Deacylation Of Phospholipids Research Articles

Cellular localization, cloning and expression of Leishmania braziliensis Phospholipase A1

Leishmaniasis is caused by several species of protozoan parasites of the genus Leishmania and represents an important global health problem. Leishmania braziliensis in particular is responsible of cutaneous and mucocutaneous forms of this parasitosis, with prevalence in Latin America. In the present work, we describe in L. braziliensis promastigotes and amastigotes the presence of a Phospholipase A1 (PLA1) activity, an enzyme that catalyses extensive deacylation of phospholipids like phosphatidylcholine. In order to deepen the knowledge about L. braziliensis PLA1, the cloning and expression of the gene that codifies for this enzyme was carried out in a baculovirus expression system with the obtaintion of a purified recombinant protein that displayed PLA1 activity. Given that this is the first molecular and functional protein characterization of a PLA1 in the Leishmania genus, we also performed a phylogenetic analysis of this gene throughout 12 species whose genome sequences were available. The results presented here will contribute to increase the knowledge about trypanosome phospholipases, which could be novel and valuable as potential targets to fight neglected diseases like Leishmaniasis.

In vitro synthesis of phospholipids with yeast phospholipase B, a phospholipid deacylating enzyme

The gene encoding the Saccharomyces cerevisiae phospholipid deacylation enzyme, phospholipase B (ScPLB1), was successfully expressed in E. coli. The enzyme (Scplb1p) was engineered to have a histidine-tag at the C-terminal end and was purified by metal (Ni) affinity chromatography. Enzymatic properties, optimal pH, and substrate specificity were similar to those reported previously. For example, deacylation activity was observed in acidic pH in the absence of Ca2+ and was additive in neutral pH in the presence of Ca2+, and the enzyme had the same substrate priority as reported previously, with the exception of PE, suggesting that yeast phospholipase B could be produced in its native structure in bacterial cells. Scplb1p retained transacylation activity in aqueous medium, and esterified lysophosphatidylcholine with free fatty acid to form phosphatidylcholine in a non-aqueous, glycerin medium. We propose that phospholipase B could serve as an additional tool for in vitro enzyme-mediated phospholipid synthesis.

Lysophosphatidylcholine acyltransferase 4 is involved in chondrogenic differentiation of ATDC5 cells

Glycerophospholipids have important structural and functional roles in cells and are the main components of cellular membranes. Glycerophospholipids are formed via the de novo pathway (Kennedy pathway) and are subsequently matured in the remodeling pathway (Lands’ cycle). Lands’ cycle consists of two steps: deacylation of phospholipids by phospholipases A2 and reacylation of lysophospholipids by lysophospholipid acyltransferases (LPLATs). LPLATs play key roles in the maturation and maintenance of the fatty acid composition of biomembranes, and cell differentiation. We examined whether LPLATs are involved in chondrogenic differentiation of ATDC5 cells, which can differentiate into chondrocytes. Lysophosphatidylcholine acyltransferase 4 (LPCAT4) mRNA expression and LPCAT enzymatic activity towards 18:1-, 18:2-, 20:4-, and 22:6-CoA increased in the late stage of chondrogenic differentiation, when mineralization occurred. LPCAT4 knockdown decreased mRNA and protein levels of chondrogenic markers as well as Alcian blue staining intensity and alkaline phosphatase activity in ATDC5 cells. These results suggest that LPCAT4 plays important roles during the transition of chondrocytes into hypertrophic chondrocytes and/or a mineralized phenotype.

Stability of an amphipathic helix-hairpin surfactant peptide in liposomes

Surfactant protein B (SP-B; 79 residues) is a member of the saposin superfamily and plays a pivotal role in lung function. The N- and C-terminal regions of SP-B, cross-linked by two disulfides, were theoretically predicted to fold as charged amphipathic helices, suggesting participation in surfactant activities. Previous studies with oxidized Super Mini-B (SMB), a construct based on the N- and C-regions of SP-B (i.e., residues 1–25 and 63–78) joined with a designer turn (-PKGG-) and two disulfides, indicated that freshly prepared SMB in lipids folded as a surface active, α-helix-hairpin. Because other peptides modeled on α-helical SP domains lost helicity and surfactant activity on storage, experiments were here performed on oxidized SMB in surfactant liposomes stored at ~2–8°C for ≤5.5years. Captive bubble surfactometry confirmed low minimum surface tensions for fresh and stored SMB preparations. FTIR spectroscopy of fresh and stored SMB formulations showed secondary structures compatible with the peptide folding as α-helix-hairpin. A homology (I-TASSER) model of oxidized SMB demonstrated a globular protein, exhibiting a core of hydrophobic residues and a surface of polar residues. Since mass spectroscopy indicated that the disulfides were maintained on storage, the stability of SMB may be partly due to the disulfides bringing the N- and C-α-helices closer. Mass spectroscopy of stored SMB preparations showed some methionine oxidation, and also partial deacylation of surfactant phospholipids to form lyso-derivatives. However, the stable conformation and activity of stored SMB surfactant suggest that the active helix-hairpin resists these chemical changes which otherwise may lead to surfactant inhibition.

