Arachidonic Acid-containing Phospholipids Research Articles

Association of Altered Plasma Lipidome with Disease Severity in COVID-19 Patients.

The severity of COVID-19 is linked to an imbalanced immune response. The dysregulated metabolism of small molecules and bioactive lipids has also been associated with disease severity. To promote understanding of the disease biochemistry and provide targets for intervention, we applied a range of LC-MS platforms to analyze over 100 plasma samples from patients with varying COVID-19 severity and with detailed clinical information on inflammatory responses (>30 immune markers). This is the third publication in a series, and it reports the results of comprehensive lipidome profiling using targeted LC-MS/MS. We identified 1076 lipid features across 25 subclasses, including glycerophospholipids, sterols, glycerolipids, and sphingolipids, among which 531 lipid features were dramatically changed in the plasma of intensive care unit (ICU) patients compared to patients in the ward. Patients in the ICU showed 1.3-57-fold increases in ceramides, (lyso-)glycerophospholipids, diglycerides, triglycerides, and plasmagen phosphoethanolamines, and 1.3-2-fold lower levels of a cyclic lysophosphatidic acid, sphingosine-1-phosphates, sphingomyelins, arachidonic acid-containing phospholipids, lactosylceramide, and cholesterol esters compared to patients in the ward. Specifically, phosphatidylinositols (PIs) showed strong fatty acid saturation-dependent behavior, with saturated fatty acid (SFA)- and monosaturated fatty acid (MUFA)-derived PI decreasing and polystaturated (PUFA)-derived PI increasing. We also found ~4000 significant Spearman correlations between lipids and multiple clinical markers of immune response with |R| ≥ 0.35 and FDR corrected Q < 0.05. Except for lysophosphatidic acid, lysophospholipids were positively associated with the CD4 fraction of T cells, and the cytokines IL-8 and IL-18. In contrast, sphingosine-1-phosphates were negatively correlated with innate immune markers such as CRP and IL-6. Further indications of metabolic changes in moderate COVID-19 disease were demonstrated in recovering ward patients compared to those at the start of hospitalization, where 99 lipid species were altered (6 increased by 30-62%; 93 decreased by 1.3-2.8-fold). Overall, these findings support and expand on early reports that dysregulated lipid metabolism is involved in COVID-19.

Characterization of chemoresistant human non-small cell lung cancer cells by metabolic and lipidomic profiling.

Lung cancer is one of the most malignant cancers and the leading cause of cancer-related deaths worldwide, while acquired chemoresistance would represent a major problem in the treatment of non-small cell lung cancer (NSCLC) because of the reduced treatment effect and increased rates of recurrence. To establish the chemoresistant NSCLC cells, doxorubicin was treated to A549 cells over 3 months at gradually increasing concentrations from 0.03 to 0.5 µM. Real-time PCR and Western blotting were employed for investigating mRNA and protein expression of the glutathione peroxidase (GPX) protein family and multidrug resistance protein 1 (MRP1) in A549 and A549/CR cells. We also employed gas chromatography mass-spectrometry and nano electrospray ionization mass-spectrometry coupled with multivariate statistical analysis to characterize the unique metabolic and lipidomic profiles of chemoresistant NSCLC cells in order to identify potential therapeutic targets. Reactive oxygen species levels were decreased, and mRNA and protein levels of GPX2 and multidrug resistance protein 1 (MRP1) were increased in A549/CR. We identified 87 metabolites and intact lipid species in A549 and A549/CR. Among these metabolites, lactic acid, glutamic acid, glycine, proline, aspartic acid, succinic acid, and ceramide, alongside the PC to PE ratio, and arachidonic acid-containing phospholipids were suggested as characteristic features of chemoresistant NSCLC cells (A549/CR). This study reveals characteristic feature differences between drug-resistance NSCLC cells and their parental cells. We suggest potential therapeutic targets in chemoresistant NSCLC. Our results provide new insight into metabolic and lipidomic alterations in chemoresistant NSCLC. This could be used as fundamental information to develop therapeutic strategies for the treatment of chemoresistant NSCLC patients.

