Intracellular- and extracellular-derived Ca influence phospholipase A2-mediated fatty acid release from brain phospholipids

Docosahexaenoic acid (DHA) and arachidonic acid (AA) are found in high concentrations in brain cell membranes and are important for brain function and structure. Studies suggest that AA and DHA are hydrolyzed selectively from the sn-2 position of synaptic membrane phospholipids by Ca²⁺-dependent cytosolic phospholipase A₂ (cPLA₂) and Ca²⁺-independent phospholipase A₂ (iPLA₂), respectively, resulting in increased levels of the unesterified fatty acids and lysophospholipids. Cell studies also suggest that AA and DHA release depend on increased concentrations of Ca²⁺, even though iPLA₂ has been thought to be Ca²⁺-independent. The source of Ca²⁺ for activation of cPLA₂ is largely extracellular, whereas Ca²⁺ released from the endoplasmic reticulum can activate iPLA₂ by a number of mechanisms. This review focuses on the role of Ca²⁺ in modulating cPLA₂ and iPLA₂ activities in different conditions. Furthermore, a model is suggested in which neurotransmitters regulate the activity of these enzymes and thus the balanced and localized release of AA and DHA from phospholipid in the brain, depending on the primary source of the Ca²⁺ signal.     

Extracellular-derived calcium does not initiate in vivo neurotransmission involving docosahexaenoic acid

In vitro studies show that docosahexaenoic acid (DHA) can be released from membrane phospholipid by Ca²⁺-independent phospholipase A₂ (iPLA₂), Ca²⁺-independent plasmalogen PLA₂ or secretory PLA_{2 (sPLA2)}, but not by Ca²⁺-dependent cytosolic PLA₂ (cPLA2), which selectively releases arachidonic acid (AA). Since glutamatergic NMDA (N-methyl-D-aspartate) receptor activation allows extracellular Ca²⁺ into cells, we hypothesized that brain DHA signaling would not be altered in rats given NMDA, to the extent that in vivo signaling was mediated by Ca²⁺-independent mechanisms. Isotonic saline, a subconvulsive dose of NMDA (25 mg/kg), MK-801, or MK-801 followed by NMDA was administered i.p. to unanesthetized rats. Radiolabeled DHA or AA was infused intravenously and their brain incorporation coefficients k*, measures of signaling, were imaged with quantitative autoradiography. NMDA or MK-801 compared with saline did not alter k* for DHA in any of 81 brain regions examined, whereas NMDA produced widespread and significant increments in k* for AA. In conclusion, in vivo brain DHA but not AA signaling via NMDA receptors is independent of extracellular Ca²⁺ and of cPLA₂. DHA signaling may be mediated by iPLA₂, plasmalogen PLA₂, or other enzymes insensitive to low concentrations of Ca²⁺. Greater AA than DHA release during glutamate-induced excitotoxicity could cause brain cell damage.     

Characterization of Phospholipase A2 Activity Enriched in the Nerve Growth Cone

Abstract: Nerve growth cones isolated from fetal rat brain exhibit in their cytosol a robust level of phospholipase A₂ activity hydrolyzing phosphatidylinositol (PI) and phosphatidylethanolamine (PE) but not phosphatidylcholine (PC). Western blot analysis with an antibody to the well-characterized cytosolic phospholipase A₂ (mol wt 85,000) reveals only trace amounts of this PC- and PE-selective enzyme in growth cones. By gel filtration on Superose 12, growth cone phospholipase A₂ activity elutes essentially as two peaks of high molecular mass, at ～65 kDa and at well over 100 kDa. Anion exchange chromatography completely separates a PI-selective from a PE-selective activity, indicating the presence of two different, apparently monoselective phospholipase A₂ species. The PI-selective enzyme, the predominant phospholipase A₂ activity in whole growth cones, is enriched greatly in these structures relative to their parent fractions from fetal brain. This phospholipase A₂ is resistant to reducing agents and is found in the cytosol as well as membrane-associated in the presence of Ca²⁺. However, its catalytic activity is Ca²⁺-independent regardless of whether the enzyme is associated with pure substrate or mixed-lipid growth cone vesicles. The PE-selective phospholipase A₂ in growth cones was studied in less detail but shares with the PI-selective enzyme several properties, including intracellular localization, the existence of cytosolic and membrane-associated forms, and Ca²⁺ independence. Our data indicate growth cones contain two high-molecular-weight forms of phospholipase A₂ that share many properties with known, Ca²⁺-independent cytosolic phospholipase A₂ species but that appear to be monoselective for PI and PE, respectively. In particular, the PI-selective enzyme may represent a new member of the growing family of cytoplasmic phospholipase A₂. The enrichment of the PI-selective phospholipase A₂ in growth cones suggests it plays a major role in the regulation of growth cone function.     