Lysophospholipid acyltransferases and eicosanoid biosynthesis in zebrafish myeloid cells

Eicosanoids derived from the enzymatic oxidation of arachidonic acid play important roles in a large number of physiological and pathological processes in humans. Many animal and cellular models have been used to investigate the intricate mechanisms regulating their biosynthesis and actions. Zebrafish is a widely used model to study the embryonic development of vertebrates. It expresses homologs of the key enzymes involved in eicosanoid production, and eicosanoids have been detected in extracts from adult or embryonic fish. In this study we prepared cell suspensions from kidney marrow, the main hematopoietic organ in fish. Upon stimulation with calcium ionophore, these cells produced eicosanoids including PGE2, LTB4, 5-HETE and, most abundantly, 12-HETE. They also produced small amounts of LTB5 derived from eicosapentaenoic acid. These eicosanoids were also produced in kidney marrow cells stimulated with ATP, and this production was greatly enhanced by preincubation with thimerosal, an inhibitor of arachidonate reacylation into phospholipids. Microsomes from these cells exhibited acyltransferase activities consistent with expression of MBOAT5/LPCAT3 and MBOAT7/LPIAT1, the main arachidonoyl-CoA:lysophospholipid acyltransferases. In summary, this work introduces a new cellular model to study the regulation of eicosanoid production through a phospholipid deacylation–reacylation cycle from a well-established, versatile vertebrate model species.

Disruption of phospholipase B‐mediated turnover upon loss of Ypk1 in S. cerevisiae

Ypk1, the yeast homolog of the human serum‐ and glucocorticoid‐induced kinase (Sgk1), is a serine/threonine protein kinase known to affect various downstream processes, including plasma membrane flippase activity and sphingolipid homeostasis. The results described here indicate that Ypk1 also impinges upon the turnover of phospholipids. Pulse‐chase experiments reveal that a ypk1 mutant exhibits alterations in phospholipid deacylation and resultant glycerophosphodiester production as compared to a wild type strain. Furthermore, loss of Plb1, a plasma membrane (PM) associated phospholipase, in a ypk1 background results in further alterations in phospholipid deacylation and a growth defect. We are in the process of examining potential explanations for the altered Plb1 activity in the ypk1 mutant. Strains in which other phospholipase B‐encoding genes are deleted in combination with YPK1 do not display a growth defect, suggesting a specific functional interaction between Plb1 and Ypk1. Preliminary studies have identified model PM proteins, including the Git1 permease, whose activity is affected by loss of Ypk1 and/or Plb1. Our data is consistent with the idea that Ypk1 and Plb1 alter plasma membrane phospholipid composition, which in turn impinges upon the activity of integral plasma membrane proteins.

Enzymological analysis of the tumor suppressor A-C1 reveals a novel group of phospholipid-metabolizing enzymes

A-C1 protein is the product of a tumor suppressor gene negatively regulating the oncogene Ras and belongs to the HRASLS (HRAS-like suppressor) subfamily. We recently found that four members of this subfamily expressed in human tissues function as phospholipid-metabolizing enzymes. Here we examined a possible enzyme activity of A-C1. The homogenates of COS-7 cells overexpressing recombinant A-C1s from human, mouse, and rat showed a phospholipase A½ (PLA½) activity toward phosphatidylcholine (PC). This finding was confirmed with the purified A-C1. The activity was Ca²⁺ independent, and dithiothreitol and Nonidet P-40 were indispensable for full activity. Phosphatidylethanolamine (PE) was also a substrate and the phospholipase A₁ (PLA₁) activity was dominant over the PLA₂ activity. Furthermore, the protein exhibited acyltransferase activities transferring an acyl group of PCs to the amino group of PEs and the hydroxyl group of lyso PCs. As for tissue distribution in human, mouse, and rat, A-C1 mRNA was abundantly expressed in testis, skeletal muscle, brain, and heart. These results demonstrate that A-C1 is a novel phospholipid-metabolizing enzyme. Moreover, the fact that all five members of the HRASLS subfamily, including A-C1, show similar catalytic properties strongly suggests that these proteins constitute a new class of enzymes showing PLA½ and acyltransferase activities.

