RESUMO
Large amounts of type II-secreted phospholipase A2 (type II sPLA2) are secreted into inflammatory synovial fluid and they are believed to induce the synthesis of lipid mediators by articular chondrocytes. Preliminary experiments showed that insulin-like growth factor-I, which counteracts cartilage degradation in arthritis, inhibits interleukin-1beta-induced type II sPLA2 gene expression in rabbit articular chondrocytes (Berenbaum, F., G. Thomas, S. Poiraudeau, G. Bereziat, M.T. Corvol, and J. Masliah. 1994. FEBS Lett. 340: 51-55). The present study showed that IL-1beta induced the sustained synthesis of prostaglandin E2 and a parallel increase in type II sPLA2 gene expression (assessed by enzymatic activity and Northern blot analysis), but no increase in cytosolic PLA2 gene expression (assessed by Northern and Western blot analysis) or cytosolic PLA2 activity in rabbit articular chondrocytes. IGF-I inhibited both IL-1beta-stimulated PGE2 synthesis and type II sPLA2 gene expression, but had no effect on cytosolic PLA2 gene expression. Nuclear run-on experiments revealed that IL-1beta stimulated the transcription rate of type II sPLA2 gene, giving rise to long-lived mRNA in cells treated with actinomycin D. IGF-I did not affect transcription rate, suggesting that it acts as a post-transcriptional step. Sucrose density gradient analysis of the translation step showed no effect of IGF-I on the entry of type II sPLA2 mRNA into the polysomal pool or on its distribution into the various polysomal complexes, suggesting that IGF-I does not act on the translation of the mRNA. Lastly, IGF-I strongly decreased the half-life of IL-1beta-induced type II sPLA2 mRNA (from 92 to 12 h), suggesting that IGF-I destabilizes mRNA. These data demonstrate that IL-1beta stimulates the transcription rate of the type II sPLA2 gene and gives rise to a very stable mRNA. In contrast, IGF-I decreases the half-life of the type II sPLA2 message.
Assuntos
Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/metabolismo , Fator de Crescimento Insulin-Like I/farmacologia , Interleucina-1/farmacologia , Fosfolipases A/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Cartilagem Articular/citologia , Células Cultivadas , DNA Complementar/genética , Dinoprostona/biossíntese , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Cinética , Dados de Sequência Molecular , Fosfolipases A/classificação , Fosfolipases A/metabolismo , Fosfolipases A2 , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Coelhos , Proteínas Recombinantes/farmacologia , Ribossomos/efeitos dos fármacos , Ribossomos/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Lyso-phospholipids exert a major injurious effect on lung cell membranes during Acute Respiratory Distress Syndrome (ARDS), but the mechanisms leading to their in vivo generation are still unknown. Intratracheal administration of LPS to guinea pigs induced the secretion of type II secretory phospholipase A2 (sPLA2-II) accompanied by a marked increase in fatty acid and lyso-phosphatidylcholine (lyso-PC) levels in the bronchoalveolar lavage fluid (BALF). Administration of LY311727, a specific sPLA2-II inhibitor, reduced by 60% the mass of free fatty acid and lyso-PC content in BALF. Gas chromatography/mass spectrometry analysis revealed that palmitic acid and palmitoyl-2-lyso-PC were the predominant lipid derivatives released in BALF. A similar pattern was observed after the intratracheal administration of recombinant guinea pig (r-GP) sPLA2-II and was accompanied by a 50-60% loss of surfactant phospholipid content, suggesting that surfactant is a major lung target of sPLA2-II. In confirmation, r-GP sPLA2-II was able to hydrolyze surfactant phospholipids in vitro. This hydrolysis was inhibited by surfactant protein A (SP-A) through a direct and selective protein-protein interaction between SP-A and sPLA2-II. Hence, our study reports an in vivo direct causal relationship between sPLA2-II and early surfactant degradation and a new process of regulation for sPLA2-II activity. Anti-sPLA2-II strategy may represent a novel therapeutic approach in lung injury, such as ARDS.
