Phospholipase A2 and its Molecular Mechanism after Spinal Cord Injury

Phospholipases A₂ (PLA₂s) are a diverse family of lipolytic enzymes which hydrolyze the acyl bond at the sn-2 position of glycerophospholipids to produce free fatty acids and lysophospholipids. These products are precursors of bioactive eicosanoids and platelet-activating factor which have been implicated in pathological states of numerous acute and chronic neurological disorders. To date, more than 27 isoforms of PLA₂ have been found in the mammalian system which can be classified into four major categories: secretory PLA₂, cytosolic PLA₂, Ca²⁺-independent PLA₂, and platelet-activating factor acetylhydrolases. Multiple isoforms of PLA₂ are found in the mammalian spinal cord. Under physiological conditions, PLA₂s are involved in diverse cellular responses, including phospholipid digestion and metabolism, host defense, and signal transduction. However, under pathological situations, increased PLA₂ activity, excessive production of free fatty acids and their metabolites may lead to the loss of membrane integrity, inflammation, oxidative stress, and subsequent neuronal injury. There is emerging evidence that PLA₂ plays a key role in the secondary injury process after traumatic spinal cord injury. This review outlines the current knowledge of the PLA₂ in the spinal cord with an emphasis being placed on the possible roles of PLA₂ in mediating the secondary SCI.     

Phospholipase A2 and Its Role in Brain Tissue

Abstract: Phospholipase A₂ (PLA₂) is the name for the class of lipolytic enzymes that hydrolyze the acyl group from the sn-2 position of glycerophospholipids, generating free fatty acids and lysophospholipids. The products of the PLA₂-catalyzed reaction can potentially act as second messengers themselves, or be further metabolized to eicosanoids, platelet-activating factor, and lysophosphatidic acid. All of these are recognized as bioactive lipids that can potentially alter many ongoing cellular processes. The presence of PLA₂ in the central nervous system, accompanied by the relatively large quantity of potential substrate, poses an interesting dilemma as to the role PLA₂ has during both physiologic and pathologic states. Several different PLA₂ enzymes exist in brain, some of which have been partially characterized. They are classified into two subtypes, CA²⁺-dependent and Ca²⁺-independent, based on their catalytic dependence on Ca²⁺. Under physiologic conditions, PLA₂ may be involved in phospholipid turnover, membrane remodeling, exocytosis, detoxification of phospholipid peroxides, and neurotransmitter release. However, under pathological situations, increased PLA₂ activity may result in the loss of essential membrane glycerophospholipids, resulting in altered membrane permeability, ion homeostasis, increased free fatty acid release, and the accumulation of lipid peroxides. These processes, along with loss of ATP, may be responsible for the loss of membrane phospholipid and subsequent neuronal injury found in ischemia, spinal cord injury, and other neurodegenerative diseases. This review outlines the current knowledge of the PLA₂ found in the central nervous system and attempts to define the role of PLA₂ during both physiologic and pathologic conditions.     

Emerging roles for phospholipase A2 enzymes in cancer

Phospholipase A₂ (PLA₂) enzymes (EC3.1.4.4) regulate the release of biologically active fatty acids and lysophospholipids from membrane phospholipid pools. These lipids are also substrates for intracellular biochemical pathways that generate potent autocrine and paracrine lipid mediators such as the eicosanoids and platelet activating factor. These factors, in turn, regulate cell proliferation, survival, differentiation, motility, tissue vascularisation, and immune surveillance in virtually all tissues, functions that are subverted by cancer cells for tumour growth and metastasis. Thus the relevance of PLA₂-dependent pathways to the genesis and progression of cancer has been of interest since their discovery and with recent technological advances, their role in tumourigenesis has become more tractable experimentally. Limited human genetic studies have not yet identified PLA₂ enzymes as classical mutated oncogenes or tumour suppressor genes. However, there is strong evidence that of the 22 identified human PLA₂ enzymes, ten of which have been studied in cancer to date, most are aberrantly expressed in a proportion of tumours derived from diverse organs. Correlative and functional studies implicate the expression of some secreted enzymes (sPLA₂s), particularly the best studied enzyme Group IIA sPLA₂ in either tumour promotion or inhibition, depending on the organ involved and the biochemical microenvironment of tumours. As in immune-mediated inflammatory pathologies, genetic deletion studies in mice, supported by limited studies with human cells and tissues, have identified an important role for Group IVA PLA₂ in regulating certain cancers. Pharmacological intervention studies in prostate cancer suggest that hGIIA-dependent tumour growth is dependent on indirect regulation of Group IVA PLA₂. Group VI calcium-independent PLA₂ enzymes have also been recently implicated in tumourigenesis with in vitro studies suggesting multiple possible roles for these enzymes. Though apparently complex, further characterization of the regulatory relationships amongst PLA₂ enzymes, lipid mediator biosynthetic enzymes and the lipid mediators they produce during tumour progression is required to define the biochemical context in which the enzymes modulate cancer growth and development.     

