BAPA-labeled proteins and sPLA2-V in LPS-treated cells. These findings give evidence of a complex TG2/sPLA2-V, suggesting the possibility that sPLA2-V is a substrate for TG2. These results demonstrated that TG2 increases produced a sustained activation of PLA2 activity, suggesting a functional interaction between these enzymes in the regulation of inflammatory response.

Assessing Phospholipase A2 Activity toward Cardiolipin by Mass Spectrometry

Cardiolipin, a major component of mitochondria, is critical for mitochondrial functioning including the regulation of cytochrome c release during apoptosis and proper electron transport. Mitochondrial cardiolipin with its unique bulky amphipathic structure is a potential substrate for phospholipase A2 (PLA2) in vivo. We have developed mass spectrometric methodology for analyzing PLA2 activity toward various cardiolipin forms and demonstrate that cardiolipin is a substrate for sPLA2, cPLA2 and iPLA2, but not for Lp-PLA2. Our results also show that none of these PLA2s have significant PLA1 activities toward dilyso-cardiolipin. To understand the mechanism of cardiolipin hydrolysis by PLA2, we also quantified the release of monolyso-cardiolipin and dilyso-cardiolipin in the PLA2 assays. The sPLA2s caused an accumulation of dilyso-cardiolipin, in contrast to iPLA2 which caused an accumulation of monolyso-cardiolipin. Moreover, cardiolipin inhibits iPLA2 and cPLA2, and activates sPLA2 at low mol fractions in mixed micelles of Triton X-100 with the substrate 1-palmitoyl-2-arachidonyl-sn-phosphtidylcholine. Thus, cardiolipin functions as both a substrate and a regulator of PLA2 activity and the ability to assay the various forms of PLA2 is important in understanding its function.

Two novel approaches targeting cancer cell membrane for tumor therapy

Disruption of normal cell function by chemicals, UV radiation or viruses can cause various cancer. Drugs that have been developed for cancer therapy bind to various targets to correct disorder cell behavior, repair damaged DNA or promote cell apoptosis. However, there is rare study that focuses on cancer cell membrane as target. We propose two approaches for achieving our goal. One is to use phospholipase A2 (PLA2) to cleave phospholipid heads of the bilayer of cancer cells. Because PLA2 has unique Ca2+ catalytic site and the pH of healthy tissue cells should be slightly alkaline at 7.2–7.5, it can be easily protected by CO32− in the form of PLA2–CaCO3. While PLA2–CaCO3 accumulate in cancer cells in the acidic microenvironment of which the pH is below 7, it could be converted to active state (PLA2-Ca2+) which can intensively damage the cancer cell membrane. The other one is to use both monoclonal antibodies and dimethylsulfoxide (DMSO). The internalization of targeted cancer cell antibodies could change the curvature of cell membrane from order state to disorder state, therefore strong detergent DMSO can destroy cancer cells at extreme low concentration. These two approaches present no harm for normal cells, therefore, drugs targeted cancer cell membrane might become a new and high effective clinical cancer therapy.

An albumin-associated PLA2-like activity inactivates surfactant phosphatidylcholine secreted from fetal type II pneumocytes

Type II pneumocytes are responsible for the synthesis and secretion of pulmonary surfactant, which reduces surface tension in lung alveoli, thus decreasing their tendency to collapse during expiration. For this effect to be sustained, the integrity of the surface-active components of surfactant must be maintained. This study has shown that, when cultured type II pneumocytes are exposed to lipoprotein-free serum (LFS), the level of lyso-phosphatidylcholine (lyso-PC) in the secreted surfactant phospholipids is markedly elevated with a concomitant decline in the level of phosphatidylcholine (PC). This effect is the result of hydrolysis of surfactant PC by a phospholipase A(2) (PLA(2))-like activity present within serum. Anion-exchange chromatography, gel filtration chromatography and preparative electrophoresis of human LFS have shown that this PLA(2)-like activity coelutes with albumin and is biochemically distinct from the secretory form of PLA(2). Furthermore, specific inhibitors of PLA(2) such as p-bromophenacyl bromide, aristolochic acid, and palmitoyl trifluoromethyl ketone do not inhibit this activity of serum. Commercially purified human serum albumin fraction V and recombinant human serum albumin (rHSA) are almost as effective as LFS in enhancing the level of lyso-PC in the media. The latter finding implies that rHSA directly generates lyso-PC from secreted PC and suggests that this PLA(2)-like activity may be an intrinsic attribute of albumin.

