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Ether lipid metabolism. Incorporation of O-hexadecyl ethanediol into rat brain lipids.

1-O-[1'-14C]Hexadecyl ethanediol was administered intracerebrally to myelinating rat brain, and incorporation of radioactivity into brain lipids was followed over a 48-h period: (1) O-Hexadecyl ethanediol was metabolized primarily through oxidative ether bond cleavage, and much of the label was recovered in phospholipid acyl groups. (2) Substantial amounts of radioactivity were also found in choline and ethanolamine phospholipids having an O-hexadecyloxyethyl glycerol backbone. This means that alkyl ethanediol was used in glycerol ether biosynthesis as are long-chain primary alcohols. (3) Acidic hydrolysis of the ethanolamine glycerophosphatide fraction yielded also labeled hexadecanol which may indicate desaturation of 1-O-hexadecyloxyethyl 2-acyl glycerophosphoryl ethanolamine to the plasmalogen analogue. (4) Small amounts of the substrate were oxidized to O-hexadecyl glycolic acid and incorporated into the phospholipids. The substrate did not serve as precursor of O-hexadecyl ethanediol phosphorylcholine or phosphorylethanolamine in the brain.

Animals↗

Distribution of phospholipids, fatty acids, and platelet factor 3 activity among subcellular fractions of human platelets.

As compared with other methods, our recently reported method for subcellular fractionation of human platelets improves the separation of mitochondria, alpha granules, and lysosomal enzyme activities. The relative purity of these fractions has led us to undertake the present study to compare the subcellular distribution of phospholipids, fatty acids, and platelet factor 3 (clot-promoting) activity. Two findings pertaining to distribution of phospholipids were entirely new. (1) In the alpha granule zone, plasmalogen phosphatidyl ethanolamine peaked at the expense of diacyl phosphatidyl ethanolamine. (2) The fatty acid composition of the membrane lysophosphatidyl choline suggested that it may have been formed by the action of platelet phospholipase A2 activity. The fatty acids of the membranes showed a markedly asymmetrical distribution in noncholine versus choline phospholipids. The latter held 94%, 72%, and 85%, respectively, of the total content of 16:0, 18:1, and 18:2 fatty acids, whereas 55% of the 18:0, 72% of 20:4, and 67% of higher polyenoic acids other than 20:4 were esterified to the noncholine group. The most important new information related to clot-promoting activity, which, on the basis of protein content, was highest in the membrane fractions, but on the basis of phospholipid content in the nonmembranous fractions. The discussion centers on possible explanations for this novel finding.

Blood Coagulation Factors↗

[Zellweger syndrome. Reports on two new cases].

INTRODUCTION: Zellweger syndrome, or cerebrohepatorenal syndrome, is the most serious form of the peroxisomal diseases. Clinically, it is characterised by the association between craniofacial dysmorphia and neurological disorders, together with the involvement of other organs. To perform a diagnosis it is advisable to follow a procedural protocol that begins with the quantification of very long chain fatty acids in an assortment of samples (serum, fibroblasts and mononucleate cells), plasmalogens, branched chain fatty acids in serum (phytanic and pristanic acids), polyunsaturated acids and bile salts. Studies conducted with neuroimaging, renal echography, skeletal X rays and biopsy samples of different tissues will provide us with information about the involvement of different organs. CASE REPORTS: The first case we report is that of a male who, from birth, presented a distinctive phenotype with very large fontanelles, important hypotonia, epileptic seizures and acute organic disorders that led to death at the age of seven weeks. The second case involved a new born male suffering from prenatally diagnosed heart disease and craniofacial dysmorphia with hypotonia. CONCLUSIONS: Given the scarce survival rate of patients, early diagnosis of this type of disease is crucial and, although complex, a study must be conducted so as to be able to provide genetic counselling.

Cerebral Cortex↗

[Nuclear receptor(s) regulating peroxisome genes].

