RESUMEN

Interleukin 1 beta (IL-1ß) induced platelet activating factor (PAF) synthesis in U-937 cells through stimulation of acetyl-CoA:lysoPAF-acetyltransferase (lyso PAF-AT) at 3 h and DTT-independentCDP-choline-1-alkyl-2-acetyl-sn-glycerol cholinophosphotransferase (PAF-CPT) at 0.5 h. The aim of this study was to investigate the effect of tyrosol (T), resveratrol (R) and their acetylated derivatives(AcDs) which exhibit enhanced bioavailability, on PAF synthesis in U-937 after IL-1ß stimulation. The specific activity of PAF enzymes and intracellular levels were measured in cell homogenates. T and R concentration capable of inducing 50% inhibition in IL-1ß effect on lyso PAF-AT was 48 µΜ ± 11 and 157 µΜ ± 77, for PAF-CPT 246 µΜ ± 61 and 294 µΜ ± 102, respectively. The same order of concentration was also observed on inhibiting PAF levels produced by IL-1ß. T was more potent inhibitor than R (p<0.05). AcDs of T retain parent compound inhibitory activity, while in the case of R only two AcDs retain the activity. The observed inhibitory effect by T,R and their AcDs, may partly explain their already reported beneficial role.

Asunto(s)

Antiinflamatorios no Esteroideos/farmacología , Antioxidantes/farmacología , Regulación hacia Abajo/efectos de los fármacos , Monocitos/efectos de los fármacos , Alcohol Feniletílico/análogos & derivados , Factor de Activación Plaquetaria/antagonistas & inhibidores , Estilbenos/farmacología , 1-Alquil-2-acetilglicerofosfocolina Esterasa/antagonistas & inhibidores , 1-Alquil-2-acetilglicerofosfocolina Esterasa/química , 1-Alquil-2-acetilglicerofosfocolina Esterasa/metabolismo , Acetilación , Acetiltransferasas/antagonistas & inhibidores , Acetiltransferasas/química , Acetiltransferasas/metabolismo , Antiinflamatorios no Esteroideos/síntesis química , Antiinflamatorios no Esteroideos/química , Antioxidantes/síntesis química , Antioxidantes/química , Línea Celular , Diacilglicerol Colinafosfotransferasa/antagonistas & inhibidores , Diacilglicerol Colinafosfotransferasa/química , Diacilglicerol Colinafosfotransferasa/metabolismo , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Interleucina-1beta/antagonistas & inhibidores , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Monocitos/inmunología , Monocitos/metabolismo , Concentración Osmolar , Alcohol Feniletílico/farmacología , Factor de Activación Plaquetaria/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Resveratrol , Estilbenos/química

RESUMEN

CDP-diacylglycerol synthases (CDS) are critical enzymes that catalyze the formation of CDP-diacylglycerol (CDP-DAG) from phosphatidic acid (PA). Here we show in vitro that the two isoforms of human CDS, CDS1 and CDS2, show different acyl chain specificities for its lipid substrate. CDS2 is selective for the acyl chains at the sn-1 and sn-2 positions, the most preferred species being 1-stearoyl-2-arachidonoyl-sn-phosphatidic acid. CDS1, conversely, shows no particular substrate specificity, displaying similar activities for almost all substrates tested. Additionally, we show that inhibition of CDS2 by phosphatidylinositol is also acyl chain-dependent, with the strongest inhibition seen with the 1-stearoyl-2-arachidonoyl species. CDS1 shows no acyl chain-dependent inhibition. Both CDS1 and CDS2 are inhibited by their anionic phospholipid end products, with phosphatidylinositol-(4,5)-bisphosphate showing the strongest inhibition. Our results indicate that CDS1 and CDS2 could create different CDP-DAG pools that may serve to enrich different phospholipid species with specific acyl chains.

Asunto(s)

Diacilglicerol Colinafosfotransferasa/metabolismo , Animales , Células COS , Chlorocebus aethiops , Citidina Difosfato Diglicéridos/metabolismo , Diacilglicerol Colinafosfotransferasa/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/metabolismo , Ácidos Fosfatidicos/química , Ácidos Fosfatidicos/metabolismo , Fosfatidilinositoles/farmacología , Transporte de Proteínas , Especificidad por Sustrato