Involvement of phospholipase A2 in the response of Solanum species to an elicitor from Phytophthora infestans

Changes in activity of phospholipase A2 (PLA2), a key enzyme in lipid metabolism and signal network in defence mechanisms, were investigated in Solanum species and Phytophthora infestans interaction. We have compared PLA2 activity in response to an elicitor, a culture filtrate (CF) derived from P. infestans, in non-host resistant Solanum nigrum var. gigantea, field resistant S. tuberosum cv Bzura and susceptible S. tuberosum clone H-8105. To elucidate the contribution of specific forms of PLA2 to plant defence mechanism reasonably selective PLA2 inhibitors, haloenol lactone suicide substrate (HELSS) and p-bromophenacyl bromide (BPB), which discriminate between Ca+2-independent PLA2 (iPLA2) and Ca+2-dependent secretory PLA2 (sPLA2), were used. The in vivo and in vitro effects of the inhibitors on PLA2 activity and on generation of reactive oxygen species (ROS) induced by CF in the studied plants were assayed. We found that PLA2 activity increased in response to CF treatment, displaying various kinetics and intensity depending on the resistance status of a given genotype. Differences among the genotypes in the effects of each inhibitor on CF-induced PLA2 activity and on ROS production may reflect the diversity of PLA2 isoforms in plants. Contrary to BPB, the inhibitory effect of HELSS was observable mainly on CF-induced PLA2 activity, which suggests that iPLA2 participates in signal transduction in defence reactions. Various effects of the two inhibitors on PLA2 activity and ROS production suggest different contribution of sPLA2 and iPLA2 to modulation of defence reactions in the interaction between Solanum genotypes and P. infestans.

Modulation of the activity of cytosolic phospholipase A2α (cPLA2α) by cellular sphingolipids and inhibition of cPLA2α by sphingomyelin

We examined the effect of the cellular sphingolipid level on the release of arachidonic acid (AA) and activity of cytosolic phospholipase A2alpha (cPLA2alpha) using two Chinese hamster ovary (CHO)-K1-derived mutants deficient in sphingolipid synthesis: LY-B cells defective in the LCB1 subunit of serine palmitoyltransferase for de novo synthesis of sphingolipid species, and LY-A cells defective in the ceramide transfer protein CERT for SM synthesis. When LY-B and LY-A cells were cultured in Nutridoma medium and the sphingolipid level was reduced, the release of AA stimulated by the Ca(2+) ionophore A23187 increased 2-fold and 1.7-fold, respectively, compared with that from control cells. The enhancement in LY-B cells was decreased by adding sphingosine and treatment with the cPLA2alpha inhibitor. When CHO cells were treated with an acid sphingomyelinase inhibitor to increase the cellular SM level, the release of AA induced by A23187 or PAF was decreased. In vitro studies were then conducted to test whether SM interacts directly with cPLA2alpha. Phosphatidylcholine vesicles containing SM reduced cPLA2alpha activity. Furthermore, SM disturbed the binding of cPLA2alpha to glycerophospholipids. These results suggest that SM at the biomembrane plays important roles in regulating the cPLA2alpha-dependent release of AA by inhibiting the binding of cPLA2alpha to glycerophospholipids.

Phospholipases A2 and platelet-activating-factor acetylhydrolase in patients with acute respiratory distress syndrome*