RB1-deficient prostate tumor growth and metastasis are vulnerable to ferroptosis induction via the E2F/ACSL4 axis.

Inactivation of the RB1 tumor suppressor gene is common in several types of therapy-resistant cancers, including metastatic castration-resistant prostate cancer, and predicts poor clinical outcomes. Effective therapeutic strategies against RB1-deficient cancers, however, remain elusive. Here we showed that RB1-loss/E2F activation sensitized cancer cells to ferroptosis, a form of regulated cell death driven by iron-dependent lipid peroxidation, by upregulating expression of ACSL4 and enriching ACSL4-dependent arachidonic acid-containing phospholipids, which are key components of ferroptosis execution. ACSL4 appeared to be a direct E2F target gene and was critical to RB1 loss-induced sensitization to ferroptosis. Importantly, using cell line-derived xenografts and genetically engineered tumor models, we demonstrated that induction of ferroptosis in vivo by JKE-1674, a highly selective and stable GPX4 inhibitor, blocked RB1-deficient prostate tumor growth and metastasis and led to improved survival of the mice. Thus, our findings uncover an RB/E2F/ACSL4 molecular axis that governs ferroptosis, and also suggest a promising approach for the treatment of RB1-deficient malignancies.

Mass spectrometry imaging identifies altered hepatic lipid signatures during experimental Leishmania donovani infection.

IntroductionSpatial analysis of lipids in inflammatory microenvironments is key to understand the pathogenesis of infectious disease. Granulomatous inflammation is a hallmark of leishmaniasis and changes in host and parasite lipid metabolism have been observed at the bulk tissue level in various infection models. Here, mass spectrometry imaging (MSI) is applied to spatially map hepatic lipid composition following infection with Leishmania donovani, an experimental mouse model of visceral leishmaniasis.MethodsLivers from naïve and L. donovani-infected C57BL/6 mice were harvested at 14- and 20-days post-infection (n=5 per time point). 12 µm transverse sections were cut and covered with norhamane, prior to lipid analysis using MALDI-MSI. MALDI-MSI was performed in negative mode on a Rapiflex (Bruker Daltonics) at 5 and 50 µm spatial resolution and data-dependent analysis (DDA) on an Orbitrap-Elite (Thermo-Scientific) at 50 µm spatial resolution for structural identification analysis of lipids.ResultsAberrant lipid abundances were observed in a heterogeneous distribution across infected mouse livers compared to naïve mouse liver. Distinctive localized correlated lipid masses were found in granulomas and surrounding parenchymal tissue. Structural identification revealed 40 different lipids common to naïve and d14/d20 infected mouse livers, whereas 15 identified lipids were only detected in infected mouse livers. For pathology-guided MSI imaging, we deduced lipids from manually annotated granulomatous and parenchyma regions of interests (ROIs), identifying 34 lipids that showed significantly different intensities between parenchyma and granulomas across all infected livers.DiscussionOur results identify specific lipids that spatially correlate to the major histopathological feature of Leishmania donovani infection in the liver, viz. hepatic granulomas. In addition, we identified a three-fold increase in the number of unique phosphatidylglycerols (PGs) in infected liver tissue and provide direct evidence that arachidonic acid-containing phospholipids are localized with hepatic granulomas. These phospholipids may serve as important precursors for downstream oxylipin generation with consequences for the regulation of the inflammatory cascade. This study provides the first description of the use of MSI to define spatial-temporal lipid changes at local sites of infection induced by Leishmania donovani in mice.