Peroxisomes are becoming more and more attractive. This interest has grown up due to some key metabolic functions (i.e. very long chain fatty acid specific beta-oxidation, prostaglandins and polyamines catabolism, and first reactions in plasmalogen synthesis, as well as biliary salts) and to their strong response to peroxisome proliferators which can promote hepatocarcinogenesis in rodents. This mini-review comments recent breakthrough which is the discovery of peroxisome proliferator activated nuclear receptors (PPAR's) from the steroid receptor superfamily. Mouse PPAR and Xenopus PPAR's (alpha, beta and upsilon) activate the rat peroxisomal acyl CoA oxidase gene by recognition of specific responsive elements containing the 5'flanking "TGACCT" repeated sequence in the -560 upstream region. Such mechanism is involved in the control of peroxisomal beta-oxidation pathway.

Animals↗

Neuronal migration depends on intact peroxisomal function in brain and in extraneuronal tissues.

Functional peroxisome deficiency, as encountered in Zellweger syndrome, causes a specific impairment of neuronal migration. Although the molecular mechanisms underlying the neuronal migration defect are at present unknown, the excess of very long chain fatty acids in brain, a consequence of peroxisomalbeta-oxidation deficiency, has often been hypothesized to play a major role. The purpose of the present study was to investigate the contribution of peroxisomal dysfunction in brain as opposed to peroxisomal dysfunction in extraneuronal tissues to the migration defect. Peroxisomes were selectively reconstituted either in brain or liver of Pex5 knock-out mice, a model for Zellweger syndrome, by tissue-selective overexpression of Pex5p. We found that both rescue strains exhibited a significant correction of the neuronal migration defect despite an incomplete reconstitution of peroxisomal function in the targeted tissue. Animals with a simultaneous rescue of peroxisomes in both tissues displayed a pattern of neuronal migration indistinguishable from that of wild-type animals on the basis of cresyl violet staining and 5',3'-bromo-2'-deoxyuridine birth-dating analysis. These data suggest that peroxisomal metabolism in brain but also in extraneuronal tissues affects the normal development of the mouse neocortex. In liver-rescued mice, the improvement of the neuronal migration was not accompanied by changes in very long chain fatty acid, docosahexaenoic acid, or plasmalogen levels in brain, indicating that other metabolic factors can influence the neuronal migration process.

Animals↗

[Specificity of endometrium phospholipids and their fatty acids changes in the development of hyperplasia].

Quantitative changes in phospholipids (PL) and fatty acids content (FA) in patients with endometrium hyperplasia have been revealed. During observing endomentrium hyperplasia process we found out on the one hand reliable decrease in phosphatidylethanolamine (PEA), phosphatidylcholine (PC) content and on the other hand increase of its lysoforms. Changes in unsaturated fatty acids (PA n-6)--arachidonic (AA) content and docosahaenoic (DHA) acids content were also shown. The decrease of PC, PEA in patients with endometrium hyperplasia is interrelated with quantitative change of plasmalogenic phospholipids PL, which is too much sensitive to oxidative reactions and at the same time able to protect sells against oxidative stress. Considerable decrease in plasmenyl PEA content on the one side and plasmenyl PC content stability on the other was revealed in patients with endometrium hyperplasia. Above mentioned lipid metabolic derangements of the endometrium are specific with respect to different classes of phospholipids and their fatty acids composition.

Animals↗

Roles of unsaturated fatty acids (especially omega-3 fatty acids) in the brain at various ages and during ageing.