Phospholipases A2 (PLA2) comprise a family of enzymes probably implicated in the development of acute respiratory distress syndrome (ARDS). The aim was to investigate PLA2 activities and characteristics in bronchoalveolar lavage (BAL) fluid, BAL cells, and plasma from patients with ARDS by a fluorometric method. Prospective, controlled study. Fourteen-bed polyvalent intensive care unit in a university hospital. A total of 31 mechanically ventilated patients, 20 with and 11 without ARDS, were studied. BAL was performed by fiberoptic bronchoscopy in mechanically ventilated patients with a controlled mechanical ventilation mode. PLA2 and platelet-activating-factor acetylhydrolase were determined in BAL fluid, cells, and plasma. For the classification of PLA2-specific inhibitors, Western blot analysis and their biochemical characteristics were used. In ARDS patients, increased PLA2 levels were detected in BAL fluid, BAL cells, and plasma compared with the control patients. PLA2 in BAL fluid was mainly type IIA secretory and cytosolic types. In plasma, type IIA secretory and cytosolic and a Ca-independent PLA2 were found. In BAL cells, a cytosolic form, probably a Ca-independent intracellular form, and a low activity of type IIA secretory PLA2 was also observed. Total PLA2 activity correlated inversely with Pao2/Fio2 ratio and positively with the mortality rate. Patients with direct ARDS exhibited higher PLA2 activity compared with patients with indirect ARDS. Platelet-activating-factor acetylhydrolase activity was higher in BAL fluid and plasma, but it was lower in BAL cells. Ca-dependent, secretory, cytosolic, and Ca-independent forms of PLA2 and platelet-activating-factor acetylhydrolase could play important roles in the development or down-regulation of inflammation in ARDS, respectively.

Structure, function and interfacial allosterism in phospholipase A2: insight from the anion-assisted dimer

Enzymes that function on membrane surfaces offer many challenges to understanding structural and functional details due to the difficulties of obtaining relevant information of the protein in a physiological environment. Focusing on this aspect of structural biology, it is important to develop conditions that mimic the interaction of membrane proteins with their binding surface and ultimately the mechanisms of action. This approach has been used to characterize the allosteric nature of secreted phospholipase A2 (PLA2) to its substrate interface. The breakthrough here was to crystallize the pancreatic group-IB PLA2 in an anion-assisted dimer with five coplanar phosphate anions bound. In the anion-assisted dimer structure one molecule of a tetrahedral mimic inhibitor and five anions are shared between the two subunits of the dimer. The sn-2-phosphate of the inhibitor, which mimics the tetrahedral intermediate of the esterolysis reaction, is bound in the active site of one subunit, and the alkyl chain extends into the active site slot of the second subunit across the subunit–subunit interface. This interface-bound structural mimic provided insight into the active site environment and specific anionic interactions to the i-face of the protein. The presence or absence of a single critical active site water, corresponds to the difference between the activated or inactivated form of the enzyme. The anion-assisted dimer structure supports a calcium coordinated nucleophilic water mechanism, with its p K a modulated by this assisting water. This working model has been further strengthened with an enzyme–product complex structure solved with the hydrolysis products of the substrate PAF also bound to the anion-assisted dimer form of PLA2. Additional confirmation of the assisting-water mechanism comes from a structure of the inactive zymogen proPLA2 also crystallized in an anion-assisted dimer. Remarkably, the assisting water present in the activated complex is absent in this proPLA2 structure.

Phospholipase A2 plays an important role in myelin breakdown and phagocytosis during wallerian degeneration

Phospholipase A2 (PLA2) hydrolyzes phosphatidylcholine to lysophosphatidylcholine and arachidonic acid. The former can induce myelin breakdown and the latter, via eicosanoids, can stimulate inflammatory responses. Immunohistochemical analysis of secreted (sPLA2) and cytosolic (cPLA2) forms of the enzyme was assessed in the injured adult rat sciatic and optic nerves. sPLA2 and cPLA2 are expressed in the first 2 weeks in the injured sciatic nerve, which correlates with rapid Wallerian degeneration in peripheral nerves. In contrast, both forms of PLA2 were not expressed in the optic nerve for the first 3 weeks after crush injury, which correlates with slow Wallerian degeneration in the central nervous system (CNS). In addition, PLA2 is not expressed in the lesioned sciatic nerve of C57BL/Wlds mutant mice in which Wallerian degeneration is severely retarded. Blocking cPLA2 in the transected sciatic nerve of C57BL/6 mice, which have a naturally occurring null mutation for the major from of sPLA2, resulted in a marked slowing of myelin and axonal degradation and phagocytosis in the distal nerve segment. These results provide direct evidence of an important role for cPLA2 in Wallerian degeneration.

Five coplanar anion binding sites on one face of phospholipase A2: relationship to interface binding.