RESUMEN

Exposure to mustard gas causes inflammatory lung diseases including acute respiratory distress syndrome (ARDS). A defect in the lung surfactant system has been implicated as a cause of ARDS. A major component of lung surfactant is dipalmitoyl phosphatidylcholine (DPPC) and the major pathway for its synthesis is the cytidine diphosphocholine (CDP-choline) pathway. It is not known whether the ARDS induced by mustard gas is mediated by its direct effects on some of the enzymes in the CDP-choline pathway. In the present study we investigated whether mustard gas exposure modulates the activity of cholinephosphotransferase (CPT) the terminal enzyme by CDP-choline pathway. Adult guinea pigs were intratracheally infused with single doses of 2-chloroethyl ethyl sulfide (CEES) (0.5 mg/kg b.wt. in ethanol). Control animals were injected with vehicles only. The animals were sacrificed at different time and the lungs were removed after perfusion with physiological saline. CPT activity increased steadily up to 4 h and then decreased at 6 h and stabilized at 7 days in both mitochondria and microsomes. To determine the dose-dependent effect of CEES on CPT activity we varied the doses of CEES (0.5-6.0 mg/kg b.wt.) and sacrificed the animals at 1 h and 4 h. CPT activity showed a dose-dependent increase of up to 2.0 mg/kg b.wt. of CEES in both mitochondria and microsomes then decreased at 4.0 mg/kg b.wt. For further studies we used a fixed single dose of CEES (2.0 mg/kg b.wt.) and fixed exposure time (7 days). Lung injury was determined by measuring the leakage of iodinated-bovine serum albumin into lung tissue and expressed as the permeability index. CEES exposure (2.0 mg/kg b.wt. for 7 days) caused a significant decrease of both CPT gene expression (approximately 1.7-fold) and activity (approximately 1.5-fold) in the lung. This decrease in CPT activity was not associated with any mutation of the CPT gene. Previously we reported that CEES infusion increased the production of ceramides which are known to modulate PC synthesis. To determine whether ceramides affect microsomal CPT activity the lung microsomal fraction was incubated with different concentrations of C(2)-ceramide prior to CPT assay. CPT activity decreased significantly with increasing dose and time. The present study indicates that CEES causes lung injury and significantly decreases CPT gene expression and activity. This decrease in CPT activity was not associated with any mutation of the CPT gene is probably mediated by accumulation of ceramides. CEES induced ceramide accumulation may thus play an important role in the development of ARDS by modulating CPT enzyme.

Asunto(s)

Diacilglicerol Colinafosfotransferasa/antagonistas & inhibidores , Inhibidores Enzimáticos/toxicidad , Pulmón/efectos de los fármacos , Gas Mostaza/análogos & derivados , Animales , Secuencia de Bases , Northern Blotting , Ceramidas/farmacología , Cartilla de ADN , Diacilglicerol Colinafosfotransferasa/genética , Cobayas , Pulmón/enzimología , Pulmón/ultraestructura , Masculino , Microscopía Electrónica , Microsomas/efectos de los fármacos , Microsomas/enzimología , Gas Mostaza/toxicidad , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

Nematodes were found to synthesize phosphorylcholine-containing molecules not present in higher organisms, i.e. phosphorylcholine-substituted glycosphingolipids and (glyco)proteins. Investigations on the biosynthesis of these structures provided first biochemical evidence for the presence of the Kennedy and Bremer-Greenberg pathways in the model organism Caenorhabditis elegans.

Asunto(s)

Caenorhabditis elegans/metabolismo , Glicoproteínas/biosíntesis , Glicoesfingolípidos/biosíntesis , Fosforilcolina/metabolismo , Animales , Radioisótopos de Carbono , Diacilglicerol Colinafosfotransferasa/antagonistas & inhibidores , Electroforesis en Gel de Poliacrilamida , Farnesol/farmacología

RESUMEN

The human choline/ethanolamine phosphotransferase 1 (CEPT1) gene codes for a dual-specificity enzyme that catalyzes the de novo synthesis of the two major phospholipids through the transfer of a phosphobase from CDP-choline or CDP-ethanolamine to DAG to form PC and PE. We used an expression system devoid of endogenous cholinephosphotransferase and ethanolaminephosphotransferase activities to assess the diradylglycerol specificity of CEPT1. A mixed micellar assay was used to ensure that the diradylglycerols delivered were not affecting the membrane environment in which CEPT1 resides. The CEPT1 enzyme displayed an apparent Km of 36 microM for CDP-choline and 4.2 mol% for di-18:1 DAG with a Vmax of 14.3 nmol min(-1) mg(-1). When CDP-ethanolamine was used as substrate, the apparent Km was 98 microM for CDP-ethanolamine and 4.3 mol% for di-18:1 DAG with a Vmax of 8.2 nmol min(-1) mg(-1). The preferred diradylglycerol substrates used by CEPT1 with CDP-choline as the phosphobase donor were di-18:1 DAG, di-16:1 DAG, and 16:0/18:1 DAG. A major difference between previous emulsion-based assay results and the mixed micelle results was a complete inability to use 16:0(O)/2:0 as a substrate for the de novo synthesis of platelet-activating factor when the mixed micelle assay was used. When CDP-ethanolamine was used as the phosphobase donor, 16:0/18:1 DAG, di-18:1 DAG, and di-16:1 DAG were the preferred substrates. The mixed micelle assay also allowed the lipid activation of CEPT to be measured, and both the cholinephosphotransferase and ethanolaminephosphotransferase activities displayed the unusual property of product activation at 5 mol%, implying that specific lipid activation binding sites exist on CEPT1. The protein kinase C inhibitor chelerythrine and the human DAG kinase inhibitor R59949 both inhibited CEPT1 activity with IC50 values of 40 microM.