Assuntos
Pneumopatias/fisiopatologia , Lisofosfatidilcolinas/metabolismo , Fosfolipases A/metabolismo , Proteolipídeos/metabolismo , Surfactantes Pulmonares/metabolismo , Doença Aguda , Animais , Técnicas Biossensoriais , Líquido da Lavagem Broncoalveolar/química , Ácidos Graxos/metabolismo , Fosfolipases A2 do Grupo II , Cobaias , Hidrólise , Indóis/farmacologia , Lipopolissacarídeos/farmacologia , Masculino , Ácido Palmítico/metabolismo , Fosfolipases A/antagonistas & inibidores , Fosfolipases A2 , Ligação Proteica , Proteína A Associada a Surfactante Pulmonar , Proteínas Associadas a Surfactantes PulmonaresRESUMO
We previously reported that the type II secreted phospholipase A2 (sPLA2) promoter from positions (-326 to +20) ([-326;+20] promoter) is negatively regulated by two adjacent regulatory elements, C (-210 to -176) and D (-247 to -210). This study examines in greater detail the way in which this negative regulation operates. Successive 5' deletions of the [-326;+20] type II sPLA2 promoter indicated that the region upstream of position -195 inhibits the transcription activity sixfold in HepG2 cells but not in HeLa cells. Although the whole [-326;-176] region decreased the activity of a heterologous thymidine kinase promoter, this effect was orientation and position sensitive. C/EBP beta, C/EBP alpha, and C/EBP delta, which bind to element C, prevented the inhibition of promoter activity. Electrophoretic mobility shift experiments identified the binding of NF1-like proteins to the [-225;-218] site, which overlaps an insulin response-like sequence, 5'-TGTTTTG-3'. This sequence bound a factor which also recognized the promoters of the apolipoproteins C-III and A-II. Substitutions preventing the binding of this factor or the NF1-like proteins did not increase the transcription activity, but substitution in the [-217;-204] sequence blocked the transcription inhibition. This sequence did not bind any double-strand binding factor, but its antisense strand is critical for the binding of single-strand binding proteins to the [-232;-191] region. We therefore suggest that these single-strand binding proteins are involved in the inhibitory mechanism.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação Enzimológica da Expressão Gênica/genética , Proteínas Nucleares/metabolismo , Fosfolipases A/genética , Regiões Promotoras Genéticas/genética , Animais , Apolipoproteínas/genética , Sequência de Bases , Proteínas Estimuladoras de Ligação a CCAAT , Carcinoma Hepatocelular , Sondas de DNA , DNA Recombinante/metabolismo , Humanos , Fígado/metabolismo , Neoplasias Hepáticas , Dados de Sequência Molecular , Neurofibromina 1 , Fosfolipases A2 , Proteínas/metabolismo , Ratos , Deleção de Sequência/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica/fisiologia , Transfecção , Células Tumorais CultivadasRESUMO
When sonicated suspensions of 1-palmitoyl-2-lysophosphatidyl-N-monomethylethanolamine (lysoPME) or 1-palmitoyl-2-lysophosphatidyl-N,N-dimethylethanolamine (lysoPDE) were incubated with rat liver microsomes and [Me-3H]AdoMet or [Me-14C]AdoMet, one methyl group was added to these lipids. With dipalmitoylphosphatidyl-N-monomethylethanolamine (PME) or dipalmitoylphosphatidyl-N,N-dimethylethanolamine (PDE) as substrates, enzymic monomethylation was also observed. However, the methylation of the lyso compounds was biphasic with an optimum at 0.75 mM lysoPME and 0.5 mM lysoPDE. In contrast to PME or PDE, the lysophospholipids produced a decrease in PC synthesis by lysoPME was reversible and was accompanied by an up to 4-fold increase in PDE synthesis. Competition experiments between lysoPME or lysoPDE and PME or PDE, together with kinetic studies, indicate a connection with methylation of both lyso- and diacylphospholipids. The same active site or sites in close proximity may serve for the second and third methylations. Hence, the presence of two acyl groups on the phospholipid molecule is not a prerequisite for N-methylation of this class of compounds. On the contrary, suspensions of phosphatidylethanolamine, or 2-lysophosphatidylethanolamine (lysoPE) with acyl chains of different degrees of saturation or with one alkenyl at the C1 position of the glycerol were not substrates for PE-N-methyltransferase; the lysoPEs were inhibitory above 0.5 mM.