Trace amounts of enhancing factor/phospholipase A2 in mouse peritoneal exudate cells

Enhancing factor (EF), a mouse phospholipase A₂ (PLA₂), has been purified from the small intestines, based on its ability to increase the binding of epidermal growth factor in a radioreceptor assay. EF/PLA₂ was found to be localized predominantly in the Paneth cells in the small intestines. Whether mouse intestinal EF/PLA₂ is identical/similar to mouse secretory PLA₂ was to be determined. Phospholipases are known to play a crucial role in the process of inflammation. This paper reports the presence of trace amounts of EF/PLA₂ in the peritoneal exudate cells. Western blot analysis of the acid extracts showed the presence of a 14 kDa immunologically cross-reactive protein. RT-PCR analysis using EF specific primers amplified a ∼700 bp product which was further confirmed to be EF-specific by nested PCR analysis and sequencing. Presence of EF in the peritoneal exudate cells could be a unique mode of transport of growth factor modulator to the site of injury to aid in regeneration/cell proliferation of damaged tissue.     

An acidic phospholipase A₂ with antibacterial activity from Porthidium nasutum snake venom

Snake venoms are complex mixtures of proteins among which both basic and acidic phospholipases A₂ (PLA₂s) can be found. Basic PLA₂s are usually responsible for major toxic effects induced by snake venoms, while acidic PLA₂s tend to have a lower toxicity. A novel PLA₂, here named PnPLA₂, was purified from the venom of Porthidium nasutum by means of RP-HPLC on a C18 column. PnPLA₂ is an acidic protein with a pI of 4.6, which migrates as a single band under both non-reducing and reducing conditions in SDS-PAGE. PnPLA₂ had a molecular mass of 15,802.6 Da, determined by ESI-MS. Three tryptic peptides of this protein were characterized by HPLC-nESI-MS/MS, and N-terminal sequencing by direct Edman degradation showing homology to other acidic PLA₂s from viperid venoms. PnPLA₂ displayed indirect hemolytic activity in agarose erythrocyte-egg yolk gels and bactericidal activity against Staphylococcus aureus in a dose-dependent manner, with a MIC and MBC of 32 μg/mL. In addition, PnPLA₂ showed a potent inhibitory effect on platelet aggregation with doses up to 40 μg/mL. This acidic PLA₂, in contrast to basic enzymes isolated from other viperid snake venoms, was not cytotoxic to murine skeletal muscle myoblasts C₂C₁₂. This is the first report on a bactericidal protein of Porthidium nasutum venom.     

Structure/Function Relationships of Adipose Phospholipase A2 Containing a Cys-His-His Catalytic Triad

Adipose phospholipase A₂ (AdPLA or Group XVI PLA₂) plays an important role in the onset of obesity by suppressing adipose tissue lipolysis. As a consequence, AdPLA-deficient mice are resistant to obesity induced by a high fat diet or leptin deficiency. It has been proposed that AdPLA mediates its antilipolytic effects by catalyzing the release of arachidonic acid. Based on sequence homology, AdPLA is part of a small family of acyltransferases and phospholipases related to lecithin:retinol acyltransferase (LRAT). To better understand the enzymatic mechanism of AdPLA and LRAT-related proteins, we solved the crystal structure of AdPLA. Our model indicates that AdPLA bears structural similarity to proteins from the NlpC/P60 family of cysteine proteases, having its secondary structure elements configured in a circular permutation of the classic papain fold. Using both structural and biochemical evidence, we demonstrate that the enzymatic activity of AdPLA is mediated by a distinctive Cys-His-His catalytic triad and that the C-terminal transmembrane domain of AdPLA is required for the interfacial catalysis. Analysis of the enzymatic activity of AdPLA toward synthetic and natural substrates indicates that AdPLA displays PLA₁ in addition to PLA₂ activity. Thus, our results provide insight into the enzymatic mechanism and biochemical properties of AdPLA and LRAT-related proteins and lead us to propose an alternate mechanism for AdPLA in promoting adipose tissue lipolysis that is not contingent on the release of arachidonic acid and that is compatible with its combined PLA₁/A₂ activity.     