アラキドン酸含有リン脂質が担う神経障害性疼痛における役割

アラキドン酸含有リン脂質が担う神経障害性疼痛における役割

Acyl-CoA synthetase 6 enriches seminiferous tubules with the ω-3 fatty acid docosahexaenoic acid and is required for male fertility in the mouse

Docosahexaenoic acid (DHA) is an ω-3 dietary-derived polyunsaturated fatty acid of marine origin enriched in testes and necessary for normal fertility, yet the mechanisms regulating the enrichment of DHA in the testes remain unclear. Long-chain ACSL6 (acyl-CoA synthetase isoform 6) activates fatty acids for cellular anabolic and catabolic metabolism by ligating a CoA to a fatty acid, is highly expressed in testes, and has high preference for DHA. Here, we investigated the role of ACSL6 for DHA enrichment in the testes and its requirement for male fertility. Acsl6-/- males were severely subfertile with smaller testes, reduced cauda epididymal sperm counts, germ cell loss, and disorganization of the seminiferous epithelium. Total fatty acid profiling of Acsl6-/- testes revealed reduced DHA and increased ω-6 arachidonic acid, a fatty acid profile also reflected in phospholipid composition. Strikingly, lipid imaging demonstrated spatial redistribution of phospholipids in Acsl6-/- testes. Arachidonic acid-containing phospholipids were predominantly interstitial in control testes but diffusely localized across Acsl6-/- testes. In control testes, DHA-containing phospholipids were predominantly within seminiferous tubules, which contain Sertoli cells and spermatogenic cells but relocalized to the interstitium in Acsl6-/- testes. Taken together, these data demonstrate that ACSL6 is an initial driving force for germ cell DHA enrichment and is required for normal spermatogenesis and male fertility.

Lysophosphatidylcholine Acyltransferase 3 Is the Key Enzyme for Incorporating Arachidonic Acid into Glycerophospholipids during Adipocyte Differentiation

Cellular membranes contain glycerophospholipids, which have important structural and functional roles in cells. Glycerophospholipids are first formed in the de novo pathway (Kennedy pathway) and are matured in the remodeling pathway (Lands’ cycle). Recently, lysophospholipid acyltransferases functioning in Lands’ cycle were identified and characterized. Several enzymes involved in glycerophospholipid biosynthesis have been reported to have important roles in adipocytes. However, the role of Lands’ cycle in adipogenesis has not yet been reported. Using C3H10T1/2, a cell line capable of differentiating to adipocyte-like cells in vitro, changes of lysophospholipid acyltransferase activities were investigated. Lysophosphatidylcholine acyltransferase (LPCAT), lysophosphatidylethanolamine acyltransferase (LPEAT) and lysophosphatidylserine acyltransferase (LPSAT) activities were enhanced, especially with 18:2-CoA and 20:4-CoA as donors. Correspondingly, mRNA expression of LPCAT3, which possesses LPCAT, LPEAT and LPSAT activities with high specificity for 18:2- and 20:4-CoA, was upregulated during adipogenesis. Analysis of acyl-chain compositions of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS) showed a change in their profiles between preadipocytes and adipocytes, including an increase in the percentage of arachidonic acid-containing phospholipids. These changes are consistent with the activities of LPCAT3. Therefore, it is possible that enhanced phospholipid remodeling by LPCAT3 may be associated with adipocyte differentiation.

Markers of Monocyte Activation Revealed by Lipidomic Profiling of Arachidonic Acid-Containing Phospholipids