Assuntos
Lisofosfolipídeos , Metiltransferases/metabolismo , Microssomos Hepáticos/enzimologia , Fosfatidiletanolaminas/metabolismo , Acilação , Animais , Sítios de Ligação , Feminino , Fosfatidil-N-Metiletanolamina N-Metiltransferase , Fosfatidiletanolamina N-Metiltransferase , Fosfolipídeos/metabolismo , Ratos , Ratos Endogâmicos , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Substrate specificity of platelet phospholipase A2 was investigated following Ca2+-dependent hydrolysis by endogenous enzyme of linoleate- or arachidonate-labelled platelet phospholipids. Alkylacyl, alkenylacyl and diacyl classes of ethanolamine and choline glycerophospholipids (GPE and GPC) were separated after their diacylglycerol derivation, and molecular species of diacyl-GPE were analyzed by HPLC. Hydrolysis of platelet ethanolamine and choline glycerophospholipids was dependent on Ca2+ and was maximal at neutral pH. In the presence of 0.2 mM Ca2+ the hydrolysis rate for [14C]arachidonate-labelled phospholipids was in the order diacyl-GPE greater than alkylacyl-GPE = diacyl-GPC = alkenylacyl-GPE greater than alkylacyl-GPC. In addition to being the best substrate at high Ca2+ concentration, diacyl-GPE could be degraded with Ca2+ concentrations in the micromolar range, concentrations which are unable to induce any degradation of diacyl-GPC. As a function of Ca2+ concentration, the hydrolysis rate of [14C]linoleate- and [14C]arachidonate-labelled diacyl-GPE or diacyl-GPC was identical. The five main molecular species of diacyl-GPE labelled with arachidonate or with linoleate were hydrolyzed at the same rate in the presence of 50 microM Ca2+. This study shows that platelet phospholipase A2 is specific for endogenous diacyl-GPE and is independent of fatty chain composition. These results are discussed in relation to the Ca2+ concentration observed in stimulated platelets and in relation to the lysophospholipid-induced specific transfer of arachidonate. They suggest that diacyl-GPE hydrolysis by phospholipase A2 could play a key role in stimulated platelets.
Assuntos
Plaquetas/enzimologia , Fosfolipases A/sangue , Fosfolipases/sangue , Fosfolipídeos/sangue , Animais , Ácido Araquidônico , Ácidos Araquidônicos/sangue , Cálcio/farmacologia , Hidrólise , Ácido Linoleico , Ácidos Linoleicos/sangue , Fosfatidilcolinas/sangue , Fosfatidiletanolaminas/sangue , Fosfolipases A2 , Ratos , Ratos EndogâmicosRESUMO
Rat platelet homogenates are able to catalyze CoA-mediated, ATP-independent transfer of arachidonic acid from platelet phospholipids to added lysophospholipids. Homogenates of platelets prelabelled with radioactive arachidonic or oleic acid were incubated in the presence of CoA and various lysophospholipids. Transfer observed with arachidonic acid-labelled platelets was dependent on the lysophospholipid added. When 1-alkenyl- or 1-acyllysophosphatidylethanolamine was used, there was a more efficient arachidonyl transfer from phosphatidylcholine than from phosphatidylinositol to the phosphatidylethanolamine fraction. Lysophosphatidylserine also accepted arachidonyl from phosphatidylcholine. Addition of lysophosphatidylcholine resulted in a decrease in the labelling of phosphatidylinositol and to a lesser extent of phosphatidylethanolamine with concomitant transfer to phosphatidylcholine. Lysophosphatidylinositol and lysophosphatic acid did not act as substrate for this transfer reaction. Free, non-radioactive arachidonic acid did not compete for the labelled arachidonic acid transfer. This pathway may play a major role in the synthesis of arachidonyl species of phosphatidylethanolamine and phosphatidylserine and for the arachidonyl transfer to the phosphatidylethanolamine plasmologen in stimulated platelets.
Assuntos
Ácidos Araquidônicos/metabolismo , Plaquetas/metabolismo , Fosfolipídeos/metabolismo , Acil Coenzima A/metabolismo , Acilação , Animais , Ácido Araquidônico , Sistema Livre de Células , Lisofosfatidilcolinas/metabolismo , Lisofosfolipídeos , Lipídeos de Membrana/metabolismo , RatosRESUMO
CoA-dependent transacylation and phospholipid hydrolysis were studied in parallel experiments using rat platelet sonicates. The decrease observed in palmitoyllyso-sn-glycero-3-phosphocholine (palmitoyllyso-GPC) transcylation as a function of Ca2+ concentration was found to be correlated with appearance of endogenous lysoderivatives. We also demonstrated that endogenously produced acyllyso-sn-glycero-3-phosphoethanolamine (acyllyso-GPE) induced CoA-dependent arachidonate transfer from diacyl-GPC. These results further argue for a two-step arachidonate release from diacyl-GPC when platelets are stimulated with thrombin.