Lipid-Dependent Modulation of Ca2+ Availability in Isolated Mossy Fiber Nerve Endings

An enhancement of glutamate release from hippocampal neurons has been implicated in long-term potentiation, which is thought to be a cellular correlate of learning and memory. This phenomenom appears to be involved the activation of protein kinase C and lipid second messengers have been implicated in this process. The purpose of this study was to examine how lipid-derived second messengers, which are known to potentiate glutamate release, influence the accumulation of intraterminal free Ca²⁺, since exocytosis requires Ca²⁺ and a potentiation of Ca²⁺ accumulation may provide a molecular mechanism for enhancing glutamate release. The activation of protein kinase C with phorbol esters potentiates the depolarization-evoked release of glutamate from mossy fiber and other hippocampal nerve terminals. Here we show that the activation of protein kinase C also enhances evoked presynaptic Ca²⁺ accumulation and this effect is attenuated by the protein kinase C inhibitor staurosporine. In addition, the protein kinase C-dependent increase in evoked Ca²⁺ accumulation was reduced by inhibitors of phospholipase A₂ and voltage-sensitive Ca²⁺ channels, as well as by a lipoxygenase product of arachidonic acid metabolism. That some of the effects of protein kinase C activation were mediated through phospholipase A₂ was also indicated by the ability of staurosporine to reduce the Ca²⁺ accumulation induced by arachidonic acid or the phospholipase A₂ activator melittin. Similarly, the synergistic facilitation of evoked Ca²⁺ accumulation induced by a combination of arachidonic acid and diacylglycerol analogs was attenuated by staurosporine. We suggest, therefore, that the protein kinase C-dependent potentiation of evoked glutamate release is reflected by increases in presynaptic Ca²⁺ and that the lipid second messengers play a central role in this enhancement of chemical transmission processes.     

Involvement of phospholipase A2 and arachidonic acid in the depolarization-evoked accumulation of Ca2+ in hippocampal mossy fiber nerve endings

Depolarization-evoked increases in intraterminal free Ca²⁺ are required for the induction of neurotransmitter release from nerve terminals. Although the mechanisms that regulate the voltage-induced accumulation of presynaptic Ca²⁺ remain obscure, there is evidence that the phospholipase-dependent accumulation of arachidonic acid, or its metabolites, may be involved. Therefore, fura-2 loaded hippocampal mossy fiber nerve endings were used to investigate the relationships between membrane depolarization, lipid metabolism and presynaptic Ca²⁺ availability. It was observed that depolarization of the nerve terminals with KCl induced an increase in intraterminal free calcium that was inhibited more than 90% by a combination of voltage-sensitive Ca²⁺ channel blockers. In addition, the K⁺-dependent effects on Ca²⁺ concentrations were attenuated in the presence of phospholipase A₂ inhibitors, but were mimicked by the phospholipase A₂ activator melittin and exogenous arachidonic acid. Both the melittin- and arachidonic acid-induced increases in presynaptic Ca²⁺ were reduced by voltage-sensitive Ca²⁺ channel blockers. The stimulatory effects of arachidonic acid appeared to be independent of its further metabolism to prostaglandins. In fact, inhibition of either cyclooxygenase or lipoxygenase pathways resulted in a potentiation of the depolarization-evoked increase in intraterminal free Ca²⁺. From these results, we propose that some portion of the depolarization-evoked increase in intraterminal free calcium depends on the activation of phospholipase A₂ and the subsequent accumulation of unesterified arachidonic acid.     