Robust Utilization of Phospholipase-Generated Metabolites, Glycerophosphodiesters, by Candida albicans: Role of the CaGit1 Permease

Glycerophosphodiesters are the products of phospholipase-mediated deacylation of phospholipids. In Saccharomyces cerevisiae, a single gene, GIT1, encodes a permease responsible for importing glycerophosphodiesters, such as glycerophosphoinositol and glycerophosphocholine, into the cell. In contrast, the Candida albicans genome contains four open reading frames (ORFs) with a high degree of similarity to S. cerevisiae GIT1 (ScGIT1) Here, we report that C. albicans utilizes glycerophosphoinositol (GroPIns) and glycerophosphocholine (GroPCho) as sources of phosphate at both mildly acidic and physiological pHs. Insertional mutagenesis of C. albicans GIT1 (CaGIT1) (orf19.34), the ORF most similar to ScGit1, abolished the ability of cells to use GroPIns as a phosphate source at acidic pH and to transport [(3)H]GroPIns at acidic and physiological pHs, while reintegration of a GIT1 allele into the genome restored those functions. Several lines of evidence, including the detection of internal [(3)H]GroPIns, indicated that GroPIns is transported intact through CaGit1. GroPIns transport was shown to conform to Michaelis-Menten kinetics, with an apparent K(m) of 28 ± 6 μM. Notably, uptake of label from [(3)H]GroPCho was found to be roughly 50-fold greater than uptake of label from [(3)H]GroPIns and roughly 500-fold greater than the equivalent activity in S. cerevisiae. Insertional mutagenesis of CaGIT1 had no effect on the utilization of GroPCho as a phosphate source or on the uptake of label from [(3)H]GroPCho. Growth under low-phosphate conditions was shown to increase label uptake from both [(3)H]GroPIns and [(3)H]GroPCho. Screening of a transcription factor deletion set identified CaPHO4 as required for the utilization of GroPIns, but not GroPCho, as a phosphate source.

Regulation and physiological roles of the phospholipase B's encoded by PLB1‐3 in Saccharomyces cerevisiae

The phospholipase Bs (PLBs) encoded by PLB1, PLB2, and PLB3, are associated with the plasma membrane, cell wall, and periplasmic space of Saccharomyces cerevisiae. PLB activity catalyzes phospholipid deacylation and the concomitant production of intracellular and extracellular glycerophosphodiesters.. The extracellular glycerophosphodiesters produced, glycerophosphocholine and glycerophosphoinositol, are transported into the cell via the permease encoded by the GIT1 gene. Whereas GIT1 is transcriptionally regulated by phosphate levels, we report here that PLB1‐3 are not similarly regulated. Alterations in RAS/cAMP signaling, however, do affect expression of PLB1‐3; PLB1 and PLB3 message levels are increased upon upregulation of RAS/cAMP signaling, whereas PLB2 message levels are decreased. The mechanism of this regulation is under study. To probe the physiological roles of Plb1‐3‐mediated phospholipid turnover, we performed phenotypic analyses. A strain in which PLB1‐3 have been deleted (a plb1‐3Δ mutant) exhibits slower growth as compared to its wild type counterpart. In addition, a plb1‐3Δ mutant exhibits increased latrunculin A sensitivity as compared to wild type, suggesting a connection between cell‐surface phospholipase B activity and stability of the actin cytoskeleton. Current studies are aimed at determining the molecular mechanisms involved in producing the observed phenotypes.