Assuntos
Aciltransferases/sangue , Trifosfato de Adenosina/metabolismo , Plaquetas/enzimologia , Cálcio/farmacologia , Lisofosfolipase/sangue , Complexos Multienzimáticos/sangue , Fosfolipases A/sangue , Fosfolipases/sangue , Animais , Fosfolipases A2 , RatosRESUMO
The appearance of individual molecular species of phospholipids in the complete sequence of the transmethylation of phosphatidylethanolamine (PE) was examined in rat liver microsomes incubated with S-adenosyl-L-[methyl-14C]methionine. Reverse-phase HPLC analysis of phosphatidylcholine (PC), phosphatidyl-N,N-dimethylethanolamine (dimethyl-PE), or phosphatidyl-N-monomethylethanolamine (monomethyl-PE) showed that radioactivity was present in the same six principal molecules; a first group is constituted by 16:0/22:6, 16:0/20:4 and 16:0/18:2 and a second one by the homologous molecules with 18:0 instead of 16:0 at the sn-1 position of glycerol. In PC, 16:0/22:6 (23% of total radioactivity) was preponderant, and 18:0/20:4 was the lowest. The ratios cpm in PC/nmol in PE were in the order: 16:0/22:6 greater than 16:0/18:2 greater than 16:0/20:4 followed by the corresponding 18:0 molecules. On the other hand, in intermediate phospholipids, incorporation of methyl groups was most marked in 18:0/20:4 (24-27% of total). 16:0/22:6 and 16:0/18:2 were low in comparison to their relative values in PC. The ratio (18:0/20:4)/(16:0/22:6) was 4.5-5.6-times higher in monomethyl-PE and dimethyl-PE than in PC. These differences were found consistently, regardless of incubation time of microsomes (2.5-60 min) and of S-adenosyl-L-methionine (AdoMet) concentration (3 or 100 microM). In liver membranes, it would therefore seem that there is a different selectivity in methyl group transfer, depending upon whether the first two steps or the third step of the reaction are considered. Side reactions, such as deacylation/reacylation, are unlikely to account for this difference, which could rather be related to the enzyme itself.
Assuntos
Microssomos Hepáticos/metabolismo , Fosfatidiletanolaminas/metabolismo , Animais , Cromatografia Gasosa , Cromatografia Líquida de Alta Pressão , Ácidos Graxos/análise , Feminino , Membranas Intracelulares/metabolismo , Metilação , Estrutura Molecular , Fosfatidilcolinas/análise , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/análise , Ratos , S-Adenosilmetionina/metabolismoRESUMO
Phospholipid acyltransferase activities of plasma membranes have been investigated with various acyl-CoA thioesters (palmitoyl, stearoyl, oleoyl, linoleoyl and arachidonoyl) with and without added lysoderivatives. Different patterns of incorporation were observed for each acyl-CoA into endogenous phosphatidylcholine and phosphatidylethanolamine. The turnover rates calculated with tracer amounts of 10 microM acyl-CoA thioesters were five times faster for the polyunsaturated than for the saturated acyl moieties of phosphatidylethanolamine and phosphatidylcholine. Arachidonoyl-CoA was the best acyl donor at low concentrations and the maximal turnover rate was observed at about 25 microM. No saturation appeared at up to 100 microM linoleoyl-CoA. Linoleoyl-CoA transacylase acylated the lyso-compounds in the following order: lysophosphatidylcholine greater than lysophosphatidylserine and lysophosphatidylinositol, while lysophosphatidylethanolamine inhibited linoleate incorporation into the phosphatidylethanolamine itself. Linoleoyl-CoA transacylation was not affected by the fatty acyl moiety at the 1-position of the lysophosphatidylcholine. The results support the view that the plasma membrane acyltransferase activity might contribute to the formation of bile phosphatidylcholines.
Assuntos
Membrana Celular/metabolismo , Fígado/metabolismo , Lisofosfolipídeos , Fosfolipídeos/metabolismo , Acilação , Aciltransferases/metabolismo , Animais , Técnicas In Vitro , Lisofosfatidilcolinas/metabolismo , Masculino , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , Fosfatidilinositóis/metabolismo , Fosfatidilserinas/metabolismo , RatosRESUMO
A homogenate of human platelets was fractionated by zonal ultracentrifugation into membranes, various granules and mitochondria. The membrane fraction was composed of two populations. The first, which represented 75% of the proteins, was rich in plasma membranes; the second, which represented the remaining 25%, was rich in microsomal membranes. Lysophospholipase was essentially localised in the cytosol. Phospholipase A1 which was only weakly bound to membranes, was mostly found in the soluble fraction (75%); the remainder was located in the plasma membranes and the mitochondria. Two-thirds of the phospholipase A2 was found in the particulate fractions.