Asunto(s)

Diacilglicerol Colinafosfotransferasa/metabolismo , Etanolaminofosfotransferasa/metabolismo , Fosfatidilcolinas/biosíntesis , Fosfatidiletanolaminas/biosíntesis , Transferasas (Grupos de Otros Fosfatos Sustitutos)/química , Transferasas (Grupos de Otros Fosfatos Sustitutos)/metabolismo , Membrana Celular/enzimología , Diacilglicerol Colinafosfotransferasa/antagonistas & inhibidores , Diglicéridos/metabolismo , Etanolaminofosfotransferasa/antagonistas & inhibidores , Humanos , Cinética , Micelas , Complejos Multienzimáticos/química , Complejos Multienzimáticos/metabolismo , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/química , Fosfatidiletanolaminas/metabolismo , Saccharomyces cerevisiae , Especificidad por Sustrato , Transferasas (Grupos de Otros Fosfatos Sustitutos)/antagonistas & inhibidores

RESUMEN

Phosphatidylcholine (PC) constitutes a major portion of cellular phospholipids and displays unique molecular species in different cell types and tissues. Inhibition of the CDP-choline pathway in most mammalian cells or overexpression of the hepatic phosphatidylethanolamine methylation pathway in hepatocytes leads to perturbation of PC homeostasis, growth arrest or even cell death. Although many agents that perturb PC homeostasis and induce cell death have been identified, the signaling pathways that mediate this cell death have not been well defined. This review summarizes recent progress in understanding the relationship between PC homeostasis and cell death.

Asunto(s)

Apoptosis/fisiología , Moléculas de Adhesión Celular , Fosfatidilcolinas/fisiología , Proteínas Supresoras de Tumor , Animales , Línea Celular , Colina/metabolismo , Colina Quinasa/antagonistas & inhibidores , Citidililtransferasa de Colina-Fosfato/antagonistas & inhibidores , Diacilglicerol Colinafosfotransferasa/antagonistas & inhibidores , Activación Enzimática , Homeostasis , Humanos , Glicoproteínas de Membrana/metabolismo , Metilación , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , Transducción de Señal , Esfingomielinas/metabolismo

RESUMEN

We have studied in vitro the effects of ethanol on the different enzymes involved in the biosynthesis of phosphatidylcholine (PC) via CDP-choline. Ethanol alters neither choline kinase (CK) nor CTP:phosphocholine cytidylyltransferase (CT) activities but, at levels higher than 50 mM, it does significantly inhibit microsomal cholinephosphotransferase (CPT) activity concomitantly with an increase in the ethanol concentration. A study of the kinetics of the reaction catalysed by CPT shows that ethanol decreases Vmax without altering Km, indicating a non-competitive inhibitory effect. An analysis of the thermodependence of CPT activity in the absence of ethanol reveals a break in the Arrhenius plot and thus a straight relationship between enzyme activity and the physico-chemical state of the microsomal membrane. Incubation of microsomes in the presence of ethanol increased the transition temperature from 25.8-28.2 degrees C. Microsomes were also incubated with n-alkanols with chain-lengths of fewer than five carbon atoms at concentrations which, according to their partition coefficients, produce equimolar levels in the membrane. Under these conditions all the alkanols caused the same inhibitory effect. All these results demonstrate that ethanol modulate the PC biosynthesis at the level of CPT activity and does not affect the CT enzyme. The inhibition found on CPT is clearly dependent on the alteration produced by ethanol on the hepatic microsomal membrane.

Asunto(s)

Citidina Difosfato Colina/metabolismo , Diacilglicerol Colinafosfotransferasa/antagonistas & inhibidores , Etanol/farmacología , Hígado/metabolismo , Fosfatidilcolinas/biosíntesis , Alcoholes/farmacología , Animales , Colina Quinasa/metabolismo , Citidililtransferasa de Colina-Fosfato/metabolismo , Diacilglicerol Colinafosfotransferasa/metabolismo , Cinética , Hígado/enzimología , Masculino , Microsomas Hepáticos/enzimología , Ratas , Ratas Sprague-Dawley , Temperatura

RESUMEN

Levels of cerebral platelet activating factor (PAF) result from the balance between the activities of its synthesizing enzyme dithiothreitol (DTT)- insensitive cholinephosphotransferase and its degradative enzyme PAF acetylhydrolase. Cerebral fractions of aged rats (19 months) displayed higher levels of PAF acetylhydrolase isoenzymes (P<0.05; n=4), unaltered levels of DTT-insensitive cholinephosphotransferase and lower PAF levels than young animals (2 months). Cerebral fractions of aged rats treated with cytidine (5') diphosphocholine displayed lower DTT-insensitive cholinephosphotransferase (55% after 8 days of treatment with 350 mg/kg per day, P<0.05; n=4), unaltered levels of PAF acetylhydrolase and lower PAF levels than untreated control animals. Thus our data would indicate that decrease of cerebral PAF may be attributed to an activation of PAF acetylhydrolase in ageing, and to an inactivation of DTT-insensitive cholinephosphotransferase in cytidine (5') diphosphocholine treated animals.

Asunto(s)

Envejecimiento/fisiología , Corteza Cerebral/enzimología , Citidina Difosfato Colina/metabolismo , Diacilglicerol Colinafosfotransferasa/metabolismo , Nootrópicos/metabolismo , Factor de Activación Plaquetaria/biosíntesis , 1-Alquil-2-acetilglicerofosfocolina Esterasa , Envejecimiento/efectos de los fármacos , Animales , Membrana Celular/efectos de los fármacos , Membrana Celular/enzimología , Corteza Cerebral/efectos de los fármacos , Citidina Difosfato Colina/farmacología , Diacilglicerol Colinafosfotransferasa/antagonistas & inhibidores , Alimentos Formulados , Isoenzimas/efectos de los fármacos , Isoenzimas/metabolismo , Masculino , Nootrópicos/farmacología , Fosfolipasas A/metabolismo , Factor de Activación Plaquetaria/efectos de los fármacos , Ratas , Ratas Wistar , Accidente Cerebrovascular/enzimología , Accidente Cerebrovascular/fisiopatología , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/enzimología