Cerebral microvessel phospholipase A2 activity in senescent mouse

Phospholipase A₂ activity was measured in cerebral microvessels isolated from 5 to 6 month (young adult) and 21 to 24 month (aged adult) old mice. Radiolabeled 1-stearoyl-2-[1-¹⁴C]arachidonyl choline phosphoglyceride was used as the enzyme substrate, and enzyme activity determined at pH 8 and pH 9. Activity in older animals was significantly less than in younger animals at both pH's. With choline phosphoglyceride as a substrate, phospholipase A₂ activity was predominantly Ca²⁺-dependent, although a small, but measurable Ca²⁺-independent component was present. Negligible production of diacylglycerol indicated little or no phospholipase C activity. These findings indicate that activity of a phospholipase A₂, which utilizes choline phosphoglyceride as a substrate, is affected by the aging process. Moreover, a change in PLA₂ activity would result in altered metabolism of specific phosphoglycerides and turnover of fatty acids at the sn-2 position in cerebral microvessels.     

Tryptophan modification of phospholipase A2 enzymes and presynaptic neurotoxins from snake venoms

Three phospholipases A₂ purified from cobra venoms and two presynaptically acting neurotoxins that exhibit phospholipase A₂ activity were subjected to tryptophan modification with 2-hydroxy-5-nitrobenzyl bromide. Associated with the modification of an increasing number of Trp residues were marked decreases in enzymatic activity and lethality, whereas antigenicity remained unchanged. The degree of exposure of tryptophanyl groups as determined by acrylamide quenching was consistent with the relative reactivity toward 2-hydroxy-5-nitrobenzyl bromide, except for Hemachatushaemachatus phospholipase A₂, which showed unusually high reactivity due to its characteristic dimeric conformation. Difference spectra of Trp-modified derivatives differed from those of their native enzymes by the presence of a new positive perturbation between 350 and 500 nm, with a maximum at 415 nm. Scatchard plots revealed only one type of binding site for Ca²⁺, and the binding abilities of the modified enzymes were not impaired. At pH 8.0, all native enzymes enhanced the emission intensity of 8-anilinonaphthalene sulfonate (ANS) dramatically, and the emission intensity of the ANS-enzyme complex increased or decreased in parallel with increasing concentration of Ca²⁺ for the respective enzyme. The Trp-modified derivatives did not enhance the emission intensity of ANS at all either in the presence or absence of Ca²⁺. By means of tryptophan modification, we were able to infer that the tryptophan residues are in the vicinity of the Ca²⁺ binding site and are directly involved in the binding with ANS. This, together with the suggestion that the hydrophobic pocket that interacts with ANS might be the site of binding of the phospholipase A₂ enzyme with the substrate, suggests that the Trp residues in phospholipase A₂ enzymes and presynaptic toxins are involved in substrate binding.     

Effect of Retinoic Acid on the Ca2+-Independent Phospholipase A2 in Nuclei of LA-N-1 Neuroblastoma Cells

LA-N-1 neuroblastoma cell cultures contain Ca²⁺-independent phospholipases A₂ hydrolyzing phosphatidylethanolamine and ethanolamine plasmalogens. These enzymes differ from each other in their molecular mass, substrate specificity, and kinetic properties. Subcellular distribution studies have indicated that the activity of these phospholipases is not only localized in the cytosol but also in non-nuclear membranes and in nuclei. The treatment of LA-N-1 neuroblastoma cell cultures with retinoic acid results in a marked stimulation of Ca²⁺-independent phospholipases A₂ hydrolyzing phosphatidylethanolamine and plasmenylethanolamine. The increase of the activities of both enzymes was first observed in nuclei followed by those present in the cytosol. No effect of retinoic acid on either phospholipase activity could be observed in non-nuclear membranes. The stimulation of these enzymes may be involved in the generation and regulation of arachidonic acid and its metabolites during differentiation.     

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.     

