RESUMEN
Glycosaminoglycans (GAGs) are complexed with plasma proteins and proteolysis of plasma reduced the protein-GAG ratio about 140-fold. After dialysis, analysis by gradient PAGE revealed heparinase-1-sensitive GAGs, thus suggesting that heparin could be among the plasma GAGs. However, after dialysis most of the plasma GAGs were still not 'free'. PAGE of peptides resistant to proteolysis showed high molecular weight bands on the two sides of the dialysis membrane despite the 3.5 kDa molecular weight cut-off. Progressive dilution of the sample allowed passage of peptides appearing as high molecular weight bands in the diffusate. We interpret this phenomenon as the presence of low molecular weight peptides that aggregate when concentrated. Peptides on both sides of the membranes bound heparin.
Asunto(s)
Glicosaminoglicanos/sangre , Glicosaminoglicanos/aislamiento & purificación , Heparina/sangre , Heparina/aislamiento & purificación , Aminoácidos/análisis , Celulosa/análogos & derivados , Celulosa/metabolismo , Electroforesis en Acetato de Celulosa , Electroforesis en Gel de Poliacrilamida , Humanos , Peso Molecular , Péptidos/sangre , Péptidos/química , Péptidos/aislamiento & purificación , Unión ProteicaRESUMEN
Human MRC5 fibroblasts, at different passages in cultures, were used as an in vitro model to assess variations and/or induction of aging parameters under basal conditions or following sublethal oxidative stress by H2O2. DNA sensitivities to oxidatively-induced breakage, rather than basal levels of damaged DNA, were significantly different between cultures at low and high population doubling level (PDL): old cells maintained most of their DNA integrity even at high concentrations of H2O2, while young cells showed more extensive DNA damage which developed in a dose-dependent fashion. However, young cells pretreated with low doses of H2O2 exhibited increased resistance against further oxidative damage to DNA thus reproducing a senescent-like profile of sensitivity. In turn, DNA from old cultures incubated in a NAD precursor-free medium was more prone to H2O2-induced strand breaks mimicking DNA sensitivity of young cells. The extent of oxidatively-induced DNA damage in MRC5 populations correlated inversely with the levels of glutathione peroxidase (GPx) activity that almost doubled when cells passed from the young to the senescent stage. In addition, H2O2-pretreatment of young cells induced an increase in GPx expression approaching old cell values and promoted also the premature appearance of neutral beta-galactosidase activity and decreased c-fos expression upon serum stimulation, both of which were assumed to be characteristic traits of the senescent phenotype.
Asunto(s)
Senescencia Celular/fisiología , Daño del ADN , Fibroblastos/metabolismo , Glutatión Peroxidasa/metabolismo , Peróxido de Hidrógeno/metabolismo , Oxidantes/metabolismo , Línea Celular , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Humanos , Peróxido de Hidrógeno/farmacología , Oxidantes/farmacologíaRESUMEN
Divergent literature data are found concerning the effect of lactate on free radical production during exercise. To clarify this point, we tested the pro- or antioxidant effect of lactate ion in vitro at different concentrations using three methods: 1) electron paramagnetic resonance (EPR) was used to study the scavenging ability of lactate toward the superoxide aion (O(2)(-).) and hydroxyl radical (.OH); 2) linoleic acid micelles were employed to investigate the lipid radical scavenging capacity of lactate; and 3) primary rat hepatocyte culture was used to study the inhibition of membrane lipid peroxidation by lactate. EPR experiments exhibited scavenging activities of lactate toward both O(2)(-). and.OH; lactate was also able to inhibit lipid peroxidation of hepatocyte culture. Both effects of lactate were concentration dependent. However, no inhibition of lipid peroxidation by lactate was observed in the micelle model. These results suggested that lactate ion may prevent lipid peroxidation by scavenging free radicals such as O(2)(-). and.OH but not lipid radicals. Thus lactate ion might be considered as a potential antioxidant agent.