Stimulated human monocytes undergo an intense trafficking of arachidonic acid (AA) among glycerophospholipidclasses. Using HPLC coupled to electrospray ionization mass spectrometry, we have characterized changes in the levels of AA-containing phospholipid species in human monocytes. In resting cells, AA was found esterified into various molecular species of phosphatidylinositol (PI), choline glycerophospholipids (PCs), and ethanolamine glycerophospholipids (PEs). All major AA-containing PC and PI molecular species decreased in zymosan-stimulated cells; however, no PE molecular species was found to decrease. In contrast, the levels of three AA-containing species increased in zymosan-activated cells compared with resting cells: 1,2-diarachidonyl-glycero-3-phosphoinositol [PI(20:4/20:4)]; 1,2-diarachidonyl-glycero-3-phosphocholine [PC(20:4/20:4)]; and 1-palmitoleoyl-2-arachidonyl-glycero-3-phosphoethanolamine [PE(16:1/20:4)]. PI(20:4/20:4) and PC(20:4/20:4), but not PE(16:1/20:4), also significantly increased when platelet-activating factor or PMA were used instead of zymosan to stimulate the monocytes. Analysis of the pathways involved in the synthesis of these three lipids suggest that PI(20:4/20:4) and PC(20:4/20:4) were produced in a deacylation/reacylation pathway via acyl-CoA synthetase-dependent reactions, whereas PE(16:1/20:4) was generated via a CoA-independent transacylation reaction. Collectively, our results define the increases in PI(20:4/20:4) and PC(20:4/20:4) as lipid metabolic markers of human monocyte activation and establish lipidomics as a powerful tool for cell typing under various experimental conditions.

Docosahexaenoic acid-induced changes in phospholipids in cortex of young and aged rats: A lipidomic analysis

The age-related decline in cognitive function has been associated with biochemical changes that can be attenuated following n-3 polyunsaturated fatty acid treatment. Dietary supplementation with docosahexaenoic acid (DHA) has been shown to reverse age-related changes in synaptic function. Here, lipidomic analyses were undertaken to examine changes in lipid classes and phospholipid species in cortical tissue of young (2–4 months) and aged (20–22 months), control- and DHA-treated (10 mg daily) rats following treatment for 8 weeks, aiming to explore the mechanism of DHA action. Dietary supplementation normalised the age-related decrease in unsaturation index, reduced the levels of arachidonic acid-containing phospholipids in both young and aged animals, and gave rise to production of new phosphatidylserine and phosphatidylinositol species. These findings suggest that DHA may mediate some of its effects through alterations in the membrane lipid composition that can consequently affect the production of pro-inflammatory mediators and signalling molecular species.

Differential Roles for Cytosolic and Microsomal Ca2+-Independent Phospholipase A2in Cell Growth and Maintenance of Phospholipids

Physiological roles of microsomal (iPLA(2)gamma) and cytosolic (iPLA(2)beta)Ca(2+)-independent phospholipase A(2) were determined in two different epithelial cell models. R- and S-enantiomers of the iPLA(2) inhibitor bromoenol lactone (BEL) were isolated and shown to selectively inhibit iPLA(2gamma) and iPLA(2beta), respectively. The effect of these enantiomers on cell growth was assessed in human embryonic kidney 293 and Caki-1 cells using 3-(4-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT). S-BEL (0-5.0 microM) decreased MTT staining 35% after 24 h compared with control cells, whereas treatment with either R-BEL or R/S-BEL induced 15% decreases. Neither R-BEL nor S-BEL induced cell death as determined by annexin V and propidium iodide staining. Transfection of cells with iPLA(2)beta siRNA reduced MTT staining approximately 35%, whereas transfection of cells with iPLA(2)gamma siRNA only decreased MTT staining 10 to 15% compared with control cells. The effect of iPLA(2)beta and iPLA(2)gamma siRNA on cell number and protein was also determined, and iPLA(2)beta siRNA decreased cell number and protein 25% compared with control cells. In contrast, iPLA(2)gamma siRNA decreased cell number, but not cellular protein, compared with control cells. Selective inhibition of iPLA(2)beta, but not iPLA(2)gamma, decreased several arachidonic acid-containing phospholipids, including 16:1-20:4, 16:0-20:4, 18:1-20:4, and 18:0-20:4 phosphatidylcholine, showing that the ability of iPLA(2)beta inhibitors to decrease cell growth correlates with their ability to decrease arachidonic acid-containing phospholipids. These data show that iPLA(2)beta inhibition results in greater decreases in cell growth and proliferation than iPLA(2)gamma, identifies specific phospholipids whose expressions are differentially regulated by iPLA(2)beta and iPLA(2)gamma, and suggests novel roles for iPLA(2)beta in cell growth.