Lysophospholipid Acyltransferases and Arachidonate Recycling in Human Neutrophils

The cycle of deacylation and reacylation of phospholipids plays a critical role in regulating availability of arachidonic acid for eicosanoid production. The major yeast lysophospholipid acyltransferase, Ale1p, is related to mammalian membrane-bound O-acyltransferase (MBOAT) proteins. We expressed four human MBOATs in yeast strains lacking Ale1p and studied their acyl-CoA and lysophospholipid specificities using novel mass spectrometry-based enzyme assays. MBOAT1 is a lysophosphatidylserine (lyso-PS) acyltransferase with preference for oleoyl-CoA. MBOAT2 also prefers oleoyl-CoA, using lysophosphatidic acid and lysophosphatidylethanolamine as acyl acceptors. MBOAT5 prefers lysophosphatidylcholine and lyso-PS to incorporate linoleoyl and arachidonoyl chains. MBOAT7 is a lysophosphatidylinositol acyltransferase with remarkable specificity for arachidonoyl-CoA. MBOAT5 and MBOAT7 are particularly susceptible to inhibition by thimerosal. Human neutrophils express mRNA for these four enzymes, and neutrophil microsomes incorporate arachidonoyl chains into phosphatidylinositol, phosphatidylcholine, PS, and phosphatidylethanolamine in a thimerosal-sensitive manner. These results strongly implicate MBOAT5 and MBOAT7 in arachidonate recycling, thus regulating free arachidonic acid levels and leukotriene synthesis in neutrophils.

Yeast Pgc1p (YPL206c) Controls the Amount of Phosphatidylglycerol via a Phospholipase C-type Degradation Mechanism

The product of the open reading frame YPL206c, Pgc1p, of the yeast Saccharomyces cerevisiae displays homology to bacterial and mammalian glycerophosphodiester phosphodiesterases. Deletion of PGC1 causes an accumulation of the anionic phospholipid, phosphatidylglycerol (PG), especially under conditions of inositol limitation. This PG accumulation was not caused by increased production of phosphatidyl-glycerol phosphate or by decreased consumption of PG in the formation of cardiolipin, the end product of the pathway. PG accumulation in the pgc1Delta strain was caused rather by inactivation of the PG degradation pathway. Our data demonstrate an existence of a novel regulatory mechanism in the cardiolipin biosynthetic pathway in which Pgc1p is required for the removal of excess PG via a phospholipase C-type degradation mechanism.

Role of Group VIA Calcium-independent Phospholipase A2 in Arachidonic Acid Release, Phospholipid Fatty Acid Incorporation, and Apoptosis in U937 Cells Responding to Hydrogen Peroxide

Group VIA calcium-independent phospholipase A2 (iPLA2) has been shown to play a major role in regulating basal phospholipid deacylation reactions in certain cell types. More recently, roles for this enzyme have also been suggested in the destruction of membrane phospholipid during apoptosis and after oxidant injury. Proposed iPLA2 roles have rested heavily on the use of bromoenol lactone as an iPLA2-specific inhibitor, but this compound actually inhibits other enzymes and lipid pathways unrelated to PLA2, which makes it difficult to define the contribution of iPLA2 to specific functions. In previous work, we pioneered the use of antisense technology to decrease cellular iPLA2 activity as an alternative approach to study iPLA2 functions. In the present study, we followed the opposite strategy and prepared U937 cells that exhibited enhanced iPLA activity by stably expressing a plasmid containing iPLA2 cDNA. Compared with control cells, the iPLA2 -overexpressing U937 cells showed elevated responses to hydrogen peroxide with regard to both arachidonic acid mobilization and incorporation of the fatty acid into phospholipids, thus providing additional evidence for the key role that iPLA2 plays in these events. Long-term exposure of the cells to hydrogen peroxide resulted in cell death by apoptosis, and this process was accelerated in the iPLA2-overexpressing cells. Increased phospholipid hydrolysis and fatty acid release also occurred in these cells. Unexpectedly, however, abrogation of U937 cell iPLA2 activity by either methyl arachidonyl fluorophosphonate or an antisense oligonucleotide did not delay or decrease the extent of apoptosis induced by hydrogen peroxide. These results indicate that, although iPLA2-mediated phospholipid hydrolysis occurs during apoptosis, iPLA2 may actually be dispensable for the apoptotic process to occur. Thus, beyond a mere destructive role, iPLA2 may play other roles during apoptosis.

Lysosomal phospholipase A1 in Trypanosoma cruzi: an enzyme with a possible role in the pathogenesis of Chagas' disease.

We found that, as in African trypanosomes, endogenous phospholipase A(1) (Plase A(1)) activity can catalyse extensive deacylation of phospholipids upon cell death in all life stages of Trypanosoma cruzi. A major lysosomal Plase A(1) was purified and characterized. The enzyme products can explain the lesions surrounding degenerating T. cruzi cells in host tissues. Thus Plase A(1) emerges as a target to block pathogenesis in trypanosomal infections.