A [Lys49]phospholipase A2 from Protobothrops flavoviridis Venom Induces Caspase-Independent Apoptotic Cell Death Accompanied by Rapid Plasma-Membrane Rupture in Human Leukemia Cells

Protobothrops flavoviridis venom contains plural phospholipase A₂ (PLA₂) isozymes. A [Lys⁴⁹]PLA₂ called BPII induced cell death in human leukemia cells. PLA2, an [Asp⁴⁹]PLA₂ that has much stronger lipolytic activity than BPII, failed to induce cell death. BPII-treated cells showed morphological changes, DNA fragmentation, and nuclear condensation. This BPII-induced apoptotic cell death was neither inhibited by inhibitors of caspases 3 and 6 nor accompanied by activation of procaspase 3, indicating that BPII-induced cell death is caspase independent. Since inactive p-bromophenacylated BPII induced cell death, BPII-induced apoptotic cell death is independent of PLA₂ lipolytic activity. Rapid externalization of phosphatidylserine in BPII-treated cells was observed for fluorescein isothiocyanate (FITC)-labeled annexin V. In the cells treated with BPII, this spread over the cell membranes, implying that the cell toxicity of BPII is mediated via its cell-surface receptor.     

Solution structure of porcine pancreatic phospholipase A2 complexed with micelles and a competitive inhibitor

Summary The three-dimensional structure of porcine pancreatic PLA₂ (PLA₂), present in a 40 kDa ternary complex with micelles and a competitive inhibitor, has been determined using multidimensional heteronuclear NMR spectroscopy. The structure of the protein (124 residues) is based on 1854 constraints, comprising 1792 distance and 62 torsion angle constraints. A total of 18 structures was calculated using a combined approach of distance geometry and restrained molecular dynamics. The atomic rms distribution about the mean coordinate positions for residues 1–62 and 72–124 is 0.75±0.09 Å for the backbone atoms and 1.14±0.10 Å for all atoms. The rms difference between the averaged minimized NMR structures of the free PLA₂ and PLA₂ in the ternary complex is 3.5 Å for the backbone atoms and 4.0 Å for all atoms. Large differences occur for the calcium-binding loop and the surface loop from residues 62 through 72. The most important difference is found for the first three residues of the N-terminal -helix. Whereas free in solution Ala¹, Leu² and Trp³ are disordered, with the -amino group of Ala¹ pointing out into the solvent, in the ternary complex these residues have an -helical conformation with the -amino group buried inside the protein. As a consequence, the important conserved hydrogen bonding network which is also seen in the crystal structures is present only in the ternary complex, but not in free PLA₂. Thus, the NMR structure of the N-terminal region (as well as the calcium-binding loop and the surface loop) of PLA₂ in the ternary complex resembles that of the crystal structure. Comparison of the NMR structures of the free enzyme and the enzyme in the ternary complex indicates that conformational changes play a role in the interfacial activation of PLA₂.     

A possible role of lysophospholipids produced by calcium-independent phospholipase A2 in membrane-raft budding and fission

Phospholipase A₂ (PLA₂) not only plays a role in the membrane vesiculation system but also mediates membrane-raft budding and fission in artificial giant liposomes. This study aimed to demonstrate the same effects in living cells. Differentiated Caco-2 cells were cultured on filter membranes. MDCK cells were challenged with Influenza virus. The MDCK cultures were harvested for virus titration with a plaque assay. Alkaline phosphatase (ALP), a membrane-raft associated glycosylphosphatidylinositol (GPI)-anchored protein, was 70% released by adding 0.2 mmol/l lysophosphatidylcholine, which was abolished by treatment with a membrane-raft disrupter, methyl-β-cyclodextrin. Activation of calcium-independent PLA₂ (iPLA₂) by brefeldin A increased the apical release of ALP by approximately 1.5-fold (p < 0.01), which was blocked by PLA₂ inhibitor bromoenol lactone (BEL). BEL also reduced Influenza virus production into the media (< 10%) in the MDCK culture. These results suggest that cells utilize inverted corn-shaped lysophospholipids generated by PLA₂ to modulate plasma membrane structure and assist the budding of raft-associated plasma membrane particles, which virus utilizes for its budding. Brush borders are enriched with membrane-rafts and undergo rapid turnover; thus, PLA₂ may be involved in the regulatory mechanism in membrane dynamism. Further, iPLA₂ may provide a therapeutic target for viral infections.     