Mechanism of Inhibition of Calcium Uptake into Sarcoplasmic Reticulum by Notexin,a Neurotoxic and Myotoxic Polypeptide

Notexin belongs to a class of snake venom neurotoxins and myotoxins that have phospholipase A₂ activity. Previous studies have shown that these toxins affect target cells differently from phospholipases that are not neurotoxic or myotoxic. Notexin inhibited the Ca²⁺ uptake into fragmented sarcoplasmic reticulum from rabbit skeletal muscle, but it did not cause an efflux of previously accumulated Ca²⁺ or inhibit the Ca²⁺–ATPase activity. It is suggested that notexin specifically binds to and decreases the conductance for Ca²⁺ of the Ca²⁺ pump and/or the conductance of a channel for an ion that facilitates Ca²⁺ transport. The K⁺ ionophore valinomycin reversed the notexin-induced inhibition of Ca²⁺ uptake into sarcoplasmic reticulum, suggesting that the molecular target of notexin could be a K⁺ channel. Two types of reconstitution experiments make it unlikely that notexin acts by degrading a minor lipid that is resistant to hydrolysis by nontoxic phospholipases A₂. Notexininactivated sarcoplasmic reticulum vesicles were reactivated (with respect to Ca²⁺ uptake) by simple solubilization with detergent and subsequent reconstitution by detergent removal. Second, notexin was still active on sarcoplasmic reticulum vesicles after >94% of the lipids were replaced by soybean phosphoglycerides during the reconstitution procedure.     

Lipids and lipolytic enzyme activities of rat heart mitochondria

The lipid composition and lipolytic enzyme activities in rat cardiac mitochondria were examined. Subsarcolemmal mitochondria were prepared by treatment of heart muscle with a Polytron tissue processor, while interfibrillar mitochondria were released by exposure of the remaining low-speed pellet to the protease, nagarse. These procedures are known to yield two functionally different populations of mitochondria. However, their phospholipid contents and compositions were identical, as were the positional distributions of the constituent fatty acids. Of the ethanolamine phospholipids, 20% were plasmalogens, and about 2% of the choline phospholipids consisted of this alkenylacyl species. Both subsarcolemmal and interfibrillar mitochondria contained a Ca²⁺-activated phospholipase A₂, as evidenced by the Ca²⁺-dependent release of unsaturated fatty acids and lysophosphatidylethanolamine from endogenous lipids. Ruthenium red prevented the activation of this enzyme by Ca²⁺, indicating that the activity is located in the matrix space or associated with the inner surface of the inner membrane. Both mitochondrial fractions produced free fatty acids and lysophosphatidylethanolamine in the absence of free Ca²⁺ apparently due to an outer membrane phospholipase A₁. The activity of this enzyme decreased with time, particularly in interfibrillar mitochondria, providing that Ca²⁺ was absent. Nagarse treatment of subsarcolemmal mitochondria resulted in a preparation with the same phospholipase A₁ properties as interfibrillar mitochondria. The possibility that differences in phospholipase A₁ properties account for some of the functional variations between the two mitochondrial types is discussed.     

Manoalide,a Phospholipase A2 Inhibitor,Inhibits Arachidonate Incorporation and Turnover in Brain Phospholipids of the Awake Rat

The Fatty Acid method was used to determine whether incorporation of plasma radiolabeled arachidonic acid into brain phospholipids is controlled by phospholipase A₂. Awake rats received an i.v. injection of a phospholipase A₂ inhibitor, manoalide (10 mg/kg), and then were infused i.v. with [1-¹⁴C]arachidonate or [³H]arachidonate. Animals were killed after infusion by microwave irradiation, and tracer distribution was analyzed in brain phospholipid, neutral lipid and acyl-CoA pools. Calcium-independent phospholipase A₂ activity in brain homogenate was reduced by manoalide, whereas phospholipase C activity was unaffected. At 60 min but not at 20 or 40 min after its injection, manoalide had significantly decreased by 50% incorporation of unesterified arachidonate into and turnover within brain phospholipids, taking into account dilution of the brain arachidonoyl-CoA pool by recycled arachidonate. Manoalide also increased by 100% the net rate of unesterified arachidonate incorporation into brain triacylglycerol. This study indicates that manoalide can be used to inhibit brain phospholipase A₂ in vivo, and that phospholipase A₂ plays a critical role in arachidonate turnover in brain phospholipids and neutral lipids.     