Among various organs, in the brain, the fatty acids most extensively studied are omega-3 fatty acids. Alpha-linolenic acid (18:3omega3) deficiency alters the structure and function of membranes and induces minor cerebral dysfunctions, as demonstrated in animal models and subsequently in human infants. Even though the brain is materially an organ like any other, that is to say elaborated from substances present in the diet (sometimes exclusively), for long it was not accepted that food can have an influence on brain structure, and thus on its function. Lipids, and especially omega-3 fatty acids, provided the first coherent experimental demonstration of the effect of diet (nutrients) on the structure and function of the brain. In fact the brain, after adipose tissue, is the organ richest in lipids, whose only role is to participate in membrane structure. First it was shown that the differentiation and functioning of cultured brain cells requires not only alpha-linolenic acid (the major component of the omega-3, omega3 family), but also the very long omega-3 and omega-6 carbon chains (1). It was then demonstrated that alpha-linolenic acid deficiency alters the course of brain development, perturbs the composition and physicochemical properties of brain cell membranes, neurones, oligodendrocytes, and astrocytes (2). This leads to physicochemical modifications, induces biochemical and physiological perturbations, and results in neurosensory and behavioural upset (3). Consequently, the nature of polyunsaturated fatty acids (in particular omega-3) present in formula milks for infants (premature and term) conditions the visual and cerebral abilities, including intellectual. Moreover, dietary omega-3 fatty acids are certainly involved in the prevention of some aspects of cardiovascular disease (including at the level of cerebral vascularization), and in some neuropsychiatric disorders, particularly depression, as well as in dementia, notably Alzheimer's disease. Recent results have shown that dietary alpha-linolenic acid deficiency induces more marked abnormalities in certain cerebral structures than in others, as the frontal cortex and pituitary gland are more severely affected. These selective lesions are accompanied by behavioural disorders more particularly affecting certain tests (habituation, adaptation to new situations). Biochemical and behavioural abnormalities are partially reversed by a dietary phospholipid supplement, especially omega-3-rich egg yolk extracts or pig brain. A dose-effect study showed that animal phospholipids are more effective than plant phospholipids to reverse the consequences of alpha-linolenic acid deficiency, partly because they provide very long preformed chains. Alpha-linolenic acid deficiency decreases the perception of pleasure, by slightly altering the efficacy of sensory organs and by affecting certain cerebral structures. Age-related impairment of hearing, vision and smell is due to both decreased efficacy of the parts of the brain concerned and disorders of sensory receptors, particularly of the inner ear or retina. For example, a given level of perception of a sweet taste requires a larger quantity of sugar in subjects with alpha-linolenic acid deficiency. In view of occidental eating habits, as omega-6 fatty acid deficiency has never been observed, its impact on the brain has not been studied. In contrast, omega-9 fatty acid deficiency, specifically oleic acid deficiency, induces a reduction of this fatty acid in many tissues, except the brain (but the sciatic nerve is affected). This fatty acid is therefore not synthesized in sufficient quantities, at least during pregnancy-lactation, implying a need for dietary intake. It must be remembered that organization of the neurons is almost complete several weeks before birth, and that these neurons remain for the subject's life time. Consequently, any disturbance of these neurons, an alteration of their connections, and impaired turnover of their constituents at any stage of life, will tend to accelerate ageing. The enzymatic activities of sytivities of synthesis of long-chain polyunsaturated fatty acids from linoleic and alpha-linolenic acids are very limited in the brain: this organ therefore depends on an exogenous supply. Consequently, fatty acids that are essential for the brain are arachidonic acid and cervonic acid, derived from the diet, unless they are synthesized by the liver from linoleic acid and alpha-linolenic acid. The age-related reduction of hepatic desaturase activities (which participate in the synthesis of long chains, together with elongases) can impair turnover of cerebral membranes. In many structures, especially in the frontal cortex, a reduction of cervonic and arachidonic acids is observed during ageing, predominantly associated with a reduction of phosphatidylethanolamines (mainly in the form of plasmalogens). Peroxisomal oxidation of polyunsaturated fatty acids decreases in the brain during ageing, participating in decreased turnover of membrane fatty acids, which are also less effectively protected against peroxidation by free radicals.

Aged↗

Peroxisomal abnormality in fibroblasts from involved skin of CHILD syndrome. Case study and review of peroxisomal disorders in relation to skin disease.