Pathway-oriented profiling of lipid mediators in macrophages

Macrophages produce various kinds of lipid mediators including eicosanoids and platelet-activating factor. Since they are produced from common precursors, arachidonic acid-containing phospholipids, regulations of metabolic pathways underlie the patterning of lipid mediator production. Here, we report a pathway-oriented profiling strategy of lipid mediators by a newly developed multiplex quantification system. We profiled mouse peritoneal macrophages in different activation states. The analysis of kinetics revealed the differences in the production time course of various lipid mediators, which also differed by the macrophage types. Scatterplot matrix analysis of the inhibitor study revealed correlations of lipid mediator species. The changes of these correlations provided estimates on the effects of lipopolysaccharide priming. We also found a highly linked production of 11-hydroxyeicosatetraenoic acid and prostaglandin E 2, implying the in vivo property of cyclooxygenase-mediated 11-hydroxyeicosatetraenoic acid production. The present approach will serve as a strategy for understanding the regulatory mechanism of lipid mediator production.

Identification of hepatic peroxisomal phospholipase A 2 and characterization of arachidonic acid-containing choline glycerophospholipids in hepatic peroxisomes

Recently, a sequence encoding a novel mammalian calcium-independent phospholipase A 2 (iPLA 2γ) was identified in the human genome and subsequently cloned and expressed in Sf9 insect cells. Unexpectedly, expression studies in recombinant systems demonstrated the usage of multiple translation initiation codons resulting in different polypeptides. Herein, we demonstrate that hepatic iPLA 2γ is localized to rat liver peroxisomes, possesses a molecular mass of 63 kDa and that peroxisomal membranes are highly enriched in arachidonic acid-containing phospholipids. Collectively, these results provide the first demonstration of iPLA 2γ in mammalian tissue and suggest the possibility that iPLA 2γ can contribute to lipid second messenger generation by hydrolysis of peroxisomal arachidonic acid-containing phospholipids.

Oxidized LDLs influence thrombotic response and cyclooxygenase 2

Oxidative modification of low-density lipoproteins (LDLs) plays a key role in the development of atherosclerosis and the onset of coronary artery disease. LDL oxidation alters the antithrombotic balance of human endothelial cells inducing surface tissue factor (TF) pathway activity, which results in enhanced fibrin deposition. Fibrinolysis, which is strictly regulated by plasminogen activator inhibitor-1 (PAL-1) and tissue-type plasminogen activator (tPA). Is also dysregulated by LDL oxidation with a net increase in the inhibitory rate. Oxidized LDLs (oxLDLs) also affect many aspects of macrophage function linked to the inflammatory response of these cells, In particular, oxLDLs downregulate inducible cyclooxigenase (Cox-2) in human monocyte-derived macrophages exposed to bacterial lipopolysaccharide. This observation may support the hypothesis that, within atheromata, the transformation macrophages into foam cells results in the attenuation of the inflammatory response, thus contributing to the progression of athrogenesis. Among lipid constituents of oxLDLs, Ox-PAPC, a mixture of oxidized arachidonic acid-containing phospholipids, prevents Cox-2 expression, suggesting that it could be considered responsible for the biological activity of oxLDLs.