Asunto(s)
Antioxidantes/farmacología , Depuradores de Radicales Libres/farmacología , Ácido Láctico/farmacología , Estrés Oxidativo/efectos de los fármacos , Animales , Células Cultivadas , Espectroscopía de Resonancia por Spin del Electrón , Técnicas In Vitro , Ácido Linoleico/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Peroxidación de Lípido/fisiología , Hígado/citología , Micelas , Estrés Oxidativo/fisiología , Ratas , Superóxidos/metabolismoRESUMEN
Membrane lipid peroxidation in rat hepatocyte cultures was induced by a 5-h incubation with either ethanol (50 mM) or the chelate iron-nitrilotriacetic acid (Fe-NTA) (100 microM). To test the oxidative stress, two indices were measured simultaneously on the same sample: extracellular free malondialdehyde (MDA) measured by HPLC with a size exclusion column, and conjugated dienes (CD) determined by second derivative spectroscopy. With ethanol, both CD and MDA gave nearly the same values of lipid peroxidation, about 135% of the control value. With Fe-NTA, both indices indicated a higher lipid peroxidation, but the MDA and CD values were different. Iron lipid peroxidation evaluated by free MDA and CD was, 290 and 230%, respectively, of the control. This discrepancy could be ascribed to an increased decomposition of hydroperoxides by iron. In addition, the ratio of cis,trans and trans,trans conjugated dienes, which reflects the cellular redox status, remained unchanged after 5 h of lipid peroxidation induced either by ethanol or iron.
Asunto(s)
Alquenos/análisis , Peroxidación de Lípido , Hígado/metabolismo , Malondialdehído/análisis , Alquenos/metabolismo , Animales , Células Cultivadas , Cromatografía Líquida de Alta Presión , Estudios de Evaluación como Asunto , Espacio Extracelular/metabolismo , Ácidos Grasos Insaturados/análisis , Ácidos Grasos Insaturados/metabolismo , Hígado/citología , Malondialdehído/metabolismo , Microquímica/métodos , Oxidación-Reducción , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Espectrofotometría Ultravioleta , Tiobarbitúricos/análisisRESUMEN
The response of normal and transformed rat hepatocytes to oxidative stress was investigated. Isolated normal rat hepatocytes and differentiated hepatoma cells (the Fao cell line was derived from the Reuber H 35 rat hepatoma) in suspension were incubated with the ADP/Fe3+ chelate for 30 min at 37 degrees C. Membrane lipid oxidation was assessed by measuring (i) free malondialdehyde (MDA) production by a high-performance liquid chromatography (HPLC) procedure, (ii) membrane fatty acid disappearance as judged by capillary gas chromatography, and (iii) alpha-tocopherol oxidation as determined by HPLC and electrochemical detection. The addition of iron led to increased MDA production in normal as well as in transformed cells, and to simultaneous consumption of polyunsaturated fatty acids (PUFA) and alpha-tocopherol. In addition, in Fao cells more alpha-tocopherol was consumed during lipid peroxidation while less PUFA was oxidized. Lipid peroxidation was lower in tumoral hepatocytes than in normal cells. This could be due to a difference in membrane lipid composition because of a lower PUFA content and a higher alpha-tocopherol level in Fao cells. During oxidation, Fao cells produced 1.5 to 2 times less MDA than normal cells, while in the tumoral cells the amount of oxidized PUFA having 3 or more double bonds was 7 to 8 times lower. Therefore, measuring MDA alone as an index of lipid peroxidation did not allow for proper comparison of the membrane lipid oxidizability of transformed cells vs. the membrane lipid oxidizability of normal cells.