BACKGROUND AND DESIGN: Peroxisomal deficiency has been described in a number of syndromes characterized by chondrodysplasia punctata, including the Conradi-Hünermann (C-H) syndrome. Because of overlapping clinical features of X-chromosome inheritance, ichthyosis, and limb-reduction defects in C-H and CHILD (congenital hemidysplasia with ichthyosiform erythroderma and limb defects) syndromes, we examined peroxisomal content using diaminobenzidine cytochemistry and peroxisomal functions in fibroblasts from involved vs uninvolved skin of CHILD syndrome. RESULTS: Fibroblasts from involved skin of a patient with CHILD syndrome accumulated cytoplasmic lipid, visualized with the fluorescent probe, nile-red. Ultrastructurally, fibroblasts of involved skin of CHILD syndrome accumulated lamellated membrane and vacuolar structures. By diaminobenzidine ultracytochemistry, fewer peroxisomes were present. Moreover, the activities of two peroxisomal enzymes, catalase and dihydroxyacetone phosphate acyltransferase, were decreased (approximately 30% of normal). However, peroxisomal oxidation of very-long-chain and branched-chain fatty acids was preserved. Moreover, plasma very-long-chain fatty acids, plasma phytanic acid, and erythrocyte plasmalogen content were normal. CONCLUSIONS: The CHILD, C-H, and rhizomelic chondrodysplasia punctata syndromes are all characterized by ichthyosis, chondrodysplasia punctata, and limb defects, as well as peroxisomal deficiency. Thus, these syndromes may be related pathogenically. Because peroxisomes are involved in prostaglandin metabolism, peroxisomal deficiency may directly contribute to the previously reported alterations in prostaglandin metabolism in fibroblasts of involved skin of fibroblasts.

Acyltransferases↗

Substrate specificity of neutral phospholipase D from rat brain studied by selective labeling of endogenous synaptic membrane phospholipids in vitro.

We have designed a novel approach for studying the specificity of neutral phospholipase D from rat brain synaptic plasma membranes for endogenous phospholipid substrates in native membranes. A procedure was established that provides synaptic membranes labeled in selected phospholipids. This labeling procedure exploits the presence of endogenous acyl-coenzyme A synthetase and acyl-coenzyme A:lysophospholipid acyltransferase in synaptosomes for acylating various lysophospholipid acceptors with radioactive fatty acid. With [3H]arachidonate for acylation and optimal concentrations of the respective lysophospholipids, membranes were labeled in either of the following phospholipids: phosphatidylcholine (93% of total label in phospholipids), 1-O-alkyl-phosphatidylcholine (87%), phosphatidylinositol (90%), phosphatidylethanolamine (85%), phosphatidylethanolamine-plasmalogen (81%) or phosphatidylserine (59%). These membranes were employed to study the substrate specificity of the neutral, oleate-activated rat brain phospholipase D. This phospholipase exhibited almost absolute specificity for the choline-phospholipids phosphatidylcholine and 1-O-alkyl-phosphatidylcholine: 0.34% of the former labeled substrate were transphosphatidylated to phosphatidylpropanol during the assay and 0.28% of the latter. Activity toward other phospholipids was barely detectable and could largely be accounted for by utilization of residual labeled phosphatidylcholine present in those preparations. The phospholipase D exhibited some preference for fatty acids in the C-2 position of phosphatidylcholine in the following order: 2-oleoyl-phosphatidylcholine (0.67% of this labeled phosphatidylcholine were converted to phosphatidylpropanol), 2-myristoyl-phosphatidylcholine (0.60%), 2-palmitoyl-phosphatidylcholine (0.46%) and 2-arachidonoyl-phosphatidylcholine (0.34%). The present approach of labeling membrane phospholipids in vitro could be useful in studies of phospholipase specificity as an alternative to the use of sonicated vesicles or mixed detergent-phospholipid micellar systems.

1-Acylglycerophosphocholine O-Acyltransferase↗

Contribution of fetal MR imaging in the prenatal diagnosis of Zellweger syndrome.

Zellweger syndrome (ZS), or cerebrohepatorenal syndrome, was the first described peroxisomal biogenesis disorder. It represents the most severe phenotype, and some of its multiple congenital anomalies can manifest prenatally. Fetal hypokinesia, renal hyperechogenicity, and cerebral ventricular enlargement are the most common reported fetal features. Single and/or late detectable manifestations account for most of the difficulties of prenatal diagnosis, as well as the limitations of ultrasonography itself. Prenatal diagnosis, however, can be achieved through (1) assays of concentrations of peroxisomal metabolites (very-long-chain fatty acids, bile acids, intermediates, plasmalogens), (2) activities of peroxisomal enzymes (dihydroacetone-phosphate acyltransferase), or (3) molecular screening techniques, if available. We report on the contribution of MR imaging to the diagnosis of ZS in 2 unrelated fetuses. MR imaging was performed in the third trimester because of cerebral ventricular enlargement diagnosed on routine sonography examinations. In both cases, MR imaging revealed ZS-characteristic abnormal cortical gyral patterns, impaired myelination, and cerebral periventricular pseudocysts. In addition, MR imaging revealed renal microcysts and hepatosplenomegaly in one case. The high level of resolution of MR imaging, which allows analysis of cerebral gyration and myelination, facilitates the prenatal diagnosis of complex polymalformative syndromes such as ZS.