Assuntos
Plaquetas/enzimologia , Fosfolipases/metabolismo , Adulto , Membrana Celular/enzimologia , Centrifugação Zonal , Citosol/enzimologia , Humanos , Lisofosfatidilcolinas , Masculino , Frações Subcelulares/enzimologiaRESUMO
The binding of spin-labeled clofibrate to native and partially delipidated lipoproteins is a rapid, linear and non-saturable process observed up to the critical micellar concentration of the drug. Low-density lipoproteins (LDL) display a lower affinity for the drug than very-low-density lipoproteins (VLDL) and high-density lipoproteins (HDL) relative to their respective specific volume. Unlike various lipophilic drugs, uptake of spin-labeled clofibrate does not correlate with lipoprotein lipid volume. Spin-labeled clofibrate binding to LDL is enhanced when the temperature increases above 25 degrees C. The binding to HDL and VLDL is less temperature-sensitive. The simulation of the ESR spectra has shown that two types of motion should be superimposed for the spin-labeled clofibrate in HDL, in LDL or in partially delipidated LDL. From 40 down to 25 degrees C for HDL and LDL, a fast anisotropic motion is observed. From 25 degrees C down to 5 degrees C, a two-component motion takes place, including a slow isotropic motion of the probe tumbling in a highly hydrophobic environment. Interactions of spin-labeled clofibrate with the apolipoproteins in HDL and LDL are assumed from the emergence of this strongly immobilized component observed when the temperature decreases. In contrast, for spin-labeled clofibrate inserted in the apolar core of VLDL, ESR shows only one component in the whole temperature range (5-40 degrees C). The location of the spin-labeled drug inside the various lipoprotein particles is discussed as a function of temperature.
Assuntos
Clofibrato/metabolismo , Lipoproteínas/metabolismo , Marcadores de Spin/metabolismo , Difusão , Espectroscopia de Ressonância de Spin Eletrônica , Humanos , Lipoproteínas HDL/metabolismo , Lipoproteínas LDL/metabolismo , Lipoproteínas VLDL/metabolismo , Matemática , Micelas , TemperaturaRESUMO
The synthesis of arachidonic acid has been investigated in fetal and pregnant rat liver microsomes in the course of the gestation. The delta 5-desaturase activity decreased 2-3 times in rat liver between the 19th and 22nd day of the pregnancy. During this period the delta 5-desaturate activity increased 3-fold in the fetal liver, exceeding the activity of the maternal liver. In contrast, the activity of the fetal delta 6-desaturase was in the same range as in pregnant rat liver and the liver of control animals and did not change between these two stages of the gestation. The elongation rate of linoleic acid in fetal liver was 2-3 times lower than in maternal liver but this increased during the pregnancy. The fatty acid activate rate was always higher than the activity of the desaturases. At the 19th day, the activity of the delta 5-desaturase was apparently the rate limiting step of arachidonic acid synthesis in fetal liver. We did not find any delta 5- and delta 6-desaturase activities or linoleic acid elongation in the placenta microsomes.
Assuntos
Ácidos Araquidônicos/biossíntese , Microssomos Hepáticos/metabolismo , Placenta/enzimologia , Animais , Ácido Araquidônico , Ácidos Graxos Dessaturases/metabolismo , Feminino , Idade Gestacional , Malonil Coenzima A/metabolismo , Microssomos Hepáticos/enzimologia , Gravidez , RatosRESUMO
The release and the mobilization of arachidonic acid from guinea-pig alveolar macrophages labeled with [1-14C]arachidonic acid for short (1 h) and long (18 h) periods and stimulated with PAF-acether (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) was studied. After short labeling periods arachidonic acid was primarily incorporated into alkylacyl- and diacylglycerophosphocholine (alkylacylGPC, diacylGPC) and glycerophosphoinositol (GPI), whereas after long labeling periods arachidonic acid was mainly incorporated into alkenylacylglycerophosphoethanolamine (alkenylacylGPE). In macrophages labeled for 1 h, PAF-acether (1 microM) induced a significant decrease in the amount of arachidonic acid esterified into diacyl- and alkylacylGPC and GPI, as well as a significant increase of arachidonate transferred into alkenylacylGPE. No significant decrease in arachidonate esterified in GPC fractions and in GPI was induced by PAF-acether in macrophages labeled for 18 h, whereas the increased transfer of the fatty acid into alkenylacylGPE was still measurable. This study shows that PAF-acether induces the release and the mobilization of newly incorporated arachidonic acid in alveolar macrophages. When cells are labeled for long periods and the majority of arachidonic acid is retained in ether-linked phospholipids, no PAF-acether-induced release of arachidonate was obtained, whereas its transfer was maintained.