RESUMEN

Induction of apoptosis in HL-60 cells, using a variety of cytotoxic drugs, resulted, in all cases, in inhibition of CDP-choline:1, 2-diacylglycerol choline phosphotransferase, leading to an accumulation of its substrate, CDP-choline, and inhibition of phosphatidylcholine biosynthesis. Incubation of the cells with phosphatidylcholine reduced the number displaying an apoptotic morphology following drug treatment, and this was inversely related to the degree to which the drugs inhibited phosphatidylcholine biosynthesis. Inhibition of choline phosphotransferase by two of the drugs, farnesol and chelerythrine, was shown to be due to direct inhibition of the enzyme, while inhibition by the other drugs, etoposide and camptothecin, could be explained by the intracellular acidification that followed induction of apoptosis.

Asunto(s)

Apoptosis/efectos de los fármacos , Diacilglicerol Colinafosfotransferasa/antagonistas & inhibidores , Fosfatidilcolinas/biosíntesis , Alcaloides , Benzofenantridinas , Camptotecina/farmacología , Colina/metabolismo , Citidina Difosfato Colina/metabolismo , Inhibidores Enzimáticos/farmacología , Etopósido/farmacología , Farnesol/farmacología , Citometría de Flujo , Células HL-60 , Humanos , Concentración de Iones de Hidrógeno , Fenantridinas/farmacología , Fosfatidilcolinas/farmacología , Fosforilcolina/metabolismo

RESUMEN

We have previously shown that, among various isoprenoids, farnesol and geranylgeraniol specifically induced actin fiber disorganization, growth inhibition, and apoptosis in human lung adenocarcinoma A549 cells (Miquel, K., Pradines, A., and Favre, G. (1996) Biochem. Biophys. Res. Commun. 225, 869-876). Here we demonstrate that isoprenoid-induced apoptosis was preceded by an arrest in G0/G1 phase. The isoprenoid effects were independent of protein prenylation and of mitogen-activated protein kinase activity. Moreover, geranylgeraniol and farnesol induced a rapid inhibition of phosphatidylcholine biosynthesis at the last step of the CDP-choline pathway controlled by choline phosphotransferase and not at the level of CTP:phosphocholine cytidylyltransferase, the key enzyme of the pathway. Inhibition of choline phosphotransferase was confirmed by in vitro assays on microsomal fractions, which clearly showed that the isoprenoids acted by competitive inhibition with the diacylglycerol binding. Exogenous phosphatidylcholine addition prevented all the biological effects of the isoprenoids, including actin fiber disorganization and apoptosis, suggesting that inhibition of phosphatidylcholine biosynthesis might be the primary event of the isoprenoid action. These data demonstrate the molecular mechanism of geranylgeraniol and farnesol effects and suggest that the mevalonate pathway, leading notably to prenylated proteins, might be linked to the control of cell proliferation through the regulation of phosphatidylcholine biosynthesis.

Asunto(s)

Apoptosis/efectos de los fármacos , Diacilglicerol Colinafosfotransferasa/antagonistas & inhibidores , Diterpenos/farmacología , Farnesol/farmacología , Fosfatidilcolinas/biosíntesis , Actinas/análisis , Unión Competitiva , División Celular/fisiología , Colina/metabolismo , Citidililtransferasa de Colina-Fosfato/metabolismo , Citidina Difosfato Colina/metabolismo , Citoesqueleto/efectos de los fármacos , Diglicéridos/metabolismo , Inhibidores Enzimáticos/farmacología , Humanos , Interfase/fisiología , Microscopía Fluorescente , Microsomas/enzimología , Fosfatidilcolinas/farmacología , Prenilación de Proteína/efectos de los fármacos , Células Tumorales Cultivadas

RESUMEN

A yeast mutant, cdg1, was isolated on the basis of an inositol excretion phenotype. This mutant exhibited pleiotropic deficiencies in phospholipid biosynthesis, including reduced levels of CDP-diacylglycerol (DAG) synthase activity (Klig, L. S., Homann, M. J., Kohlwein, S. D., Kelley, M. J., Henry, S. A., and Carman, G. M. (1988) J. Bacteriol. 170, 1878-1886). In this study we present evidence that the molecular basis for the inositol excretion phenotype is a G305/A305 point mutation (Cys102 --> Tyr substitution) within the CDS1 gene (encodes CDP-DAG synthase) of this mutant. Expression of CDP-DAG synthase activity from a plasmid-borne copy of the CDS1 gene in the cdg1 mutant was not down-regulated, and this expression also corrected the inositol excretion phenotype. Introduction of the above mutated gene (CDS1*) controlled by its endogenous promoter on a single copy plasmid into a cds1-null background reconstituted a transformant with the cdg1 phenotype, including reduced CDP-DAG synthase activity, elevated phosphatidylserine synthase activity, and inositol excretion into the growth medium. Expression of CDS1* in a single copy in the cdg1 mutant raised CDP-DAG synthase activity from 15 to 30% of derepressed wild-type yeast levels but still did not correct the inositol excretion phenotype. CDP-DAG synthase activity was not regulated in response to precursors of phospholipid biosynthesis in the cdg1 mutant either with or without a trans copy of the CDS1* gene. An open reading frame was identified 5' to the CDS1 locus, YBR0314, which also resulted in inositol excretion when present in trans in multiple copies.