Phospholipase from Trypanosoma brucei releases arachidonic acid by sequential sn-1, sn-2 deacylation of phospholipids

Previously, we showed that arachidonic acid (AA) stimulates Ca 2+ currents in pathogenic Trypanosoma brucei (Eintracht J, Maathai R, Mellors A, Ruben L. Calcium entry in Trypanosoma brucei is regulated by phospholipase A 2 and arachidonic acid. Biochem. J 1998;336:659–666). Here we examine the mechanism used by T. brucei to release AA from the sn-2 position of diacyl glycero-phospholipids. We report that T. brucei accomplishes this feat in the apparent absence of phospholipase A 2 (PLA 2). Instead, deacylation is initiated at the sn-1 position, followed by acyl migration and hydrolysis with LysoPLA. Neither whole cell homogenates nor enriched protein fractions could release AA from substrates whose sn-1 position contained a non-hydrolyzable alkyl ether linkage. These same fractions however, released AA from ester linked phospholipids, and TLC analysis of the reaction products supported the sequential deacylation process. The release of sn-2 AA from 1-palmitoyl-2-[1- 14C]arachidonyl- sn-glycero-3-PC was linear up to 90 min at an average rate of 50 nmol min −1 mg −1. sn-2 AA was processed more efficiently than sn-2 palmitate. The reaction was also greatest for: LysoPC>diacyl-PC ( sn-1 labeled)>diacyl-PC ( sn-2 labeled). Product formation was sensitive to polar head group, and PI was processed at less than 10% the rate of PC or PE. The enzymatic deacylation was inhibited by the serine specific reagent, methyl arachidonyl fluorophosphonate (MAFP) and the cysteine reagent N-ethylmaleimide (NEM). Both NEM and MAFP inhibited LysoPLA activity under conditions where there was little effect on PLA 1 activity. Overall, we conclude that T. brucei can release AA from diacyl glycero-phospholipids by a sequential deacylation process. Two independent active sites appear to be involved. Interestingly, a high percentage of inner leaflet phospholipids are protected from degradation since they occur in the non-hydrolyzable 1-alkyl ether form.

The Assembly of Very Low Density Lipoproteins in Rat Hepatoma McA-RH7777 Cells Is Inhibited by Phospholipase A2Antagonists

In McA-RH7777 cells, the oleate-stimulated assembly and secretion of very low density lipoproteins (VLDL) was associated with enhanced deacylation of phospholipids, which was markedly decreased by inactivation of the cellular phospholipase A(2). Treatment of the cells with antagonists or antisense oligonucleotide of the Ca(2+)-independent phospholipase A(2) (iPLA(2)) significantly inhibited secretion of apoB100-VLDL and triglyceride. Similar inhibitory effect of the iPLA(2) antagonists was observed on apoB48-VLDL secretion, but secretion of high density lipoprotein particles (such as apoAI- and apoB48-high density lipoprotein) or proteins in general was unaffected. The iPLA(2) antagonist did not affect the synthesis of apoB100 or triglyceride, nor did it affect the activities of phospholipase D, phosphatidate phosphohydrolase, or microsomal triglyceride transfer protein. Inactivation of iPLA(2) resulted in impaired apoB100-VLDL assembly as shown by decreased apoB100-VLDL and triglyceride within the microsomal lumen, with concomitant increase in apoB100 association with the microsomal membranes. The inhibitory effect of iPLA(2) antagonists on apoB100-VLDL assembly/secretion could be abated by pretreatment of cells with oleate. Analysis of molecular species of microsomal phosphatidylcholine and phosphatidylethanolamine by electron spray tandem mass spectrometry revealed that the enrichment of oleoyl moieties was altered by the treatment of iPLA(2) antagonist. These results suggest that the oleate-induced VLDL assembly/secretion may depend upon the establishment of membrane glycerolipids enriched in oleoyl chain, a process mediated by the iPLA(2) activity.