Abortion, Eugenic↗

[Phospholipid composition of synaptosomes from the brains of normal rabbits and rabbits poisoned with phospho-organic cholinesterase inhibitors].

The authors studied quantitative and qualitative composition of phospholipids isolated by fractionation in the sucrose density gradient. The diacyl-aminophospholipid (APL) content of synaptosomes was lower than that of the brain homogenates. On the other hand, the plasmologen APL, phosphoinositide and phosphatidylcholine content was greater in the synaptosomes. Strong excitation of the CNS induced with organo phosphorus inhibitors resulted in a 25% decrease of diacyl APL contnet and a 17% elevation of plasmalogen APL content in the whole brain homogenate. The phospholipid composition of synaptosomes remained unchanged, however.

Animals↗

Biochemical perturbations of BW 91Y (3-deazaadenosine) on human neutrophil chemotactic potential and lipid metabolism.

The studies reported here were designed to examine the chemotactic potential, arachidonic acid (AA) metabolism and phospholipid transmethylation in peripheral blood neutrophils from patients with rheumatoid arthritis (RA): a) prior to treatment with BW 91Y (3-deazaadenosine), b) after 4 weeks when half the patients were on active medication and half were on placebo, and c) after 4 weeks at which time all patients were on active medication. The authors demonstrate that BW 91Y in vitro at 600 pg/ml caused a decrease in chemotactic potential as measured by the leading front (LF) assay in neutrophils from both normal volunteers (p less than 0.025) and RA patients. They also demonstrate that BW 91Y caused a significant increase in production of [3H]LTB4 (LTB = leukotriene B) in ionophore-stimulated neutrophils from both normal (p less than 0.025) and RA patients (p less than 0.050) as compared to initial values. BW 91Y caused decreased incorporation and percent distribution of [3H]AA into phosphatidylcholine (PC), with a resultant increase in percent distribution into phosphatidylethanolamine (PE). There was also an increased release of [3H]AA from the PE fraction in BW 91Y-treated cells in response to ionophore stimulation. BW 91Y was found to exhibit a dose-dependent (10(-7) to 10(-4) g/ml) inhibition of the uptake and incorporation of L-[methyl-3H]methionine into the cellular lipids, while at low doses (10(-9) to 10(-5) g/ml) it stimulated the significant uptake and incorporation of [methyl-14C]choline chloride into PC. Although the total cellular content and percent composition of PC remained unchanged, it was found that BW 91Y caused a slight decrease in PC plasmalogens and an apparent increase in the 1,2-diacyl-glycerophosphatidylcholine (-GPC). BW 91Y was found, however, to have no effect on the amount or stimulated metabolism of the ether-linked 1-O-alkyl-2-acyl-GPC. As further evidence for this, the authors demonstrate that BW 91Y has no effect on the ionophore-stimulated production of [14C]acetate-labelled 1-O-alkyl-2-acetyl-GPC, or [14C]PAF.

Arachidonic Acids↗

Incorporation and distribution of epoxyeicosatrienoic acids into cellular phospholipids.