Asunto(s)

Diacilglicerol Colinafosfotransferasa/metabolismo , Inositol/metabolismo , Saccharomyces cerevisiae/metabolismo , Diacilglicerol Colinafosfotransferasa/antagonistas & inhibidores , Prueba de Complementación Genética , Fosfatidilcolinas/biosíntesis , Fosfatidilinositoles/biosíntesis , Mutación Puntual , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética

RESUMEN

1. A phospholipase A2 (PLA2) represents the key enzyme in the remodelling pathway of platelet-activating factor (PAF) synthesis in human polymorphonuclear (PMN) leucocytes. 2. PLA2 activation is also the rate-limiting step for the release of the arachidonic acid utilized for the synthesis of leukotrienes in stimulated leucocytes; however, it is unknown whether the PLA2s involved in the two biosynthetic pathways are identical. 3. Cloricromene (8-monochloro-3-beta-diethylaminoethyl-4-methyl-7-ethoxy- carbonylmethoxy coumarin) is an antithrombotic coumarin derivative which inhibits platelet and leucocyte function and suppresses arachidonic acid liberation by interfering with PLA2 activation. 4. The aim of the present study was to assess whether chloricromene inhibits PAF synthesis by stimulated human polymorphonuclear leucocytes (PMNs). 5. Cloricromene (50-500 microM) inhibited in a concentration-dependent manner the release of PAF, as measured by h.p.l.c. bioassay, from A23187-stimulated PMNs. Significant inhibition (45%) of PAF-release was obtained with 50 microM cloricromene and the IC50 was 85 microM. Mepacrine (500 microM), a non-specific PLA2 inhibitor, strikingly reduced PAF release. 6. The incorporation of [3H]-acetate into [3H]-PAF induced by serum-treated zymosan in human PMNs was also inhibited concentration-dependently by cloricromene, with an IC50 of 105 microM. Mepacrine also suppressed [3H]-acetate incorporation into [3H]-PAF. 7. Cloricromene did not affect the activities of the enzymes involved in PAF-synthesis acetyltransferase or phosphocholine transferase. 8. Our data demonstrate that cloricromene, an inhibitor of PLA2-activation in human leucocytes, reduces the synthesis of PAF by stimulated PMNs. This finding has a twofold implication: the PLA2s (or the mechanisms that regulate their activation) involved in PAF synthesis and arachidonate release in human leucocytes are either identical or else indistinguishable by their sensitivity to cloricromene; the inhibition of PAF release by activated leucocytes may contribute to the antithrombotic and anti-ischaemic activities exerted by cloricromene.

Asunto(s)

Cromonar/análogos & derivados , Neutrófilos/metabolismo , Fosfolipasas A/antagonistas & inhibidores , Factor de Activación Plaquetaria/biosíntesis , Inhibidores de Agregación Plaquetaria/farmacología , Acetiltransferasas/metabolismo , Calcimicina/farmacología , Cromonar/farmacología , Diacilglicerol Colinafosfotransferasa/antagonistas & inhibidores , Activación Enzimática/efectos de los fármacos , Humanos , Técnicas In Vitro , Indicadores y Reactivos , Ionóforos/farmacología , Neutrófilos/efectos de los fármacos , Fosfolipasas A/metabolismo , Fosfolipasas A2

RESUMEN

We have studied the synthesis of phospholipids in hepatocytes isolated from chronically ethanol-treated rats by using isotopically labelled serine, ethanolamine, and choline as exogenous precursors. Our results demonstrate that ethanol induces specific effects on the biosynthesis of phosphatidylethanolamine and phosphatidylcholine via CDP-derivatives and also on the synthesis of phosphatidylserine via the Ca(++)-dependent base-exchange reaction. Thus, the synthesis of phosphatidylethanolamine from [3-H]ethanolamine and the incorporation of [3H]serine into phosphatidylserine were clearly higher in hepatocytes from ethanol-treated rats compared to controls. The synthesis of phosphatidylcholine from [methyl-14C]choline, on the other hand, decreased markedly, suggesting a specific inhibition of cholinephosphotransferase activity. We have also demonstrated that the phosphatidylcholine levels are markedly decreased in hepatocytes isolated from chronically ethanol-treated rats as a consequence of the lower phosphatidylcholine biosynthesis. The decrease in the incorporation of radioactivity from choline to betaine, which we also found, is interpreted as being the result of a higher use of betaine as methyl donor instead of methionine to maintain the hepatic S-adenosylmethionine levels in chronic alcoholism.