Group-Specific Assays That Distinguish between the Four Major Types of Mammalian Phospholipase A2

Phospholipase A2(PLA2) constitutes a diverse superfamily of enzymes which catalyze the deacylation of phospholipids. At least four types of PLA2are potentially involved in arachidonic acid release in cells and tissues. Since all of them catalyze the same enzymatic reaction, it is difficult to distinguish between them in mixtures of enzymes normally present in biological samples. Utilizing specific properties of each PLA2, we have designed distinct assay procedures which selectively and sensitively detect each type: Group VI Ca2+-independent PLA2, Group IV cytosolic Ca2+-dependent PLA2, Groups V and IIA secreted PLA2s. Each specific assay procedure is selective for a particular PLA2type by at least fourfold and as high as four orders of magnitude relative to the other three enzymes. All assays can detect PLA2activity with as low as subnanogram quantities of enzyme. Importantly, these assays are able to differentiate and quantitate the biochemically and structurally related enzymes, Group IIA and V sPLA2s in crude biological samples. Employing this system, we have found that iPLA2is the dominant PLA2in rat brain, and cPLA2is the most abundant PLA2in P388D1macrophages and human amnionic WISH cells.

Enzymatic degradation of polar lipids in Vigna unguiculata leaves and influence of drought stress

14C‐labelled polar lipids (monogalactosyl‐diacylglycerol [MGDG], digalactosyl‐diacylglycerol [DGDG], phosphatidylcholine [PC] and phosphatidylglycerol [PG]), purified from Vigna unguiculata leaves, were used as substrates to study the lipolytic activities of Vigna unguiculata leaf extracts. Analysis of the radioactive degradation products revealed the presence of at least three enzyme activities contributing to the hydrolysis of the four main leaf membrane lipids: Lipolytic acyl hydrolase (LAH) activities responsible for the deacylation of galactolipids and phospholipids, phospholipase D (PLD, EC 3.1.4.4) activity which gives rise to phosphatidic acid, and as suggested by the presence of diacylglycerols in minor quantities after phospholipid hydrolysis, phosphatidate phosphohydrolase (PAP, EC 3.1.3.4) and/or phospholipase C (PLC, EC 3.1.4.3.) activity. Under the conditions described in the present paper, the presence of phospholipase A (PLA1, EC 3.1.1.3 and PLA2, EC 3.1.1.4) activities remains hypothetical, due to the absence of lysophospholipids. LAH and PLD were partially soluble and partially associated with the membranes. When Vigna unguiculata plants were submitted to drought, the enzymatic degradation of galactolipids and phospholipids increased. The stimulation of lipolytic activities was greater in the drought‐sensitive cultivar of Vigna unguiculata (cv. 1183) than in the drought‐tolerant (cv. EPACE‐1) one. In cv. 1183, MGDG‐ and DGDG‐LAH activities in the membrane fractions were dramatically stimulated at a rather moderate water deficit (−0.75 MPa). A sharp increase in membrane phospholipolytic activities was also observed at mild drought stress (−1.2 MPa). In contrast, in cv. EPACE‐1, the stimulation of lipolytic activities was less drastic and occurred at lower leaf water potentials (below −1.2 MPa for galactolipases, and below −1.4 MPa for phospholipases). Our results confirm the presence in leaves of higher plants of a very active LAH acting on galactolipids, whereas PLD is the main enzyme responsible for the degradation of phospholipids, particularly when plants are submitted to drought stress. The differences in stimulation of lipolytic activities between the two Vigna cultivars was in accordance with the different levels of membrane lipid degradation shown previously and could explain their different capacity to sustain drought.

Heterogeneity of P2u- and P2y-purinergic receptor regulation of phospholipases in MDCK cells.

We have characterized the signaling pathways of purinergic receptors present on the renal epithelial cell line, Madin-Darby canine kidney (MDCK, D1 subclone). Several lines of evidence are consistent with the conclusion that coexisting P2u and P2y receptors release arachidonic acid and metabolites (AA) from MDCK-D1 cells: 1) relative potencies of nucleotide analogues, 2) blockade of P2y agonist- but not P2u agonist-mediated release by suramin, and 3) additivity by 2-methylthio-ATP and UTP. Differences exist between the signaling pathways of the two receptors: pertussis toxin treatment partially inhibits P2u- but not P2y-mediated AA release, and P2y (but not P2u) receptors appear to stimulate D-myo-inositol 1,4,5-trisphosphate production. P2u-receptor occupancy results in both homologous and heterologous desensitization; P2y-receptor occupancy elicits only homologous desensitization. Both receptors stimulate phosphatidylcholine hydrolysis via phospholipase C activation. However, AA release appears to result from phospholipid deacylation by phospholipase A2 activation, rather than from alternate pathways that may include PLC activation. These results demonstrate for the first time that two subtypes of P2-purinergic receptors, P2u and P2y receptors, coexist on a single renal epithelium cell type and that these two receptor subtypes can promote AA release, probably via activation of PLA2.