We report the structures of the crystallographic dimer of porcine pancreatic IB phospholipase A(2) (PLA2) with either five sulfate or phosphate anions bound. In each structure, one molecule of a tetrahedral mimic MJ33 [1-hexadecyl-3-(trifluoroethyl)-sn-glycero-2-phosphomethanol] and the five anions are shared between the two subunits of the dimer. The sn-2-phosphate of MJ33 is bound in the active site of one subunit (A), and the alkyl chain extends into the active site slot of the second subunit (B) across the subunit-subunit interface. The two subunits are packed together with a large hydrophobic and desolvated surface buried between them along with the five anions that define a plane. The anions bind by direct contact with two cationic residues (R6 and K10) per subunit and through closer-range H-bonding interactions with other polarizable ligands. These features of the "dimer" suggest that the binding of PLA2 to the anionic groups at the anionic interface may be dominated by coordination through H-bonding with only a partial charge compensation needed. Remarkably, the plane defined by the contact surface is similar to the i-face of the enzyme [Ramirez, F., and Jain, M. K. (1991) Proteins: Struct., Funct., Genet. 9, 229-239], which has been proposed to make contact with the substrate interface for the interfacial catalytic turnover. Additionally, these structures not only offer a view of the active PLA2 complexed to an anionic interface but also provide insight into the environment of the tetrahedral intermediate in the rate-limiting chemical step of the turnover cycle. Taken together, our results offer an atomic-resolution structural view of the i-face interactions of the active form of PLA2 associated to an anionic interface.

Group IIA phospholipase A2 mediates lung injury in intestinal ischemia-reperfusion.

To assess the mechanistic role of group IIA phospholipase A2 (PLA2) in the process of local and distant organ injury after intestinal ischemia-reperfusion. Intestinal ischemia-reperfusion produces lung injury by a mechanism that involves PLA2 activation, but it is unclear which isozyme is responsible for this phenomenon. Group IIA PLA2, one of the secreted forms of PLA2, is known to play a pivotal role in a variety of inflammatory reactions. Rats underwent 45 minutes of superior mesenteric artery occlusion in the presence and absence of pretreatment with group IIA PLA2 inhibitor, S-5920/LY315920Na (20 mg/kg, subcutaneously, 30 minutes before clamping). At 2 hours of reperfusion, intestinal and lung leak was assessed by 125I-albumin tissue/blood ratio, and liver injury was estimated by serum alanine aminotransferase. PLA2 activities in tissues and sera were quantitated by phosphatidyl-glycerol/sodium cholate mixed micelle assay. PLA2 activities in tissues were also measured after in vitro preincubation with EDTA, S-5920/LY315920Na, or antirat group IIA PLA2 antibody. Intestinal ischemia-reperfusion provoked intestinal leak, liver injury, and lung leak, whereas tissue PLA2 activity was decreased in the intestine, unchanged in the liver, and increased in the lung. Serum PLA2 activities were increased in the portal and systemic circulation during ischemia. Pretreatment with S-5920/LY315920Na eliminated PLA2 activities in all tissues and sera and only abolished lung leak. The in vitro experiment revealed that most of the intestinal and lung PLA2 activities were inhibited by EDTA, S-5920/LY315920Na, and antirat group IIA PLA2 antibody, but hepatic PLA2 activity was not. Intestinal ischemia-reperfusion appears to produce lung injury by a mechanism that involves group IIA PLA2 activation. Intestinal ischemia-reperfusion is likely to promote intestinal and hepatic injury independent of group IIA PLA2.

Control of the chemical step by leucine-31 of pancreatic phospholipase A2.

A well-defined region of pancreatic and other secreted phospholipase A2 (PLA2), which we call the i-face, makes a molecular contact with the interface to facilitate and control the events and processivity of the interfacial catalytic turnover cycles. The structural features of the i-face and its allosteric relationship to the active site remain to be identified. As a part of the calcium binding (26-34) loop, Leu-31 is located on the surface near the substrate binding slot of PLA2. Analysis of the primary rate and equilibrium parameters of the Leu-31 substitution mutants of the pig pancreatic PLA2 shows that the only significant effect of the substitution is to impair the chemical step at the zwitterionic interface in the presence of added NaCl, and only a modest effect is seen on kcat at the anionic interface. Leu-31 substitutions have little effect on the binding of the enzyme to the interface; the affinity for certain substrate mimics is modestly influenced in W3F, L31W double mutant. The fluorescence emission results with the double mutant show that the microenvironment of Trp-31 is qualitatively different at the zwitterionic versus anionic interfaces. At both of the interfaces Trp-31 is not shielded from the bulk aqueous environment as it remains readily accessible to acrylamide and water. The NaCl-induced change in the Trp-31 emission spectrum of the double mutant on the zwitterionic interface is similar to that seen on the binding to the anionic interface. Together, the kinetic and spectroscopic results show that the form of PLA2 at the zwitterionic interface (Ez) is distinguishably different from the catalytically more efficient form at the anionic interface (Ea). This finding provides a structural basis for the two-state model for kcat activation by the anionic interface. In conjunction with earlier results we suggest that neutralization of certain cationic residues of PLA2 exerts a control on the calcium loop through residue 31.