The different regioisomers of epoxyeicosatrienoic acids derived from cytochrome P-450 monooxygenase are readily esterified into phospholipids of mastocytoma cells. Incorporation of 14,15-epoxyeicosatrienoic acid was concentration-dependent, with Km = 1.1 microM and Vmax = 36 pmol/min/10(7) cells. Half-maximal incorporation occurred in 30 min, reaching a steady-state concentration of 470 pmol/10(6) cells. This was slightly lower than the values for arachidonic acid (665 pmol/10(6) cells) or 5-hydroxyeicosatetraenoic acid (554 pmol/10(6) cells). The distribution of 14,15-epoxyeicosatrienoic acid was preferential in the order phosphatidylethanolamine greater than phosphatidylcholine greater than phosphatidylinositol greater than phosphatidyl serine much greater than neutral lipids plus fatty acids. This contrasted with 5(S)-hydroxyeicosatetraenoic acid, which was distributed primarily into phosphatidylcholine. Fast atom bombardment/tandem mass spectrometry facilitated identification of molecular species containing epoxyeicosatrienoic acids without relying on radioisotopes. Phosphatidylethanolamine plasmalogens with 16:1 or 18:2 at the sn-1 position, or an 18:0 acyl group, and phosphatidylcholine with 16:0 alkyl ether or an acyl group at the sn-1 position incorporated all possible epoxyeicosatrienoic acid regioisomers. Under basal conditions, cells eliminated 14,15-cis-epoxyeicosatrienoic acid slowly with a half-life of 34.9 +/- 7 h. Cells stimulated with calcium ionophore A23187 eliminated 14,15-epoxyeicosatrienoic acid rapidly. It was notable that its rate of release from phosphatidylcholine and phosphatidylinositol exceeded that for arachidonic acid. A coenzyme A-independent transacylase also catalyzed the transfer of epoxyeicosatrienoic acids from mastocytoma cell membranes into 1-palmitoyl-2-lysophosphatidylcholine. The cellular incorporation, release, and distribution of epoxyeicosatrienoic acids is distinctive and contrasts with most other eicosanoids, suggesting that these compounds may have both autocoid and nonautocoid functions.

8,11,14-Eicosatrienoic Acid↗

Incorporation of D-[3-3H, U-14C] glucose into glycerolipid via acyl dihydroxyacetone phosphate untransformed and viral-transformed BHK-21-c13 fibroblasts.

Untransformed BHK-21-c13 fibroblasts as well as 4 polyoma-transformed strains were incubated with D-[U-14C,3-3H]glucose. This substrate generates intracellular labeled glycerol, and also [4-3H]NADPH via the phosphogluconate oxidative pathway. The latter selectively transfers hydrogen to C-2 of glycerol in glycerolipid via the acyl dihydroxyacetone phosphate pathway. After incubation, the distribution of radioactivity and the ratios of 3H/14C at the three positions of recovered glycerol were determined in sn-glycerol 3-phosphate, saponifiable glycerolipids, alkyl ether glycerolipids, and plasmalogens. In each of the cell types examined, 3H in the sn-1 position of glycerol in the recovered ether-containing glycerolipids was negligible, yet this position contained most of the recovered 3H in sn-glycerol 3-phosphate and saponifiable glycerolipids. The 3H/14C ratio in position 2 of glycerol, measured at various incubation times, was from 5- to 200-fold greater in the saponifiable glycerolipids than in free sn-glycerol 3-phosphate. The ratio in position 2 of ether-containing glycerolipids was the same or greater than that in the saponifiable glycerolipids in all of the cell types employed. A similar pattern in the 3H/14C ratio was observed when BHK-21-c13 cells were incubated with D-[U-14C,1-3H]glucose. These observations demonstrate significant participation of the acyl dihydroxyacetone phosphate pathway in glycerolipid synthesis in BHK cells.

Cell Line↗

[Alkoxyl lipids--relics of the organic world: their formation, biotransformation and evolution].

The data on discovery, biosynthesis and distribution of alcoxylipids in different kingdoms of the organic world, their physiologic role and biologic action are given. A conception of possible abiotic synthesis of alcoxylipids on the early stages of the Earth evolution and the ways of their biotransformation is considered. The first biologic membranes are suggested to contain primarily alcoxylipids. During the appearing of photosynthetic mechanism and its improving the alcoxylipids of biomembranes oxidized in plasmalogens and then into diacyl lipids. The possibilities to use alcoxylipids as chemical makers of biomembranes for chemotaxonomy are under discussion.

Alcohols↗

[Change in the lipid composition of the inner mitochondrial membranes in rat organs during adaptation to heat].