Asunto(s)
Ácidos Grasos/metabolismo , Peroxidación de Lípido , Neoplasias Hepáticas Experimentales/metabolismo , Vitamina E/metabolismo , Animales , Cromatografía Líquida de Alta Presión , Ácidos Grasos Insaturados/metabolismo , Hierro/farmacología , Masculino , Malondialdehído/metabolismo , Lípidos de la Membrana/metabolismo , Ratas , Ratas Sprague-Dawley , Células Tumorales CultivadasRESUMEN
Ethanol-induced lipid peroxidation was studied in primary rat hepatocyte cultures supplemented with ethanol at the concentration of 50 mM. Lipid peroxidation was assessed by two indices: (1) conjugated dienes by second-derivative UV spectroscopy in lipid extract of hepatocytes (intracellular content), and (2) free malondialdehyde (MDA) by HPLC-UV detection and quantitation for the incubation medium (extracellular content). In cultures supplemented with ethanol, free MDA increased significantly in culture media, whereas no elevation of conjugated diene level was observed in the corresponding hepatocytes. The cellular pool of low-mol-wt (LMW) iron was also evaluated in the hepatocytes using an electron spin resonance procedure. An early increase of intracellular LMW iron (< or = 1 hr) was observed in ethanol-supplemented cultures; it was inhibited by 4-methylpyrazole, an inhibitor of alcohol dehydrogenase, whereas alpha-tocopherol, which prevented lipid peroxidation, did not inhibit the increase of LMW iron. Therefore, the LMW iron elevation was the result of ethanol metabolism and was not secondarily induced by lipid hydroperoxides. Thus, ethanol caused lipid peroxidation in rat hepatocytes as shown by the increase of free MDA, although no conjugated diene elevation was detected. During ethanol metabolism, an increase in cellular LMW iron was observed that could enhance conjugated diene degradation.
Asunto(s)
Etanol/metabolismo , Etanol/farmacología , Hierro/metabolismo , Peroxidación de Lípido , Hígado/metabolismo , Animales , Células Cultivadas , Deferoxamina , Espectroscopía de Resonancia por Spin del Electrón , Fomepizol , Cinética , Peroxidación de Lípido/efectos de los fármacos , Hígado/efectos de los fármacos , Malondialdehído/metabolismo , Pirazoles/farmacología , Ratas , Factores de Tiempo , Vitamina E/farmacologíaRESUMEN
Quantitative data from experiments of gene expression are often normalized through levels of housekeeping genes transcription by assuming that expression of these genes is highly uniform. This practice is being questioned as it becomes increasingly clear that the level of housekeeping genes expression may vary considerably in certain biological samples. To date, the validation of reference genes in aging has received little attention and suitable reference genes have not yet been defined. Our aim was to evaluate the expression stability of frequently used reference genes in human peripheral blood mononuclear cells with respect to aging. Using quantitative RT-PCR, we carried out an extensive evaluation of five housekeeping genes, i.e. 18s rRNA, ACTB, GAPDH, HPRT1 and GUSB, for stability of expression in samples from donors in the age range 35-74 years. The consistency in the expression stability was quantified on the basis of the coefficient of variation and two algorithms termed geNorm and NormFinder. Our results indicated GUSB be the most suitable transcript and 18s the least for accurate normalization in PBMCs. We also demonstrated that aging is a confounding factor with respect to stability of 18s, HPRT1 and ACTB expression, which were particularly prone to variability in aged donors.
Asunto(s)
Envejecimiento/genética , Expresión Génica , Genes , Hipoxantina Fosforribosiltransferasa/genética , Hipoxantina Fosforribosiltransferasa/normas , Adulto , Anciano , Algoritmos , Humanos , Hipoxantina Fosforribosiltransferasa/metabolismo , Leucocitos Mononucleares/metabolismo , Persona de Mediana Edad , ARN Mensajero/genética , Estándares de Referencia , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodosRESUMEN
We measured both pyridine nucleotide levels and ribonucleotide reductase-specific activity in Yoshida ascites hepatoma cells as a function of growth in vivo and during recruitment from non-cycling to cycling state in vitro. Oxidized nicotinamide adenine dinucleotide (NAD+) and reduced nicotinamide adenine dinucleotide (NADP) levels remained unchanged during tumour growth, while NADP+ and reduced nicotinamide adenine dinucleotide phosphate (NADPH) levels were very high in exponentially growing cells and markedly decreased in the resting phase. Ribonucleotide reductase activity paralleled NADP(H) (NADP+ plus NADPH) intracellular content. The concomitant increase in both NADP(H) levels and ribonucleotide reductase activity was also observed during G1-S transition in vitro. Cells treated with hydroxyurea showed a comparable correlation between the pool size of NADP(H) and ribonucleotide reductase activity. On the basis of these findings, we suggest that fluctuations in NADP(H) levels and ribonucleotide reductase activity might play a critical role in cell cycle regulation.