Role of membrane associated serine esterase in the activation of phospholipase A2 by calcium ionophore (A23187) in pulmonary arterial smooth muscle cells

Exposure of rabbit pulmonary arterial smooth muscle cells to 10 M of the calcium ionophore A23187 dramatically stimulates cell membrane-associated phospholipase A₂ activity and arachidonic acid release. In addition, A23187 also enhances cell membrane-associated serine esterase activity. Serine esterase inhibitors phenylmethylsulfonylfuoride and diisopropyl fluorophosphate prevent the increase in serine esterase and phospholipase A₂ activities and arachidonic acid release caused by A23187. A23187 still stimulated serine esterase and phospholipase A₂ activities and arachidonic acid release in cells pretreated with nominal Ca²⁺ free buffer. Treatment of the cell membrane with A23187 does not cause any appreciable change in serine esterase and phospholipase A₂ activities. Pretreatment of the cells with actinomycin D or cycloheximide did not prevent the increase in the cell membrane associated serine esterase and phospholipase A₂ activities, and arachidonic acid release caused by A23187. These results suggest that (i) a membrane-associated serine esterase plays an important role in stimulating the smooth muscle cell membrane associated phospholipase A₂ activity (ii) in addition to the presence of extracellular Ca²⁺, release of Ca²⁺ from intracellular storage site(s) by A23187 also appears to play a role in stimulating the cell membrane-associated serine esterase and phospholipase A₂ activities, and (iii) the increase in the cell membrane-associated serine esterase and phospholipase A₂ activities does not appear to require new RNA or protein synthesis.Abbreviations A23187 calcium ionophore - AA arachidonic acid - PMSF phenylmethyl sulfonylfuoride - DFP diisopropyl-fluorophosphate - DMEM Dulbecco's modified Eagles medium - FCS fetal calf serum - PBS phosphate buffered saline - HBPS Hank's buffered physiological saline - PLA₂ phospholipase A₂     

Phospholipase A2 and arachidonic acid in Alzheimer's disease

Essential fatty acids (EFA) play a critical role in the brain and regulate many of the processes altered in Alzheimer's disease (AD). Technical advances are allowing for the dissection of complex lipid pathways in normal and diseased states. Arachidonic acid (AA) and specific isoforms of phospholipase A₂ (PLA₂) appear to be critical mediators in amyloid-β (Aβ)-induced pathogenesis, leading to learning, memory, and behavioral impairments in mouse models of AD. These findings and ongoing research into lipid biology in AD and related disorders promise to reveal new pharmacological targets that may lead to better treatments for these devastating conditions.     

Involvement of calcium-independent phospholipase A<Subscript>2</Subscript> in regulation of Ca<Superscript>2+</Superscript> signals induced by a calmodulin inhibitor in rat thymocytes

Previous studies have shown that micromolar concentrations of calmodulin inhibitor calmidazolium induce fast activation of nonselective Ca²⁺ channels in plasma membranes of Ehrlich ascites carcinoma cells (Zinchenko, V.P., Kasymov, V.A., Li, V.V., and Kaimachnikov, N.P., Biofizika (Rus.), 2005, vol. 50 (6), pp. 1055–1069). In order to detect this type of Ca²⁺ channels in other cells and to establish common regulatory mechanisms, we studied calmidazolium effects on rat thymocytes. It was found that calmidazolium induces biphasic increases in Ca²⁺ content in cytosol of rat thymocytes due to Ca²⁺ entry from external medium and reflects the activity of nonselective Ca²⁺ channels permeable for Mn²⁺ and Ni²⁺ ions. The rate and the amplitude of the fast phase are decreased, while those of the slow phase are increased in the presence of specific inhibitors of Ca²⁺-independent phospholipase A₂ (bromoenol lactone and palmitoyl trifluoromethyl ketone). The rate and the amplitude of the fast phase are also inhibited by arachidonic acid and the lipoxygenase inhibitor nordihydroguaiaretic acid, while the Ca²⁺-dependent phospholipase A₂ inhibitor bromophenacyl bromide, the cyclooxygenase inhibitor indomethacin, the specific store-operated Ca²⁺ channel inhibitor gadolinium and the phospholipase C inhibitor U73122 have no such effect. The rate of the fast phase only slightly depends on temperature, while that of the slow phase shows a strong temperature dependence and increases with a rise in temperatures (Q ₁₀ = 2). The amplitude of the fast phase of the Ca²⁺ signal increases with a decrease of temperatures due to prolongation of the maximum activity of the Ca²⁺ channel. The data obtained suggest that iPLA₂ is an intermediate link in the activation of calmidazolium-induced nonselective Ca²⁺ channels. The iPLA₂ products lysophospholipids and arachidonic acid activate and inhibit Ca²⁺ channels, respectively. The fact that these compounds manifest different affinities for Ca²⁺ channels shed additional light on the mechanisms of biphasic Ca²⁺ elevation in thymus cell cytosol and prolongation of the active state of Ca²⁺ channels at low temperatures.     