Essential role of p38 MAPK in caspase-independent, iPLA2-dependent cell death under hypoxia/low glucose conditions

The mechanisms of cell death induced by hypoxia or ischemia are not yet fully understood. We have previously demonstrated that cell death induced by hypoxia occurs independently of caspases, and is mediated by phospholipase A₂ (PLA₂).Here, we show that p38 mitogen-activated protein kinase is activated under hypoxia. A selective inhibitor of p38 or decrease in the p38alpha protein level prevents hypoxia-induced cell death. The p38 inhibitor abolishes PLA₂ activation by hypoxia, indicating that p38 acts upstream of PLA₂. The antioxidant N-acetyl-cysteine inhibits activation of p38 and cell death induced by hypoxia, indicating that reactive oxygen species (ROS) are responsible for p38 activation. These results demonstrate that the ROS/p38/PLA₂ signaling axis has a crucial role in caspase-independent cell death induced by hypoxia.     

Platelet‐activating factor: role in long‐term potentiation and in brain damage

Brain platelet‐activating factor (PAF) is a lipid mediator involved in neurotransmission and in LTP. It has been reported that the induction of LTP by high frequency stimulation increases the activity of the enzymes responsible for its synthesis by a still unknown mechanism ( 1 ). One of the two biosynthetic pathways is Ca²⁺‐dependent and transforms a membrane ether phospholipid into PAF by a sequence of two reactions being the first one, catalyzed by a phospholipase A₂ (PLA₂), rate limiting. Overproduction of PAF, taking place in pathological conditions, contributes to brain damage. Various PLA₂s are present in brain tissue and, particularly, sPLA₂‐IIA is very likely involved in the production of PAF as its expression increases in pathological conditions. Recently, we have found the release of sPLA₂‐IIA from rat brain cortex mitochondria and its association with nuclear membranes, which might be an intracellular target for the enzyme.     

Role of protein kinase C in oxidant — mediated activation of phospholipase A2 in rabbit pulmonary arterial smooth muscle cells

The present study was undertaken to test the hypothesis that activation of cell membrane associated protein kinase C (PKC) plays a role in stimulating cell membrane associated phospholipase A₂ (PLA₂) activity, and subsequent liberation of arachidonic acid (AA) under exposure of rabbit pulmonary arterial smooth muscle cells to the oxidant hydrogen peroxide (H₂O₂). Exposure of the smooth muscle cells to H₂O₂ dose-dependently stimulates [¹⁴C] AA release, and enhances the cell membrane associated PLA₂ activity. Pretreatment of the cells with protein kinase C (PKC) inhibitors H₇ and sphingosine prevent the cell membrane associated PLA₂ activity, and AA release caused by H₂O₂. Treatment of the smooth muscle cells with H₂O₂ stimulates the cell membrane associated PKC activity. Pretreatment of the cells with an antioxidant vitamin E prevents H₂O₂ caused stimulation of the cell membrane associated PKC activity. The cell membrane associated PLA₂ and PKC activities correlate linearly. These results suggest that H₂O₂ caused stimulation of the smooth muscle cell membrane associated PLA₂ activity, and subsequent liberation of AA can occur through an increase in the activity of the cell membrane associated PKC. (Mol Cell Biochem122: 9–15, 1993)Abbreviations AA Arachidonic Acid - PLA₂ Phospholipase A₂ - PKC Protein Kinase C - PBS Phosphate Buffered Saline - HBPS Hank's Buffered Physiological Saline - HEPES 4-(2-Hydroxyethyl)-1-Piperazine N-2-Ethanesulfonate - FCS Fetal Calf Serum - ATP Adenosine Triphosphate - H₇ 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine - DMEM Dulbecco's Modified Eagles Medium - TCA Trichloroacetic Acid     

N49 phospholipase A2, a unique subgroup of snake venom group II phospholipase A2

A novel phospholipase A₂ (PLA₂) with Asn at its site 49 was purified from the snake venom of Protobothrops mucrosquamatus by using SP-Sephadex C25, Superdex 75, Heparin-Sepharose (FF) and HPLC reverse-phage C₁₈ chromatography and designated as TM-N49. It showed a molecular mass of 13.875 kDa on MALDI-TOF. TM-N49 does not possess enzymatic, hemolytic and hemorrhagic activities. It fails to induce platelet aggregation by itself, and does not inhibit the platelet aggregation induced by ADP. However, it exhibits potent myotoxic activity causing inflammatory cell infiltration, severe myoedema, myonecrosis and myolysis in the gastrocnemius muscles of BALB/c mice. Phylogenetic analysis found that that TM-N49 combined with two phospholipase A₂s from Trimeresurus stejnegeri, TsR6 and CTs-R6 cluster into one group. Structural and functional analysis indicated that these phospholipase A₂s are distinct from the other subgroups (D49 PLA₂, S49 PLA₂ and K49 PLA₂) and represent a unique subgroup of snake venom group II PLA₂, named N49 PLA₂ subgroup.     