Regulation of Cytosolic Phospholipase A2 in Hen Granulosa Cells by Transforming Growth Factors at Different Stages of Follicular Development1

Phospholipase A2 (PLA2) is a key enzyme involved in the release of arachidonic acid and subsequent production of prostaglandins (PGs), which play important roles in regulating ovarian function, including mitogenic signalling by transforming growth factor (TGF) alpha. Cytosolic PLA2 (cPLA2), a newly identified member of the PLA2 family, is distinct from the well-characterized, secreted 14-kDa group I and II PLA2 in that cPLA2 selectively cleaves arachidonic acid-containing phospholipids at the sn-2 position. Our previous studies have demonstrated that TGF alpha stimulates and TGF beta suppresses PG production in hen granulosa cells during ovarian follicular development. The present work extends these findings. A cPLA2 is present as a 100-kDa protein doublet and has a transcript size of 3.2 kilobases (kb) in hen granulosa cells from the largest (F1) and the fifth and sixth (F5-6) preovulatory follicles. Treatment of these cells with TGF alpha caused a shift from an electrophoretically fast-migrating to slow-migrating protein, a phenomenon sensitive to inhibitors of serine/threonine kinase as well as mitogen-activated protein (MAP) kinase pathways and indicative of phosphorylation and activation of cPLA2. In contrast, TGF beta suppressed cPLA2 expression, as evidenced by a marked decrease of cPLA2 mRNA abundance and protein content, but failed to prevent the mobility shift of cPLA2 induced by TGF alpha. Consistent with the influence of this growth factor on PG production observed previously and in contrast to the action of TGF alpha, the inhibition of cPLA2 by TGF beta is more pronounced in granulosa cells at the early stage of follicular development. Our results demonstrate, for the first time, the regulation of granulosa cell cPLA2 at the transcriptional and posttranscriptional levels at different stages of follicular development. We propose that the activation by TGF alpha and suppression by TGF beta of cPLA2 may be important regulatory mechanisms in the control of granulosa cell PG production and thereby the mitogenic response of the cells to the growth factors during ovarian follicular development.

Induction of heme oxygenase-1 inhibits the monocyte transmigration induced by mildly oxidized LDL.

Heme catabolic processes produce the antioxidants biliverdin and bilirubin, as well as the potent prooxidant free iron. Since these products have opposing effects on oxidative stress, it is not clear whether heme catabolism promotes or inhibits inflammatory processes, including atherosclerotic lesion formation. Heme oxygenase (HO) catalyzes the rate-limiting step of heme catabolism. We used cocultures of human aortic endothelial cells and smooth muscle cells to examine the possible role of HO in early atherosclerosis. Heme oxygenase-1 (HO-1), the inducible isoform of HO, was highly induced by mildly oxidized LDL, and augmented induction was observed with hemin pretreatment. This augmented HO-1 induction resulted in the reduction of monocyte chemotaxis in response to LDL oxidation. Conversely, inhibition of HO by a specific inhibitor, Sn-protoporphyrin IX, enhanced chemotaxis. Furthermore, pretreatment with biliverdin or bilirubin, the products of HO, reduced chemotaxis. Oxidized phospholipids in the mildly oxidized LDL appear to be responsible for HO-1 induction, since oxidized but not native arachidonic acid-containing phospholipids also induced HO-1. These results suggest that HO-1 induced by mildly oxidized LDL may protect against the induction of inflammatory responses in artery wall cells through the production of the antioxidants biliverdin and bilirubin.

Structure, function and regulation of Ca 2+-sensitive cytosolic phospholipase A2 (cPLA2)

The 85-kDa cytosolic PLA2 (cPLA2) is present in many cells and tissues and its unusual functional properties and catalytic mechanism are being elucidated. Notably, cPLA2 becomes catalytically active in the presence of free Ca 2+ concentrations as present in stimulated cells and preferentially cleaves arachidonic acid-containing phospholipids. A variety of agonists, growth factors and cytokines, as well as stressful stimuli activate cPLA2 to hydrolyze cellular phospholipids thereby liberating fatty acids and lysophospholipids and providing the precursor substrates for the biosynthesis of eicosanoids and platelet-activating factor. These products of cPLA2 contribute to inflammatory and degenerative disease states and cPLA2 is therefore an attractive target for the development of novel therapies.