THE DIFFERENTIATION OF PHOSPHOLIPASE A1 AND A2 IN RAT AND HUMAN NERVOUS TISSUES

—Lipid-free extracts of rat and human brain have been prepared and shown to contain phospholipase A₁ and A₂ activities and a lysophospholipase. The phospholipase Aj activity has pH optima of 4·2 and 4·6 in rat and human brain, respectively; it can be partially purified and isolated in high yields by dialysing the extracts at low pH. The purified preparations hydrolyse the ester bond at the 1-position in lecithin, phosphatidyl-ethanolamine and phosphatidylserine, but have little or no action on triglyceride or cholesterol ester. An assay system for the enzyme is described. Phospholipase A₂ activity is optimal at pH 5·5 in rat brain extracts and at pH 5·0 in extracts of human brain. The phospholipase A₂ activity of human cerebral cortex is largely unaffected by heating extracts at 70°C for 5 min, whereas this treatment substantially inactivates phospholipase A₁ and completely destroys lysophospholipase. Phospholipase A₁ is widely distributed in both grey and white matter of human brain and is also present in peripheral nerve. Phospholipase A₂ activity is lower than A₁ in all regions of the CNS examined so far, and is absent from peripheral nerve. Neither enzyme appears to require Ca²⁺ but both are inhibited by di-isopropylfluorophosphate (DFP, 2 × 10^?6 m) and thus differ from phospholipase A of pancreas. These studies confirm that the phospholipase A₁ and A₂ activities in brain are due to separate enzymes.     

ATPase and phosphatase activities from human red cell membranes: II. The effects of phospholipases on Ca2+-dependent enzymic activities

Summary Treatment of human red cell membranes with pure phospholipase A₂ results in a progressive inactivation of both Ca²⁺-dependent and (Ca²⁺+K⁺)-dependent ATPase and phosphatase activities. When phospholipase C replaces phospholipase A₂, Ca²⁺-dependent ATPase activity and Ca²⁺-dependent phosphorylation of red cell membranes are lost, while Ca²⁺-dependent phosphatase activity is enhanced and its apparent affinity for Ca²⁺ is increased about 20-fold. Activation of Ca²⁺-dependent phosphatase following phospholipase C treatment was not observed in sarcoplasmic reticulum preparation. Phospholipase C increases the sensitivity of the phosphatase to N-ethylmaleimide but has little effect on the kinetic parameters relating the phosphatase activity to substrate and cofactors, suggesting that no extensive structural disarrangement of the Ca²⁺-ATPase system has occurred after incubation with phospholipase C.     

Purification and characterization of a cytosolic Ca<Superscript>2+</Superscript>-independent phospholipase A<Subscript>2</Subscript> from bovine brain