Small-molecule inhibitors as potential therapeutics and as tools to understand the role of phospholipases A2

Phospholipase A₂ (PLA₂) enzymes are involved in various inflammatory pathological conditions including arthritis, cardiovascular and autoimmune diseases. The regulation of their catalytic activity is of high importance and a great effort has been devoted in developing synthetic inhibitors. We summarize the most important small-molecule synthetic PLA₂ inhibitors developed to target each one of the four major types of human PLA₂ (cytosolic cPLA₂, calcium-independent iPLA_2, secreted sPLA_2, and lipoprotein-associated LpPLA₂). We discuss recent applications of inhibitors to understand the role of each PLA₂ type and their therapeutic potential. Potent and selective PLA₂ inhibitors have been developed. Although some of them have been evaluated in clinical trials, none reached the market yet. Apart from their importance as potential medicinal agents, PLA₂ inhibitors are excellent tools to unveil the role that each PLA₂ type plays in cells and in vivo. Modern medicinal chemistry approaches are expected to generate improved PLA₂ inhibitors as new agents to treat inflammatory diseases.     

A chromogenic substrate for solid-phase detection of phospholipase A2

A method for solid-phase detection of phospholipase A₂ (PLA₂) was developed. The method uses 1-octanoyloxynaphthalene-3-sulfonic acid, which was found to be a good substrate of PLA₂. The substrate is hydrolyzed by PLA₂ into 1-naphthol-3-sulfonic acid, which is spontaneously coupled with coexisting diazonium salt to form a red-purple azo dye. Streptomyces and bovine pancreatic PLA₂ spotted on a nitrocellulose membrane could be detected by this method with considerable sensitivity. In addition, colonies of recombinant Escherichia coli producing bacterial PLA₂ were distinguishable from those producing an inactive mutant PLA₂, facilitating high-throughput screening in directed evolution of the enzyme.     

Crystal structure of crotoxin reveals key residues involved in the stability and toxicity of this potent heterodimeric β-neurotoxin

The crystal structure of crotoxin, a potent presynaptic neurotoxin from Crotalus durissusterrificus, was solved at 1.35 Å resolution. It shows the architecture of the three disulfide-linked polypeptide chains (α, β, and γ) of the acidic subunit CA noncovalently complexed with the basic phospholipase A₂ (PLA₂) subunit CB. The unique structural scaffold of the association of the CA and CB subunits indicates that posttranslational cleavage of the pro-CA precursor is a prerequisite for the assembly of the CA-CB complex. These studies provide novel structural insights to explain the role of the CA subunit in the mechanism of action of crotoxin. The crystal structure of the highly toxic and stable CA₂CBb complex crystallized here allows us to identify key amino acid residues responsible for significant differences in the pharmacological activities of the two classes of crotoxin complexes. In particular, we show that critical residues Trp31 and Trp70 of the CBb subunit establish intermolecular polar contacts with Asp99 and Asp89, respectively, of the β-chain of CA₂ and contribute to the stability and toxicity of the CA₂CBb complex. These interactions also lead to decreased PLA₂ activity by partially blocking substrate access to the catalytic dyad and by masking several interfacial binding surface residues important for PLA₂ interaction with phospholipids.Identification of the binding interface between the CA subunits and the CB subunits of crotoxin is important for the structure-based design of antineurotoxic inhibitors. Since crotoxin displays numerous physiological functions, including antitumoral properties, knowledge of its three-dimensional structure will be useful for the understanding of these diverse effects.     