Assuntos
Macrófagos/metabolismo , Fosfolipídeos/metabolismo , Fator de Ativação de Plaquetas , Alvéolos Pulmonares/metabolismo , Animais , Ácido Araquidônico , Ácidos Araquidônicos/metabolismo , Cobaias , Técnicas In Vitro , Macrófagos/análise , Macrófagos/efeitos dos fármacos , Masculino , Fosfolipídeos/análise , Alvéolos Pulmonares/efeitos dos fármacosRESUMO
Primary cultures of adult rat hepatocytes were used to compare the uptake and esterification of essential polyunsaturated fatty acids (18:2, 20:3 and 20:4 of the n-6 series) with those of palmitic and oleic acids. The uptake of unesterified fatty acids was linearly related to the free fatty acid/albumin molar ratio for 14 h and did not depend on the unbound free fatty acid level. Whatever the initial free fatty acid/albumin molar ratio, it dropped to 0.5 +/- 0.1 mM after 14 h, thus showing that hepatocytes have a high capacity for clearing free fatty acids from the medium at high free fatty acid/albumin molar ratios. The free fatty acid uptake become saturable when the free fatty acid and albumin concentrations were raised and the free fatty acid/albumin ratio remained constant. This strongly suggests that albumin-hepatocyte interaction mediates free fatty acid uptake. This uptake was identical whatever the fatty acid tested and did not depend on the relative amounts of fatty acids when they were added simultaneously. Triacylglycerol accumulation and synthesis, monitored by labelled fatty acids, were related to the free fatty acid/albumin molar ratio and exhibited no specificity for the series of fatty acids tested. Triacylglycerols were enriched in all the fatty acids tested by up to 60%, and fatty acid incorporation into diacylglycerols and triacylglycerols reflected the free fatty acid composition of the medium. By contrast, neither the level nor the synthesis of phospholipids varied with free fatty acid/albumin, but the rate of phospholipid turnover depended on the fatty acids tested. Accumulation of these acids was smaller in phospholipids than in triacylglycerols. When linoleic and arachidonic acids were added together, phospholipids (especially phosphatidylethanolamine and phosphatidylinositol) were more enriched in arachidonic acid than triacylglycerols. This might be due to the specificity for fatty acid of the enzymes involved in phospholipid metabolism.
Assuntos
Ácidos Graxos Essenciais/metabolismo , Fígado/metabolismo , Animais , Ácido Araquidônico , Ácidos Araquidônicos/metabolismo , Células Cultivadas , Esterificação , Ácidos Graxos não Esterificados/metabolismo , Ácido Linoleico , Ácidos Linoleicos/metabolismo , Masculino , Ácido Oleico , Ácidos Oleicos/metabolismo , Ácido Palmítico , Ácidos Palmíticos/metabolismo , Fosfolipídeos/metabolismo , Ratos , Ratos Endogâmicos , Albumina Sérica/metabolismo , Triglicerídeos/metabolismoRESUMO
Since it has been earlier reported that D-galactosamine induces an inhibition of palmitoylcarnitine transferase I and a depletion of mitochondrial phospholipids which were both prevented by clofibrate, an evaluation of the effects of these drugs on mitochondrial fatty acid composition was made. Galactosamine does not alter the fatty acid pattern of these fatty acids whereas clofibrate induces a 2-fold increase in monounsaturated/saturated fatty acids ratio and a 10-fold decrease of the 20:4 (n - 6)/20:3 (n - 6) ratio in phosphatidylcholine. These alterations suggest an increase of delta 9-desaturation and a decrease of delta 5-desaturation. To determine whether the drug-induced changes in mitochondrial phospholipids has an effect on the physical properties of the membrane, the lipid structural order of mitochondrial preparations was studied using the lipophilic probes DPH and TMA-DPH. Mitochondrial isolated either from galactosamine- or clofibrate-treated rats showed a decrease in fluorescence polarization, indicating an overall decrease in lipid structural order. This alteration is more drastic when both drugs are administered. This phenomenon suggests drastic changes in the bulk phase of inner mitochondrial membrane lipids after treatments and could explain the altered kinetic properties of palmitoylcarnitine transferase I.