Asunto(s)
Neoplasias Hepáticas Experimentales/metabolismo , NADP/metabolismo , NAD/metabolismo , Ribonucleótido Reductasas/metabolismo , Animales , Ciclo Celular , División Celular , Línea Celular , Hidroxiurea/farmacología , Técnicas In Vitro , RatasRESUMEN
Pyridine and adenine nucleotide levels were measured in Friend erythroleukaemia cells (FELC) stimulated to growth and induced to differentiate by hexamethylene bisacetamide (HMBA) and N'-methylnicotinamide (N'-MNAM). A three- to fourfold increase in the NADP(H) was found to parallel cell growth stimulation in both the presence and absence of differentiation inducers. NAD(H) increased about twofold in control and to a minor extent in HMBA-treated FELC but did not vary significantly in N'-MNAM-treated cells. ATP was significantly higher in control cells stimulated to growth than in resting ones, but it did not vary in inducer-treated cells. These data confirm the relationship between high NADP(H) levels and cell resumption to growth; moreover they show that NAD(H) pool reduction and NAD/NADH ratio rise are associated with the process of FELC differentiation. The activities of NAD pyrophosphorylase and NAD kinase are much more enhanced in growth-stimulated FELC than in resting ones. On the other hand transition from the quiescent to the proliferative state was accompanied by a decrease in the activity of poly(ADP-ribose) polymerase. A decrease in poly(ADP-ribose) polymerase activity was also found in differentiated cells in contrast to controls.
Asunto(s)
Nucleótidos de Adenina/metabolismo , Virus de la Leucemia Murina de Friend/aislamiento & purificación , Leucemia Eritroblástica Aguda/metabolismo , Leucemia Eritroblástica Aguda/patología , NADP/metabolismo , NAD/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol) , Acetamidas/farmacología , Adenosina Trifosfato/metabolismo , Animales , Diferenciación Celular/fisiología , División Celular/fisiología , Hematínicos/farmacología , Leucemia Eritroblástica Aguda/microbiología , Ratones , Niacinamida/análogos & derivados , Niacinamida/farmacología , Nicotinamida-Nucleótido Adenililtransferasa/análisis , Nicotinamida-Nucleótido Adenililtransferasa/metabolismo , Fosfotransferasas/análisis , Fosfotransferasas/metabolismo , Poli(ADP-Ribosa) Polimerasas/análisis , Poli(ADP-Ribosa) Polimerasas/metabolismo , Células Tumorales Cultivadas/química , Células Tumorales Cultivadas/metabolismo , Células Tumorales Cultivadas/patologíaRESUMEN
Conditions to induce and parameters to evaluate sublethal oxidative stress of cultured human fibroblasts have been investigated in the attempt to identify markers for a more accurate quantification of cell injury. Sublethal oxidative stress was obtained by treating fibroblasts with 0.5 mM H2O2 in DMEM plus 5% FCS for times not exceeding 60 min. Under these conditions cells remained viable throughout long-term incubation, showing no appreciable release of cytosolic enzymes into the medium. On the contrary, exposures of fibroblasts to 0.5 mM H2O2 for times > 60 min induced a lethal cell injury which was fully expressed 2 days later by massive monolayer wasting and leakage of cytosolic components. Early metabolic effects of sublethal stress consisted of a rapid and significant fall of both ATP and NAD+ pools. Concomitantly, there was a moderate increase (about threefold) in both ADP-ribosyl transferase activity and free [Ca2+]i, while the specific activity of glyceraldehyde-3-phosphate dehydrogenase was partially decreased upon treatment. Oxidative injury also caused delayed effects consisting of a large depression of both protein and DNA synthesis. However, while the former was partially restored within 10 days of incubation, the latter remained severely impaired, as encountered in a growth-arrested population. Microfilaments of H2O2-treated cells appeared to be morphologically altered due to partial fragmentation of cytoskeleton actin which, however, was still maintained in the polymerized form as F-actin. Moreover, sublethally injured fibroblasts exhibited a reduced adhesiveness to plastic once they were detached and reseeded into new dishes. Relative adhesion efficiencies (number of adherent cells at 16 h as a percentage of seeded cells) were found to correlate inversely with times of exposure to H2O2. This finding allowed the identification of a biological parameter which showed itself to be very sensitive to oxidative stress and was also useful for developing an assay to grade sublethal injury to fibroblasts.