Molecular cloning and characterization of a venom phospholipase A2 from the bumblebee Bombus ignitus

Phospholipase A₂ (PLA₂) is one of the main components of bee venom. Here, we identify a venom PLA₂ from the bumblebee, Bombus ignitus. Bumblebee venom PLA₂ (Bi-PLA₂) cDNA, which was identified by searching B. ignitus venom gland expressed sequence tags, encodes a 180 amino acid protein. Comparison of the genomic sequence with the cDNA sequence revealed the presence of four exons and three introns in the Bi-PLA₂ gene. Bi-PLA₂ is an 18-kDa glycoprotein. It is expressed in the venom gland, cleaved between the residues Arg44 and Ile45, and then stored in the venom sac. Comparative analysis revealed that the mature Bi-PLA₂ (136 amino acids) possesses features consistent with other bee PLA₂s, including ten conserved cysteine residues, as well as a highly conserved Ca²⁺-binding site and active site. Phylogenetic analysis of bee PLA₂s separated the bumblebee and honeybee PLA₂ proteins into two groups. The mature Bi-PLA₂ purified from the venom of B. ignitus worker bees hydrolyzed DBPC, a known substrate of PLA₂. Immunofluorescence staining of Bi-PLA₂-treated insect Sf9 cells revealed that Bi-PLA₂ binds at the cell membrane and induces apoptotic cell death.     

Cellulase elicitor induced accumulation of capsidiol in Capsicum annumm L. suspension cultures

When growth-phase cell suspension cultures of Capsicum annuum were treated with cellulase-elicitor preparation at 3 μg/ml, the level of capsidiol was transiently increased in the culture media rather than in the cells reaching its maximum approx 24 h after treatment. With methyl jasmonate it took 18 h. Elicitor treatment doubled phospholiphase A₂ (PLA₂) activity but simultaneous treatment with aristolochic acid, a PLA₂ inhibitor, inhibited sesquiterpenoid accumulation as well as PLA₂ activity. Mastoparan, a G protein activator, treatment also increased PLA₂ activity and capsidiol production. Taken together, the present study shows that induction of capsidiol production in the C. annuum is mediated by PLA₂ activation.     

The Cytotoxic Effect of Bromoenol Lactone,a Calcium-independent Phospholipase A<Subscript>2</Subscript> Inhibitor,on Rat Cortical Neurons in Culture

This study characterized the phospholipase A₂ (PLA₂) activity in cerebral cortex of fetal rat brain and investigated effects of chemical inhibition of Ca²⁺-independent PLA₂ (iPLA₂) on neurite outgrowth and cell development of cortical neurons in vitro. The PLA₂ activity in fetal brain was insensitive to a Ca²⁺-chelator EGTA and was significantly impaired by an iPLA₂ inhibitor, bromoenol lactone (BEL). Following treatment with BEL, cortical neurons showed acute loss of neurites and impaired cell body, which were clearly dose- and time-dependent. Nuclear staining revealed nuclear regression (shrinkage), but not fragmentation, in BEL-treated cells. The cytotoxic effect of BEL was additive with arachidonic acid (AA) and AA alone also induced neurite demise. BEL treatment resulted in increased production of prostaglandin E₂. Overall data suggest that iPLA₂, a primary PLA₂ isoform in cerebral cortex, displays a housekeeping role in development and neurite outgrowth in cortical neurons in vitro probably via maintaining phospholipid membrane remodeling rather than generating free fatty acids and lysophospholipids.     

Recent progress in phospholipase A₂ research: from cells to animals to humans

Mammalian genomes encode genes for more than 30 phospholipase A₂s (PLA₂s) or related enzymes, which are subdivided into several classes including low-molecular-weight secreted PLA₂s (sPLA₂s), Ca²⁺-dependent cytosolic PLA₂s (cPLA₂s), Ca²⁺-independent PLA₂s (iPLA₂s), platelet-activating factor acetylhydrolases (PAF-AHs), lysosomal PLA₂s, and a recently identified adipose-specific PLA. Of these, the intracellular cPLA₂ and iPLA₂ families and the extracellular sPLA₂ family are recognized as the “big three”. From a general viewpoint, cPLA₂α (the prototypic cPLA₂) plays a major role in the initiation of arachidonic acid metabolism, the iPLA₂ family contributes to membrane homeostasis and energy metabolism, and the sPLA₂ family affects various biological events by modulating the extracellular phospholipid milieus. The cPLA₂ family evolved along with eicosanoid receptors when vertebrates first appeared, whereas the diverse branching of the iPLA₂ and sPLA₂ families during earlier eukaryote development suggests that they play fundamental roles in life-related processes. During the past decade, data concerning the unexplored roles of various PLA₂ enzymes in pathophysiology have emerged on the basis of studies using knockout and transgenic mice, the use of specific inhibitors, and information obtained from analysis of human diseases caused by mutations in PLA₂ genes. This review focuses on current understanding of the emerging biological functions of PLA₂s and related enzymes.