Assuntos
Clofibrato/farmacologia , Galactosamina/toxicidade , Mitocôndrias Hepáticas/efeitos dos fármacos , Animais , Cardiolipinas/fisiologia , Ácidos Graxos/metabolismo , Polarização de Fluorescência , Membranas Intracelulares/efeitos dos fármacos , Masculino , Fluidez de Membrana , Lipídeos de Membrana/fisiologia , Fosfatidiletanolaminas/metabolismo , Fosfolipídeos/metabolismo , RatosRESUMO
Modification of chloride conductance by bradykinin in epithelial cells has been attributed to an activation of protein kinase A resulting from adenylcyclase stimulation by arachidonic acid cyclooxygenase products. The results presented here compare tracheal epithelial cell lines from one control and two cystic fibrosis patients which were immortalized by transfection with the SV40 large T oncogene. The three cell lines presented the same arachidonic acid content, turnover and mobilisation under basal conditions. Bradykinin stimulated the release of arachidonic acid and the synthesis of cyclooxygenase derivatives (mainly PGE2). The cell line from the cystic fibrosis patient bearing a phenylalanine 508 deletion, which is the major form of the disease, showed a higher bradykinin-induced arachidonic acid release than either control cells or cells from a patient presenting a minor form of the disease. This higher sensitivity suggests a dysregulation of phospholipase A2 stimulation in cystic fibrosis cells and was confirmed on non-immortalized tracheal epithelial cells in primary culture and on skin fibroblasts from patients bearing the same mutation. This defect is associated with a potentiation of cholera toxin pretreatment on cAMP content of delta F508 cell line. The impaired control of arachidonic acid release cannot be attributed to an increased number of bradykinin binding sites, since this increase was similar in the two cystic fibrosis cell lines.
Assuntos
Ácido Araquidônico/metabolismo , Bradicinina/farmacologia , Fibrose Cística/metabolismo , Linhagem Celular/efeitos dos fármacos , Linhagem Celular/metabolismo , Toxina da Cólera , Meios de Cultura/análise , AMP Cíclico/análise , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Canais Iônicos/metabolismo , Proteínas de Membrana/metabolismo , Mutação , Fosfolipídeos/metabolismo , Quinacrina/farmacologia , Pele/metabolismo , Traqueia/metabolismo , Fatores de Virulência de BordetellaRESUMO
Primary cultures of rat hepatocytes were incubated in the presence of high-density lipoproteins (HDL) labelled with [1-14C]oleyl or [1-14C]linoleyl cholesteryl ester. Labelled HDL were prepared by selective delipidation with heptane, relipidation and sequential ultracentrifugations. Hepatocytes took up cholesteryl esters and cholesteryl ether their non-hydrolizable analog, at the same rate. The uptake increased with time, the cholesteryl ester/protein ratio and the amount of added HDL. It was not dependent on the nature of acyl chain or on the nature of the bond. The uptake did not depend on a specific interaction between HDL and cell membranes, since cholesteryl ester was taken up from HDL to the same extent as from albumin complexes. Linoleic and oleic acids released from cholesteryl esters taken up by hepatocytes were mainly reesterified into phosphatidylcholine and triacylglycerols. Linoleic acid was preferentially channelled into PC. A portion of these lipids were secreted by hepatocytes during a 24-h reincubation in a medium devoid of lipoprotein. Nearly the same amount of radioactivity was recovered in secreted phospholipids as in secreted triacylglycerols, in contrast with hepatocytes labelled with free fatty acids which secreted very little radioactivity as phospholipids. From these results and the high content in polyunsaturated fatty acids of cholesteryl esters, one can hypothesize that hepatic cholesteryl ester uptake may contribute to biliary phosphatidylcholine production, and therefore to polyunsaturated fatty acid sparing.