Asunto(s)

Alcoholismo/metabolismo , Colina/metabolismo , Etanolaminas/metabolismo , Hígado/metabolismo , Fosfolípidos/biosíntesis , Serina/metabolismo , Animales , Radioisótopos de Carbono , Células Cultivadas , Diacilglicerol Colinafosfotransferasa/antagonistas & inhibidores , Diacilglicerol Colinafosfotransferasa/metabolismo , Etanolamina , Masculino , Fosfatidilcolinas/biosíntesis , Fosfatidiletanolaminas/biosíntesis , Fosfatidilserinas/biosíntesis , Técnica de Dilución de Radioisótopos , Ratas , Ratas Sprague-Dawley , Valores de Referencia , Tritio

RESUMEN

While steady-state kinetic parameters (metabolite pools, Km and activation energies) are partially known for the enzymes involved in phosphatidylcholine synthesis and degradation in mammalian brain, they are not available for the nervous system of lower vertebrates or invertebrates. Since the extent of evolutionary development of an enzyme is not known a priori, we evaluated the kinetic and thermodynamic parameters of choline kinase, CTP:phosphocholine cytidylyltransferase, choline phosphotransferase and glycerophosphorylcholine phosphodiesterase in squid (Loligo pealei) optic lobe, dogfish (Mustelus canis) and rat brain. For all these enzyme activities, basic similarities in Km and inhibitor effect were found. The same was true for the activation energies Ea, with the exception of squid choline kinase and dogfish cytidylyltransferase. Treatment of microsomal membranes with phospholipase C sharply inhibited cytidylyltransferase activity in all three animal species. In dogfish brain, glycerophosphorylcholine phosphodiesterase activity was undetectable. Our results are consistent with the notion that the kinetic properties of the enzyme activities leading to the preservation of the phosphatidylcholine membranous pool may have appeared early in metazoan evolution and been fully conserved in mammals.

Asunto(s)

Evolución Biológica , Encéfalo/enzimología , Decapodiformes/metabolismo , Cazón/metabolismo , Lóbulo Óptico de Animales no Mamíferos/enzimología , Fosfatidilcolinas/metabolismo , Animales , Colina Quinasa/antagonistas & inhibidores , Colina Quinasa/química , Colina Quinasa/metabolismo , Citidililtransferasa de Colina-Fosfato , Diacilglicerol Colinafosfotransferasa/antagonistas & inhibidores , Diacilglicerol Colinafosfotransferasa/química , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Cinética , Nucleotidiltransferasas/antagonistas & inhibidores , Nucleotidiltransferasas/química , Nucleotidiltransferasas/metabolismo , Hidrolasas Diéster Fosfóricas/química , Hidrolasas Diéster Fosfóricas/metabolismo , Ratas , Termodinámica , Fosfolipasas de Tipo C/farmacología

RESUMEN

Alveolar type II cell injury by phagocytic cell-derived reactive oxygen metabolites represents a potential mechanism for the altered surfactant metabolism found in patients with the adult respiratory distress syndrome (ARDS). Previous studies demonstrated altered surfactant phospholipid metabolism after sublethal oxidant exposure. In this study, we measured intracellular ATP levels and the activities of several enzymes involved in surfactant phospholipid biosynthesis after sublethal H2O2 exposure of cultured rat alveolar type II cells. Intracellular ATP levels were reduced by 46.6% after exposure to 75 microM H2O2. The activity of CTP:phosphorylcholine cytidyltransferase was unchanged after H2O2 exposure when measured in whole cell homogenates. However, when measured in the microsomal fraction, cytidyltransferase activity significantly fell after exposure of type II cells to 75 microM H2O2. Activity in the cytosolic fractions remained unchanged. Similarly, microsomal cholinephosphotransferase was reduced after H2O2 exposure. We conclude that H2O2 decreases surfactant phosphatidylcholine biosynthesis independently of its ability to deplete intracellular ATP content. These deleterious effects may partially explain the diminished alveolar surfactant observed in patients with ARDS.

Asunto(s)

Inhibidores Enzimáticos/farmacología , Peróxido de Hidrógeno/farmacología , Fosfolípidos/biosíntesis , Alveolos Pulmonares/metabolismo , Surfactantes Pulmonares/biosíntesis , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Citidililtransferasa de Colina-Fosfato , Diacilglicerol Colinafosfotransferasa/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/administración & dosificación , Peróxido de Hidrógeno/administración & dosificación , Masculino , Nucleotidiltransferasas/antagonistas & inhibidores , Concentración Osmolar , Fosfatidilcolinas/biosíntesis , Alveolos Pulmonares/citología , Ratas , Ratas Sprague-Dawley

RESUMEN

1. The effect of brefeldin A (BFA) on generation of transport vesicles, synthesis of phosphoglycerides, sphingosine and ceramides, and utilization of the sphingolipid precursors in the formation of sphingomyelin and glycosphingolipids in Golgi was investigated. 2. In the presence of 5-10 micrograms/ml BFA, the incorporation of [3H]palmitate into glycerides, phosphoglycerides and sphingolipids decreased 45-60%, and the production of endoplasmic reticulum transport vesicles was reduced 30-50%. 3. In Golgi membranes, the presence of 5-10 micrograms/ml BFA in the mixture, assembled to generate Golgi vesicles, evoked inhibitory effect on the synthesis of sphingomyelin, glycosphingolipids and phosphatidylcholine. On average, the synthesis of the sphingolipids and phosphatidylcholine and production of Golgi transport vesicles declined to 30-40%. 4. Addition of 5-10 micrograms/ml BFA to the assay mixture prepared to measure the activity of cytidylyltransferase, phosphocholine diacylglyceroltransferase, and serine palmitoyltransferase, caused up to 50% inhibition of the enzymes involved in the synthesis of phosphatidylcholine and up to 70% inhibition of the enzyme generating 3-ketosphinganine. 5. The results suggest that BFA inhibits the synthesis of phosphoglycerides and sphingolipids. This, at first, is displayed in reduced production of endoplasmic reticulum and Golgi transport vesicles, while the depletion of sphingolipids abrogates the identity of Golgi membranes.