Under conditions of adaptation to heating lipid composition in mitochondrial membranes of rat inner tissues was altered as follows: an increase in relative concentration of plasmalogenous forms of phospholipids (kidney, heart) and in content of saturated fatty acids (liver tissue), a decrease in the index of fatty acids unsaturation and in the ratio of fatty acids omega-3/omega-6. The alterations observed enabled the membranes to keep sufficient amount of liquidity essential for functional activity of mitochondria in heating.

Adaptation, Physiological↗

Suicide inhibition of canine myocardial cytosolic calcium-independent phospholipase A2. Mechanism-based discrimination between calcium-dependent and -independent phospholipases A2.

The majority of phospholipase A2 activity in myocardium is calcium-independent and selective for hydrolysis of plasmalogen substrate (Wolf, R. A., and Gross, R. W. (1985) J. Biol. Chem. 260, 7295-7303; Hazen, S. L., Stuppy, R. J., and Gross, R. W. (1990) J. Biol. Chem. 265, 10622-10630). Accordingly, identification of an inhibitor which selectively targets calcium-independent phospholipases A2 would facilitate elucidation of the biologic significance of this class of intracellular phospholipases. We now report that the haloenol lactone, (E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one (Compound 1), is a potent, irreversible, mechanism-based inhibitor of myocardial calcium-independent phospholipase A2 which is greater than 1000-fold specific for inhibition of myocardial calcium-independent phospholipase A2 in comparisons with multiple calcium-dependent phospholipases A2. Mechanism-based inhibition of myocardial cytosolic calcium-independent phospholipase A2 by Compound 1 was established by demonstrating: 1) time-dependent irreversible inactivation; 2) covalent binding of [3H]Compound 1 to the purified phospholipase A2; 3) ablation of covalent binding of [3H]Compound 1 after chemical inactivation of phospholipase A2 enzymic activity; 4) identical inhibition of myocardial phospholipase A2 by Compound 1 in the absence or presence of nucleophilic scavengers; 5) Compound 1 is a substrate for myocardial calcium-independent phospholipase A2 resulting in the generation of the electrophilic alpha-bromomethyl ketone; 6) phospholipase A2 inhibition requires the in situ generation of the reactive electrophile (i.e. neither the alpha-bromomethyl ketone nor the diproteoenol lactone analog are inhibitory); and 7) concomitant attenuation of the inhibitory potency and the extent of covalent adduct formation in the presence of saturating substrate. Collectively, these results demonstrate that the haloenol lactone, Compound 1, is a substrate for, covalently binds to, and irreversibly inhibits canine myocardial cytosolic calcium-independent phospholipase A2.

Animals↗

Differential turnover of phospholipid acyl groups in mouse peritoneal macrophages.

Phospholipid acyl turnover was assessed in mouse peritoneal exudate cells which consisted primarily of macrophages. The cells were incubated for up to 5 h in media containing 40% H218O, and uptake of 18O into ester carbonyls of phospholipids was determined by gas chromatography-mass spectrometry of hydrogenated methyl esters. The uptake was highest in choline phospholipids and phosphatidylinositol, less in ethanolamine phospholipids, and much less in phosphatidylserine. Acyl groups at the sn-1 and sn-2 positions of diacyl glycerophospholipids, including arachidonic and other long-chain polyunsaturated fatty acids, acquired 18O at about the same rate. Acyl groups of alkylacyl glycerophosphocholine exhibited lower rates of 18O uptake, and acyl groups of ethanolamine plasmalogens (alkenylacyl glycerophosphoethanolamines) acquired only minimal amounts of 18O within 5 h, indicating a low average acyl turnover via free fatty acids. Pulse experiments with exogenous 3H-labeled arachidonic acid supported the concept that acylation of alkenyl glycerophosphoethanolamine occurs by acyl transfer from other phospholipids rather than via free fatty acids and acyl-CoA. The 18O content of intracellular free fatty acids increased gradually over a 5-h period, whereas in extracellular free fatty acids it reached maximal 18O levels within the first hour. Arachidonate and other long-chain polyunsaturated fatty acids were found to participate readily in deacylation-reacylation reactions but were present only in trace amounts in the free fatty acid pools inside and outside the cells. We conclude that acyl turnover of macrophage phospholipids through hydrolysis and reacylation is rapid but tightly controlled so that appreciable concentrations of free arachidonic acid do not occur.

Acylation↗