Structural Identification by Mass Spectrometry of Oxidized Phospholipids in Minimally Oxidized Low Density Lipoprotein That Induce Monocyte/Endothelial Interactions and Evidence for Their Presence in Vivo

Entry of monocytes into the vessel wall is an important event in atherogenesis. Previous studies from our laboratory suggest that oxidized arachidonic acid-containing phospholipids present in mildly oxidized low density lipoproteins (MM-LDL) can activate endothelial cells to bind monocytes. In this study, biologically active oxidized arachidonic acid-containing phospholipids were produced by autoxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (Ox-PAPC) and analyzed by liquid chromatography and electrospray ionization mass spectrometry in conjuction with biochemical derivatization techniques. We have now determined the molecular structure of two of three molecules present in MM-LDL and Ox-PAPC that induce monocyte-endothelial interactions. These lipids were identified as 1-palmitoyl-2-(5-oxovaleryl)-sn-glycero-3-phosphocholine (m/z 594.3) and 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (m/z 610.2). These two molecules were produced by unambiguous total synthesis and found to be identical by analytical techniques and bioactivity assays to those present in MM-LDL and Ox-PAPC. Evidence for the importance of all three oxidized phospholipids in vivo was suggested by their presence in fatty streak lesions from cholesterol-fed rabbits and by their immunoreactivity with natural antibodies present in ApoE null mice. Overall, these studies suggest that specific oxidized derivatives of arachidonic acid-containing phospholipids may be important initiators of atherogenesis.

Pharmacology of the pyrroloimidazole, SK&F 105809—I: Inhibition of inflammatory cytokine production and of 5-lipoxygenase- and cyclooxygenase-mediated metabolism of arachidonic acid

SK&F 105809 {2-(4- methylsulfinylphenyl)-3-(4- pyridyl)-6,7- dihydro-[5H]- pyrrolo[l,2-a] imidazole} was determined to be a prodrug for the sulfide metabolite SK&F 105561 {2-(4- methyltniophenyl)-3-(4- pyridyl)-6,7- dihydro-[5H]- pyrrolo[1,2-a] imidazole} which inhibited interleukin-1 (IL-1) production in vitro and both 5-lipoxygenase (5-LO) and prostaglandin H (PGH) synthase activities in vitro and ex vivo. SK&F 105561 inhibited partially purified 5-LO with a half-maximal concentration ( ic 50) of 3 μM. This inhibition was reversible, independent of preincubation time, and dependent on the concentration of the substrate arachidonic acid. SK&F 105561 also inhibited purified PGH synthase with the potency dependent on the level of peroxidase activity. The ic 50 was 100 μM in the absence of peroxidase activity, whereas an ic 50 of 3 μM was observed in the presence of peroxidase activity. Using human monocytes, SK&F 105561 inhibited A23187-stimulated prostaglandin E 2 (PGE 2) and leukotriene B 4 (LTB 4) production with ic 50 values of 0.1 and 2 μM, respectively. In addition, IL-1 production by lipopolysaccharide-stimulated human monocytes was also inhibited ( ic 50 2 μM). Oral administration of SK&F 105809 to rats resulted in a dose-related generation of SK&F 105561 and in the inhibition of thromboxane B 2 and LTB 4 production ex vivo with a half-maximal dose ( ed 50) of 15 and 60 mg/kg, respectively. SK&F 105561 showed weak inhibitory activity on 12-lipoxygenase with an ic 50 of greater than 200 μM. Neither SK&F 105561 nor SK&F 105809 inhibited the stimulated-turnover of arachidonic acid-containing phospholipids in human monocytes or the activity of cell-free phospholipases A 2 and C. Moreover, neither SK&F 105561 nor SK&F 105809 antagonized the binding of LTB 4 or leukotriene D 4 to membrane receptors. From these results, SK&F 105561, the active principle of SK&F 105809, acts as an inhibitor of both inflammatory cytokine and eicosanoid production.