5-Oxo-eicosanoids and Hematopoietic Cytokines Cooperate in Stimulating Neutrophil Function and the Mitogen-activated Protein Kinase Pathway

The newly defined eicosatetraenoates (ETEs), 5-oxoETE and 5-oxo-15(OH)-ETE, share structural motifs, synthetic origins, and bioactions with leukotriene B4 (LTB4). All three eicosanoids stimulate Ca2+ transients and chemotaxis in human neutrophils (PMN). However, unlike LTB4, 5-oxoETE and 5-oxo-15(OH)-ETE alone cause little degranulation and no superoxide anion production. However, we show herein that, in PMN pretreated with granulocyte-macrophage or granulocyte colony-stimulating factor (GM-CSF or G-CSF), the oxoETEs become potent activators of the last responses. The oxoETEs also induce translocation of secretory vesicles from the cytosol to the plasmalemma, an effect not requiring cytokine priming. To study the mechanism of PMN activation in response to the eicosanoids, we examined the activation of mitogen-activated protein kinase (MAPK) and cytosolic phospholipase A2 (cPLA2). PMN expressed three proteins (40, 42, and 44 kDa) that reacted with anti-MAPK antibodies. The oxoETEs, LTB4, GM-CSF, and G-CSF all stimulated PMN to activate the MAPKs and cPLA2, as defined by shifts in these proteins' electrophoretic mobility and tyrosine phosphorylation of the MAPKs. However, the speed and duration of the MAPK response varied markedly depending on the stimulus. 5-OxoETE caused a very rapid and transient activation of MAPK. In contrast, the response to the cytokines was rather slow and persistent. PMN pretreated with GM-CSF demonstrated a dramatic increase in the extent of MAPK tyrosine phosphorylation and electrophoretic mobility shift in response to 5-oxoETE. Similarly, 5-oxoETE induced PMN to release some preincorporated [14C]arachidonic acid, while GM-CSF greatly enhanced the extent of this release. Thus, the synergism exhibited by these agents is prominent at the level of MAPK stimulation and phospholipid deacylation. Pertussis toxin, but not Ca2+ depletion, inhibited MAPK responses to 5-oxoETE and LTB4, indicating that responses to both agents are coupled through G proteins but not dependent upon Ca2+ transients. 15-OxoETE and 15(OH)-ETE were inactive while 5-oxo-15(OH)-ETE and 5(OH)-ETE had 3- and 10-fold less potency than 5-oxoETE, indicating a rather strict structural specificity for the 5-keto group. LY 255283, a LTB4 antagonist, blocked the responses to LTB4 but not to 5-oxoETE. Therefore, the oxoETEs do not appear to operate through the LTB4 receptor. In summary, the oxoETEs are potent activators of PMN that share some but not all activities with LTB4. The response to the oxoETEs is greatly enhanced by pretreatment with cytokines, indicating that combinations of these mediators may be very important in the pathogenesis of inflammation.

Curcumin, a major component of food spice turmeric ( Curcuma longa) inhibits aggregation and alters eicosanoid metabolism in human blood platelets

In traditional medicine, Ayurveda, several spices and herbs are held to possess medicinal properties. Earlier we have reported that extracts from several spices, including turmeric, inhibit platelet aggregation and modulate eicosanoid biosynthesis. Due to their eicosanoid-modulating property, it was suggested that the spices may serve to provide clues to drugs directed to arachidonic acid (AA) pathway enzymes as pharmacological targets. Curcumin, a major component of turmeric, inhibited platelet aggregation induced by arachidonate, adrenaline and collagen. This compound inhibited thromboxane B 2 (TXB 2) production from exogenous [ 14C] arachidonate in washed platelets with a concomitant increase in the formation of 12-lipoxygenase products. Moreover, curcumin inhibited the incorporation of [ 14C]AA into platelet phospholipids and inhibited the deacylation of AA-labelled phospholipids (liberation of free AA) on stimulation with calcium ionophore A23187. Curcumin's anti-inflammatory property may, in part, be explained by its effects on eicosanoid biosynthesis.