Assuntos
Ésteres do Colesterol/metabolismo , Ácidos Graxos/metabolismo , Lipoproteínas HDL/metabolismo , Fígado/metabolismo , Animais , Apolipoproteínas/análise , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Fígado/citologia , Ratos , Ratos EndogâmicosRESUMO
Secreted phospholipases A(2) is a family of small molecular weight and calcium-dependent enzymes of which the members list is presently growing. Among these enzymes, the synovial type IIA and the type V phospholipases A(2) are involved in inflammation. Although their actual mechanism is still a subject of debate, new therapeutic strategies can result from the knowledge of the regulations of their gene expression. The human genes of the type IIA and type V phospholipases A(2) are located on the chromosome 1 at close positions and transcribed in reverse orientations. These genes can therefore be regulated by common elements but only the regulation of the type IIA phospholipase A(2) gene expression has been extensively studied. Pro-inflammatory cytokines upregulate while the growth factors downregulate the type IIA phospholipase A(2) gene expression. Interleukin-6 and interleukin-1beta exert their effects at least partially at the transcriptional level. The transcriptional regulation of the type IIA phospholipase A(2) gene is cell- and species-specific. The activity of the human promoter is controlled by the CAAT-enhancer binding protein (C/EBP) factors while that of the rat promoter is regulated by nuclear factor kappaB (NF-kappaB) and C/EBPs. Furthermore, the human promoter is constitutively repressed in hepatocytes by single strand DNA binding proteins whose effects are relieved by C/EBP factors while the glucocorticoid receptor interacts with C/EBPs in chondrocytes to achieve full basal and interleukin-1beta-stimulated transcription activity. Other factors like CTF/NF1 and Sp1 might be involved in the regulation of both the rat and human promoter. Peroxisome proliferator-activated receptors could contribute to the stimulation of the rat promoter by NF-kappaB in vascular smooth muscle cells. The study of the coactivators and coinhibitors associated to these transcription factors will give a better understanding of the diversity and complexity of the transcriptional regulations of the type IIA phospholipase A(2) gene.
Assuntos
Regulação Enzimológica da Expressão Gênica , Inflamação/enzimologia , Fosfolipases A/genética , Animais , Sequência de Bases , Sítios de Ligação , Citocinas/metabolismo , Substâncias de Crescimento/metabolismo , Humanos , Dados de Sequência Molecular , Fosfolipases A/metabolismo , Regiões Promotoras Genéticas , Alinhamento de Sequência , Transdução de Sinais , TATA Box , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
In order to study the effect of linoleyl enrichment of platelet membranes upon adenylate cyclase activity and membrane fluidity, manipulations of platelet phospholipids are carried out with phosphatidylcholine-loaded high-density lipoproteins (HDL) or phospholipid-exchange protein and phospholipid-cholesterol mixed vesicles. Incubation with HDL does not appear to be valuable for this purpose. On the other hand, phospholipid-exchange protein and mixed vesicles can be used successfully. Phospholipid-exchange protein stimulated 3-fold the spontaneous exchange of 2-linoleylphosphatidylcholine between the vesicles and the platelets. Linoleyl enrichment of platelets by dilinoleylphosphatidylcholine is about 25% and by 2-linoleylphosphatidylcholine is about 45-50%. The unsaturation index remains constant when the enrichment is performed using dilinoleylphosphatidylcholine but it increases with 2-linoleylphosphatidylcholine. Basal and prostaglandin E1-stimulated adenylate cyclase activities are not modified by dilinoleylphosphatidylcholine, while they increase significantly in the case of 2-linoleylphosphatidylcholine. There is no significant variation in diphenyl hexatriene fluorescence polarization parameters, either with dilinoleylphosphatidylcholine or with 2-linoleylphosphatidylcholine.
Assuntos
Adenilil Ciclases/sangue , Plaquetas/metabolismo , Membrana Celular/metabolismo , Lipoproteínas HDL/sangue , Lipossomos , Fluidez de Membrana , Fosfatidilcolinas/sangue , Humanos , Ácidos Linoleicos/sangue , Ácidos Linoleicos/síntese químicaRESUMO
A partial rat apo E-beta-galactosidase fusion protein was produced in Escherichia coli Y1089 infected with recombinant lambda GT11 obtained by immunoscreening of a rat liver cDNA library with an anti-rat LDL antiserum. Partial cDNA overlapped the apo E mRNA sequence coding for apo E binding domain towards the LDL(B/E) receptor up to codon for Arg-139. Fusion protein specifically bound to human fibroblasts. The high-affinity component exhibited a Kd of 5 x 10(-8) M and 4.1 x 10(5) sites per cell. Fusion protein binding to fibroblasts was mediated by their apo E moiety and not by beta-galactosidase since: (1) specific binding of fusion protein was competed out by human LDL; (2) beta-galactosidase did not compete with fusion protein binding; and (3) human fibroblasts from a patient with familial hypercholesterolemia, deficient in LDL(B/E) receptor, bound fusion protein 10-times lower than control fibroblasts. It was demonstrated that partial fusion protein retained the functional activity of the native apo E. However, compared to full-length native or engineered apo E, fusion protein was able to bind fibroblasts without being complexed with phospholipids. Fusion proteins might be a useful tool for studying the functional efficiency of the LDL(B/E) receptor and for mapping residues and domains involved in the binding process.