Lactating goat mammary gland cells in culture

1. Isolated mammary gland cells were cultured embedded in collagen gels or as monolayers on floating collagen gels. Under these conditions the cells were able to grow for at least 6 weeks during five passages. Growth was sustained in M199/F12 (1:1) supplemented with insulin, hydrocortisone, epidermal growth factor, tri-iodothyronine, estradiol and bovine serum albumin. 2. The cells secreted lactose into the medium in significant amounts throughout the culture period. 3. Prolactin had a slightly stimulatory effect as had fetal bovine serum on growth and protein synthesis, but none of these factors were obligatory in this respect. Insulin-like growth factor I (Somatomedin C) could replace high concentrations of insulin whereas bovine growth hormone had no detectable effect. 4. Depending on the hormone content of the medium and the age of the culture, different labelling patterns of the arachidonic acid-containing phospholipids were observed. The effect of prolactin on phosphatidyl inositol and arachidonic acid metabolism was studied.

Receptor-mediated activation of phospholipase A2 and arachidonic acid release in signal transduction

Neurotransmitters, hormones, cytokines, growth factors and other ligands transmit their specific messages by binding to receptor molecules on the cell surface. The ligand-bound receptors activate effector systems such as adenylate cyclase, cyclic nueleotide phosphodiesterase, ion channels, and phospholipases via membrane-associated transducing systems to generate second messengers. Receptors activate effector systems via signal transducing elements called GTPbinding proteins (G-proteins) which are heterotrimers composed of a-, Band y-subunits. When a receptor binds ligands, the G-protein disassociates into the aand Bysubunits [ 11. The disassociation of subunits is caused by the exchange of GTP for GDP which is bound to the a-subunit. Specific a-subunits are associated with the inhibition of adenylate cyclase and activation of cyclic GMP phosphodiesterase, voltage-sensitive ion channels, adenylate cyclase and phospholipase C. The a-subunits have endogenous GTPase activity which hydrolyses the bound GTP to GDP, resulting in the reassociation of the a and By-subunits and subsequent termination of the signal [ 11. Phospholipase A, as an effector enzyme can mediate receptor-induced arachidonic acid release regulated by an unknown G-protein [2]. Phospholipase A, hydrolyses arachidonic acid-containing phospholipids, such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserinc, phosphatidylinositides, phosphatidic acid and plasmalogcns, to generate free arachidonic acid and lysophospholipids. The arachidonic acid is further metabolized by cyclo-oxygenase to prostaglandin and thromboxanes; by lipoxygenase to leukotricnes and hydroxyeicosatetraenoic acids ( HETEs), and by epoxygenases to epoxides. Metabolites of arachidonic acid havc many important biological actions [3, 41. Our intcrest in phospholipase A, as a receptor-mediated effector enzyme stemmed from the initial observation that the chcmotactic peptide, fMetLeuPhe, liberated free arachidonic acid from neutrophils prelabeled with IT-arachidonic acid 151. A direct association between the amount o f arachidonic acid released by NetLeuPhe and the extent of chemotaxis was also demonstrated. Several pathways exist for the receptor-mediated generation o f arachidonic acid. The most direct involves coupling o f the receptor protein with phospholipasc A, activation through a G-protein of unknown character 161. Indirect routes havc been demonstrated involving the releasc of arachidonic acid from diacylglycerol by diglyceridc lipase 171. Diacylglycerol can be generated from the action of phospholipasc C on several phospholipids such as phosphatidylcholine and phosphatidylinositol. Alternatively, arachidonic acid can he released from phosphatidic acid by diglyceride lipase. Phosphatidic acid can be produced from the hydrolysis o f phospholipids by phospholipase D 181. In many studies, receptor-mediated release of arachidonic acid was found to be calcium dependent, while phospholipase C and D are partially or insensitive to calcium [9, lo].