Role of various phospholipases A2 and inhibitors in the pathogenesis and prevention of pancreatic acinar cell necrosis: studies with isolated rat pancreatic acini.

Phospholipase A2 (PLA2) may play a central role in the pathogenesis of pancreatic acinar cell necrosis. Several questions, however, are unsolved: Is acinar cell necrosis caused by PLA2 derived from infiltrating leukocytes or from pancreatic PLA2 itself? Does PLA2 cause cellular lysis by the release of lysolecithin from lecithin or by generation of free radicals? The aims of this study were to determine which form of PLA2 is responsible for cellular damage and how to inhibit its action. Isolated rat pancreatic acini were prepared by collagenase digestion. Newly synthesized proteins were labeled by 35S-methionine. Acini were incubated in buffer to which various factors, such as porcine pancreatic PLA2 or bee venom PLA2, homogenates of either leukocytes or pancreatic homogenates, all with or without lecithin and with or without potential inhibitors (aprotinin, 4-bromophenacylbromide, BM 16.2115, quinacrine, various analogs of arachidonic acid), or free radicals (hydrogen peroxide, xanthine/ xanthine oxidase) with or without allo-purinol or dismutase/catalase were added. Cellular destruction was measured by the release of radiolabeled proteins. PLA2 alone, free radicals, and granulocytes were not harmful to acini within 30 min of incubation. Free radicals caused significant release of radiolabeled proteins only after 3 h of incubation; this release could be inhibited by scavengers. Incubation of pancreatic acini with PLA2 in combination with lecithin caused rapid release of radiolabeled proteins. Addition of high concentrations of enterokinase activated pancreatic homogenates both alone and with lecithin caused release of cellular proteins, suggesting that pancreatic PLA2 uses lecithin from pancreatic membranes as substrate. Almost all tested potential inhibitors of PLA2 were unable to prevent the destruction caused by either pancreatic or bee venom PLA2 and lecithin. However, HK 42, a polyunsaturated fatty acid analog, was able to reduce dose dependently the release of acinar proteins caused by pancreatic PLA2 and lecithin. Pancreatic PLA2 and not PLA2 from infiltrating leukocytes may play a role in pancreatic acinar cell necrosis. Cellular lysis is caused upon the action of lysolecithin and probably not via the action of free radicals.

Functional coupling and differential regulation of the phospholipase A2-cyclooxygenase pathways in inflammation.

Prostaglandins generated by the phospholipase A2 (PLA2)/cyclooxygenase (COX) pathway are well known to mediate diverse intracellular and extracellular effects that regulate mammalian development, vascular function, renal physiology, parturition, and immune responses to infection or wounding. In immune-mediated diseases and in certain cancers, this pathway is aberrantly up-regulated and excessive prostaglandin production contributes to the pathology. It is now known that there are two isoforms of COX and multiple secreted and intracellular PLA2 enzymes. The use of isoform-specific inhibitors, coupled with antisense and in vivo gene deletion experiments, has identified independent pathways of arachidonic acid metabolism, which are differentially regulated at the levels of gene expression, protein phosphorylation, and cellular localization. There is cross-talk between the pathways at the level of PLA2 and substrate supply to the two isoforms of COX is apparently compartmentalized. Knockout studies have shown that the two COX isoforms play independent roles in immediate and delayed agonist-induced prostaglandin synthesis. Cytosolic PLA2-alpha is essential for both responses. Inducible secreted forms of PLA2 are, as yet, not essential for either response with the exception of the in vitro murine mast cell immediate response and instances of murine macrophage prostaglandin synthesis. These enzymes amplify the delayed response and are likely to modulate the severity of immune-mediated diseases.