Asunto(s)

Ciclopentanos/farmacología , Retículo Endoplásmico/metabolismo , Glicerofosfatos/biosíntesis , Aparato de Golgi/efectos de los fármacos , Lípidos de la Membrana/biosíntesis , Esfingolípidos/biosíntesis , Aciltransferasas/antagonistas & inhibidores , Autorradiografía , Brefeldino A , Fraccionamiento Celular , Ceramidas/biosíntesis , Citidililtransferasa de Colina-Fosfato , Diacilglicerol Colinafosfotransferasa/antagonistas & inhibidores , Electroforesis en Gel de Poliacrilamida , Retículo Endoplásmico/efectos de los fármacos , Glicosilación , Aparato de Golgi/metabolismo , Nucleotidiltransferasas/antagonistas & inhibidores , Fosfatidilcolinas/biosíntesis , Serina C-Palmitoiltransferasa , Esfingosina/metabolismo

RESUMEN

The control of phosphatidylcholine biosynthesis in the hamster liver was examined. Livers of hamsters fasted for 24 and 48 h were perfused with labelled choline. Under both fasting conditions, the incorporation of labelled choline into phosphatidylcholine was reduced. After 48 h of fasting, the 52% reduction in phosphatidylcholine biosynthesis was caused by changes in several factors including a diminishing rate of choline uptake and severe reductions in the pool sizes of ATP and CTP (to 33-37% control values) which resulted in a decrease in the pools of choline-containing metabolites. The activation of cytidylyltransferase after 48 h of fasting might be regarded as a compensatory mechanism for the maintenance of phosphatidylcholine biosynthesis. After 24 h of fasting, a 25% reduction in phosphatidylcholine biosynthesis was observed. The ATP and CTP levels were decreased but the reduction was not severe enough to affect the choline uptake or the labelling of the phosphocholine fraction. The activities of the cytidylyltransferase remained unchanged but an accumulation of labelled CDP-choline was detected. Although choline-phosphotransferase activity was not changed in the microsomes, the enzyme activity was attenuated in the postmitochondrial fraction. Further analysis revealed that cholinephosphotransferase in the liver was inhibited by an endogenous inhibitor in the cytosol which was later identified as argininosuccinate. The level of argininosuccinate was elevated during fasting and the change quantitatively accounted for the attenuation of cholinephosphotransferase activity. The inhibition of choline-phosphotransferase by argininosuccinate, coupled with a substantial decrease in the diacylglycerol level, would provide the hamster liver with an immediate mechanism for the transient modulation of phosphatidylcholine biosynthesis during short-term fasting.

Asunto(s)

Ácido Argininosuccínico/farmacología , Diacilglicerol Colinafosfotransferasa/metabolismo , Privación de Alimentos/fisiología , Hígado/enzimología , Fosfatidilcolinas/metabolismo , Animales , Ácido Argininosuccínico/aislamiento & purificación , Compartimento Celular , Colina/metabolismo , Colina Quinasa/análisis , Citidililtransferasa de Colina-Fosfato , Cricetinae , Diacilglicerol Colinafosfotransferasa/antagonistas & inhibidores , Diacilglicerol Colinafosfotransferasa/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Masculino , Mesocricetus , Nucleotidiltransferasas/análisis , Fracciones Subcelulares/enzimología

RESUMEN

The solubilization and partial purification of cholinephosphotransferase (CDPcholine:1,2-diacylglycerol cholinephosphotransferase, EC 2.7.8.2) from rat liver microsomes were examined in the presence of ionic (sodium deoxycholate), nonionic (Triton X-100, n-octylglycoside), or zwitter ionic (CHAPS) detergents. Among the four detergents tested, only sodium deoxycholate was found to be an efficient solubilizer of cholinephosphotransferase activity from microsomal membranes, whereas the other three detergents caused irreversible inactivation of the enzyme at the solubilization step. Addition of phospholipids at the solubilization step, or after solubilization of the membrane proteins, could not preserve or reconstitute activity to any extent. The sodium deoxycholate-solubilized activity was partially purified by gel permeation chromatography (Superose 12HR). The partially purified preparation appeared to consist of a large aggregate containing phospholipids; further dissociation of the protein-phospholipid complex caused complete inactivation of the enzyme. The partially purified cholinephosphotransferase showed a specific activity of 100-130 nmol/min/mg protein, which is the highest activity reported to date from any tissue source; this amounts to a 4-fold enrichment of cholinephosphotransferase activity from the original KCl-washed rat liver microsomes. Ethanolaminephosphotransferase (CDPethanolamine:1,2-diacylglycerol ethanolaminephosphotransferase, EC 2.7.8.1) activity was copurified and 6-fold enriched with a total recovery of 60%. During the purification of cholinephosphotransferase activity, a putative endogenous inhibitor of cholinephosphotransferase was also solubilized and was isolated from the microsomal membranes. This heat-labile, nondialyzable inhibitor was shown to act specifically on cholinephosphotransferase and not on ethanolaminephosphotransferase. Further characterization of the inhibitory activity revealed that it may act at the binding step of the cholinephosphotransferase to its lipid substrate, diacylglycerol.