The Ca²⁺-independent phospholipase A₂ (iPLA₂) subfamily of enzymes is associated with arachidonic acid (AA) release and the subsequent increase in fatty acid turnover. This phenomenon occurs not only during apoptosis but also during inflammation and lymphocyte proliferation. In this study, we purified and characterized a novel type of iPLA₂ from bovine brain. iPLA₂ was purified 4,174-fold from the bovine brain by a sequential process involving DEAE-cellulose anion exchange, phenyl-5PW hydrophobic interaction, heparin-Sepharose affinity, Sephacryl S-300 gel filtration, Mono S cation exchange, Mono Q anion exchange, and Superose 12 gel filtration. A single peak of iPLA₂ activity was eluted at an apparent molecular mass of 155 kDa during the final Superose 12 gel-filtration step. The purified enzyme had an isoelectric point of 5.3 on twodimensional gel electrophoresis (2-DE) and was inhibited by arachidonyl trifluoromethyl ketone (AACOCF₃), Triton X-100, iron, and Ca²⁺. However, it was not inhibited by bromoenol lactone (BEL), an inhibitor of iPLA₂, and adenosine triphosphate (ATP). The spot with the iPLA₂ activity did not match with any known protein sequence, as determined by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) analysis. Altogether, these data suggest that the purified enzyme is a novel form of cytosolic iPLA₂.     

Group VIA Ca-independent phospholipase A2 (iPLA2β) and its role in β-cell programmed cell death

Activation of phospholipases A₂ (PLA₂s) leads to the generation of biologically active lipid mediators that can affect numerous cellular events. The Group VIA Ca²⁺-independent PLA₂, designated iPLA₂β, is active in the absence of Ca²⁺, activated by ATP, and inhibited by the bromoenol lactone suicide inhibitor (BEL). Over the past 10–15 years, studies using BEL have demonstrated that iPLA₂β participates in various biological processes and the recent availability of mice in which iPLA₂β expression levels have been genetically-modified are extending these findings. Work in our laboratory suggests that iPLA₂β activates a unique signaling cascade that promotes β-cell apoptosis. This pathway involves iPLA₂β dependent induction of neutral sphingomyelinase, production of ceramide, and activation of the intrinsic pathway of apoptosis. There is a growing body of literature supporting β-cell apoptosis as a major contributor to the loss of β-cell mass associated with the onset and progression of Type 1 and Type 2 diabetes mellitus. This underscores a need to gain a better understanding of the molecular mechanisms underlying β-cell apoptosis so that improved treatments can be developed to prevent or delay the onset and progression of diabetes mellitus. Herein, we offer a general review of Group VIA Ca²⁺-independent PLA₂ (iPLA₂β) followed by a more focused discussion of its participation in β-cell apoptosis. We suggest that iPLA₂β-derived products trigger pathways which can lead to β-cell apoptosis during the development of diabetes.     

Amiodarone — an inhibitor of phospholipase activity: a comparative study of the inhibitory effects of amiodarone,chloroquine and chlorpromazine

Amiodarone, an antiarrhythmic drug, like chloroquine and chlorpromazine, is a tertiary amine with amphiphilic properties. Chloroquine and chlorpromazine are known inhibitors of phospholipases. All three drugs produce characteristic microcorneal deposits consistent with lysosomal accumulations of phospholipid. Similar lysosomal bodies were found in leukocytes of 15 patients on chronic amiodarone treatment as well as 3 patients each on chloroquine and chlorpromazine, suggestive of widespread systemic inhibition of lysosomal phospholipases. These lysosomal inclusions were similar in morphology, irrespective of the drug given, and were of four types: multilamellar, amorphous dense, amorphous light, or a combination of 2 or more of the preceding types. There was no simple relationship between the number of inclusion bodies per cell and the cumulative dose of amiodarone (r=0.02) or amiodarone serum levels (r=0.11). An in vitro assay was used to compare the effects of the three drugs on Ca²⁺-dependent phospholipase A and C activities. Phospholipase A₂ activity was inhibited in a dose-dependent fashion (1–8 mg/assay) by all three drugs in the order: chlorpromazine > amiodarone > chloroquine. The inhibitory effect on phospholipase C was more pronounced with all three drugs, producing almost total inhibition at 8 mg/assay. In a Ca²⁺-independent lysosomal phospholipase A system, amiodarone had a greater effect, producing 85% inhibition at 1.2 mg/assay. These observations suggest that amiodarone, like other cationic amphiphiles, induces a generalized phospholipidosis by inhibiting phospholipid catabolism. Its therapeutic and toxic effects may be due to its ability to modulate both Ca²⁺-dependent membrane phospholipases and Ca²⁺-independent acid phospholipases.