The therapeutic potential for phospholipase A2 inhibitors

Modulation of pro-inflammatory lipid mediator production by inhibition of phospholipase A2 (PLA2) activity remains a potential target for development of new drugs for the treatment of rheumatoid arthritis or other inflammatory diseases. Evidence now exists that more than one isoform of PLA2, including types IIa and V 14 and 85 kDa PLA2, is required for production of lipid mediators. Specific isoforms may be associated with production of either the prostaglandin or leukotriene class of lipid mediators in different cell types such as monocytes and neutrophils. Characterisation of isoform-selective inhibitors in models of inflammatory disease, such as rat adjuvant arthritis or phorbol myristate acetate (PMA)-induced mouse ear oedema, suggests that both 14 kDa and 85 kDa PLA2 may contribute to the development of the disease state and that both forms of PLA2 may be targets for modulation of inflammatory disease. Much work remains to clarify fully the relative roles of different PLA2 in the aetiology of inflamm...

Identification and characterization of several forms of phospholipase A2 in mouse epidermal keratinocytes

The tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) stimulated the release of arachidonic acid (AA) from mouse keratinocytes. A distinct difference was observed between the fatty acid release profile elicited by TPA and other stimuli. These findings led to the investigation of keratinocyte phospholipase A2 (PLA2), which catalyzes the release of sn-2 fatty acids from membrane phospholipids and regulates the production of eicosanoids. We characterized and identified several forms of PLA2 in mouse keratinocytes, a cytosolic or cPLA2 and two secretory or sPLA2s in the membrane. The PLA2 in keratinocyte cytosol is sensitive to heating and acid treatment, while resistant to reducing reagent. The PLA2 in keratinocyte membrane is resistant to heating and acid treatment, while sensitive to reducing reagent. These characteristics suggested the presence of a cPLA2 and at least one type of sPLA2. Inhibitor data further confirmed the identities of these PLA2s. The cPLA2 was activated by TPA, and appeared to be responsible for the majority of the specific release of AA observed in mouse keratinocytes treated with TPA. The calcium ionophore A23187, and 4α-TPA did not elicit the selectivity towards AA observed with TPA. The release of linoleic acid (LA) and oleic acid (OA) from A23187- and 4α-TPA-treated keratinocytes suggests activation of sPLA2. These activities may be due to the existence of both type I and type II sPLA2, as both were identified by polymerase chain reactions. In conclusion, keratinocytes express several forms of phospholipase A2 that differ in their substrate specificities and mechanisms of activation, resulting in distinct agonist-specific fatty acid release profiles.—LiStiles, B., H-H. Lo, and S. M. Fischer. Identification and characterization of several forms of phospholipase A2 in mouse epidermal keratinocytes.

Reduced fertility and postischaemic brain injury in mice deficient in cytosolic phospholipase A2.

Phospholipase A2 (PLA2) enzymes are critical regulators of prostaglandin and leukotriene synthesis and can directly modify the composition of cellular membranes. PLA2 enzymes release fatty acids and lysophospholipids, including the precursor of platelet-activating factor, PAF, from phospholipids. Free fatty acids, eicosanoids, lysophospholipids and PAF are potent regulators of inflammation, reproduction and neurotoxicity. The physiological roles of the various forms of PLA2 are not well defined. The cytosolic form, cPLA2, preferentially releases arachidonic acid from phospholipids and is regulated by changes in intracellular calcium concentration. We have now created 'knockout' (cPLA2-/-) mice that lack this enzyme, in order to evaluate its physiological importance. We find that cPLA2-/- mice develop normally, but that the females produce only small litters in which the pups are usually dead. Stimulated peritoneal macrophages from cPLA2-/- animals did not produce prostaglandin E2 or leukotriene B4 or C4. After transient middle cerebral artery occlusion, cPLA2-/- mice had smaller infarcts and developed less brain oedema and fewer neurological deficits. Thus cPLA2 is important for macrophage production of inflammatory mediators, fertility, and in the pathophysiology of neuronal death after transient focal cerebral ischaemia.

Properties and regulation of cytosolic phospholipase A2.

Properties and regulation of cytosolic phospholipase A2.