Asunto(s)
Fibroblastos/efectos de los fármacos , Peróxido de Hidrógeno/toxicidad , Estrés Oxidativo , Citoesqueleto de Actina/efectos de los fármacos , Adenosina Trifosfato/metabolismo , Biomarcadores , Calcio/metabolismo , Adhesión Celular/efectos de los fármacos , Supervivencia Celular , Células Cultivadas , Proteínas del Citoesqueleto/metabolismo , ADN/biosíntesis , Fibroblastos/enzimología , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , NAD/metabolismo , Biosíntesis de ProteínasRESUMEN
The recruitment into the cycling state of resting Yoshida AH 130 hepatoma cells was studied with respect to its dependence on respiration in an experimental system wherein the overall energy requirement for this recruitment can be supplied by the glycolytic ATP. The G1-S transition of these cells, unaffected by 2,4-dinitrophenol (DNP) at concentrations which uncouple the respiratory phosphorylation, is impaired either by blocking the electron flow to oxygen by antimycin A or by adding an excess of some oxidizable substrates, chiefly pyruvate and oxalacetate. An experimental analysis, focused on pyruvate activity, showed that the inhibition of cell recruitment into S is not related to the depressing effects of this substrate on aerobic glycolysis of tumor cells, nor is it modified by forcing, in the presence of DNP, pyruvate oxidation through the tricarboxylic acid cycle as well as the overall oxygen consumption. Addition of suitable concentrations of preformed purine bases (mainly adenine), completely removes the block of the G1-S transition produced either by the excess of oxidizable substrates or by antimycin A. These findings indicate the existence of a respiration-linked step in purine metabolism, which restricts the above transition and is equally impaired by blocking the respiratory chain or by saturating it with an excess of reducing equivalents derived from unrelated oxidations. The inhibitory effects of pyruvate and antimycin A can be largely removed by the addition of folate and tetrahydrofolate, suggesting that the respiration-linked restriction point of tumor cell cycling involves the folate metabolism and its connections to purine synthesis.
Asunto(s)
Interfase , Neoplasias Hepáticas Experimentales/patología , Consumo de Oxígeno , Purinas/metabolismo , Adenina/farmacología , Animales , Antimicina A/farmacología , Ácido Fólico/farmacología , Glucólisis , Interfase/efectos de los fármacos , Neoplasias Hepáticas Experimentales/metabolismo , Oxaloacetatos/farmacología , Consumo de Oxígeno/efectos de los fármacos , Piruvatos/metabolismo , Piruvatos/farmacología , Ácido Pirúvico , Ratas , Ratas Endogámicas , Tetrahidrofolatos/farmacologíaRESUMEN
New methods based on ultraviolet and infrared spectroscopy were developed to quantify oxidized and unoxidized fatty acyl esters (FAE) in cells. For this study, rat hepatocyte cultures (2.5 x 10(6) cells) were submitted to an oxidative stress by a 5-h incubation with iron(III) chelated with nitrilotriacetic acid (100 microM). Control hepatocytes were incubated under the same conditions except in the absence of iron. After cell lipid extraction, oxidized FAE were evaluated by the second derivative of the conjugated-diene (CD) spectrum, which exhibited minima at 233 and 242 nm ascribed to trans,trans (t,t) and cis,trans (c,t) CD isomers, respectively. These minima were quantified in arbitrary units as d2 A/d lambda 2; hydroperoxide concentration was determined using a linear regression curve obtained from autoxidized linoleic acid micelles. Total (oxidized and unoxidized) FAE were measured by Fourier transform infrared spectroscopy using the absorption band at 1740 cm-1. A highly significant correlation coefficient (r = 0.992) was found for the standard curve performed with glycerol trioleate expressed as nanomoles fatty acid equivalents. The extent of lipid oxidation could be estimated by the sum of minima at 233 and 242 nm which allowed the calculation of hydroperoxide concentrations. The amount of oxidized FAE was related to the amount of total FAE in the same sample. The ratio of minima at 242 nm (c,t isomers) and 233 nm (t,t isomers) could provide an evaluation of cell antioxidant capacity. A decrease of this ratio would indicate a large depletion of radical termination antioxidants.