Asunto(s)

Diacilglicerol Colinafosfotransferasa/química , Microsomas Hepáticos/enzimología , Animales , Cromatografía en Gel , Ácido Desoxicólico , Detergentes/farmacología , Diacilglicerol Colinafosfotransferasa/antagonistas & inhibidores , Diacilglicerol Colinafosfotransferasa/aislamiento & purificación , Diálisis , Diglicéridos/farmacología , Electroforesis en Gel de Poliacrilamida , Femenino , Concentración de Iones de Hidrógeno , Magnesio/farmacología , Masculino , Manganeso/farmacología , Octoxinol , Polietilenglicoles/farmacología , Cloruro de Potasio/farmacología , Ratas , Ratas Wistar , Solubilidad

RESUMEN

Cholinephosphotransferase (CPT) and ethanolaminephosphotransferase (EPT) are the enzymes catalyzing the last step of the de novo pathway for phosphatidylcholine and phosphatidylethanolamine synthesis, respectively. A major limitation for the complete characterization of the reactions catalyzed by the two enzymes derives from their poor stability in detergent-containing buffers. CPT is heavily inactivated, when native membranes are solubilized using a series of detergents, whereas EPT activity is better preserved during solubilization. An investigation of the factors which could play a role in preserving both enzymes from inactivation was carried out. The dramatic loss of enzymatic activities occurring upon dilution of solubilized membranes with detergent-containing buffers can be reduced by supplementing the dilution medium with phospholipids. The addition of Mn2+ ions to the dispersion buffer increases the stability of both enzymes. The procedure previously described for solubilizing EPT from rat brain microsomes has been modified on the basis of this evidence. Microsomes were solubilized in buffered detergent solutions containing Mn2+ ions and both CPT and EPT were partially purified in their active form by anion-exchange chromatography.

Asunto(s)

Diacilglicerol Colinafosfotransferasa/metabolismo , Etanolaminofosfotransferasa/metabolismo , Animales , Encéfalo/enzimología , Cromatografía por Intercambio Iónico , Detergentes , Diacilglicerol Colinafosfotransferasa/antagonistas & inhibidores , Diacilglicerol Colinafosfotransferasa/aislamiento & purificación , Estabilidad de Enzimas , Membranas Intracelulares/enzimología , Microsomas/enzimología , Octoxinol , Polietilenglicoles , Ratas , Solubilidad

RESUMEN

The biosynthesis of platelet-activating factor (PAF), a phospholipid autocoid with potent ulcerogenic properties that is produced in secretory exocrine glands by physiological secretagogues, was assessed in microsomal preparations of glandular gastric mucosa. For this purpose, 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine (lyso-PAF):acetyl-CoA acetyltransferase (EC 2.3.1.67); the enzymes of the 'de novo' pathway: 1-O-alkyl-2-lyso-sn-glycero-3-phosphate (alkyl-lyso-GP):acetyl-CoA acetyltransferase and 1-O-alkyl-2-acetyl-sn-glycerol (alkylacetyl-G):CDP-choline cholinephosphotransferase (EC 2.7.8.16); and some enzymes involved in the catabolism of PAF and lyso-PAF were assayed. Only the enzymes of the 'de novo' pathway and small amounts of PAF acetylhydrolase, phospholipase A2 and a lysophospholipase D acting on either lipids could be detected in the gastric preparations, whereas lyso-PAF:acetyl-CoA acetyltransferase activity was undetectable. The specific activity of alkyl-lyso-GP:acetyl-CoA acetyltransferase in the gastric mucosa was about one-tenth of that found in spleen microsomes and its apparent Km for acetyl-CoA was 454 microM compared with 277 microM in spleen microsomes. Glandular mucosa homogenates contained preformed PAF at a concentration of 2.7 +/- 0.7 ng equivalents of PAF (hexadecyl)/mg of protein. When gastric microsomes were incubated with micromolar concentrations of fatty acids (arachidonic, palmitic and oleic) prior to the assay of dithiothreitol (DTT)-insensitive cholinephosphotransferase, a dose-dependent reduction in the formation of PAF was observed, arachidonic acid being the most potent inhibitor, followed by linoleic acid (only tested on spleen microsomes) and oleic acid. By contrast, 1,2-diolein and phosphatidylcholine (dipalmitoyl) showed no or little effect. These results indicate that glandular gastric mucosa can produce PAF through the 'de novo' pathway, and that fatty acids, especially unsaturated, can reduce that synthesis by modulating the expression of DTT-insensitive cholinephosphotransferase.

Asunto(s)

Ácidos Grasos/farmacología , Mucosa Gástrica/metabolismo , Factor de Activación Plaquetaria/biosíntesis , 1-Alquil-2-acetilglicerofosfocolina Esterasa , Acetiltransferasas/metabolismo , Animales , Ácido Araquidónico , Ácidos Araquidónicos/farmacología , Diacilglicerol Colinafosfotransferasa/antagonistas & inhibidores , Mucosa Gástrica/efectos de los fármacos , Técnicas In Vitro , Cinética , Fosfatidilcolinas/farmacología , Fosfolipasas A/metabolismo , Fosfolipasas A2 , Hidrolasas Diéster Fosfóricas/metabolismo , Fosfotransferasas/metabolismo , Ratas , Ratas Endogámicas