Asunto(s)
Ésteres/análisis , Ácidos Grasos/análisis , Animales , Quelantes/metabolismo , Quelantes/farmacología , Ésteres/metabolismo , Ácidos Grasos/metabolismo , Análisis de Fourier , Hierro/metabolismo , Hierro/farmacología , Peroxidación de Lípido , Hígado/química , Hígado/citología , Microquímica/métodos , Ácido Nitrilotriacético/metabolismo , Ácido Nitrilotriacético/farmacología , Oxidación-Reducción , Ratas , Ratas Sprague-Dawley , Espectrofotometría Infrarroja/métodos , Espectrofotometría Ultravioleta/métodosRESUMEN
We studied urokinase-type plasminogen activator (u-PA)-dependent chemotaxis and DNA synthesis in both human fibroblasts and LB6 mouse fibroblasts transfected with human u-PA receptor (u-PAR) gene (LB6 clone 19). Both cell lines have receptors for the amino-terminal fragment of u-PA (u-PA-ATF). We observed that u-PA and u-PA-ATF stimulated chemotactic migration of both LB6 clone 19 cells and human fibroblasts, which could be impaired by down-regulation of protein kinase C (PKC) with phorbol myristate acetate (PMA). While LB6 clone 19 cells were unable to undergo mitosis following exposure to either u-PA or u-PA-ATF, human fibroblasts were stimulated to mitosis by exogenous addition of native u-PA, and u-PA-ATF was ineffective. The mitogenic activity of u-PA on human fibroblasts could also be impaired by down-regulation of PKC with PMA. We studied second messenger formation following u-PAR stimulation. Neither inositol lipid metabolism nor intracellular Ca2+ content were affected, while an increase of diacylglycerol (DAG) generation was observed. Such DAG formation was related to de novo synthesis from glucose and was dependent on ligand-receptor interaction. Both u-PA-ATF and the native u-PA molecule were able to stimulate DAG formation, u-PA being from three to fourfold more efficient than ATF. These data suggest that u-PAR stimulation per se is sufficient to trigger DAG formation. The native molecule confers on the cell an additional stimulus, possibly related with the activation of a u-PA-catalytic site-dependent substrate. Such stimulation allows the cell to reach the DAG threshold level required to trigger DNA synthesis.
Asunto(s)
Fibroblastos/metabolismo , Receptores de Superficie Celular/metabolismo , Sistemas de Mensajero Secundario , Transfección , Activador de Plasminógeno de Tipo Uroquinasa/metabolismo , Animales , Movimiento Celular , Células Cultivadas , Quimiotaxis/efectos de los fármacos , Células Clonales , Regulación hacia Abajo , Fibroblastos/citología , Humanos , Ratones , Mitógenos/metabolismo , Fragmentos de Péptidos/metabolismo , Proteína Quinasa C/metabolismo , Receptores de Superficie Celular/química , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Acetato de Tetradecanoilforbol/farmacología , Transducción GenéticaRESUMEN
The tRNA content and aminoacyl-tRNA synthetases of regenerating liver in the phase of rapid growth were compared with those of livers from both intact and sham-operated rats. At 48 h after hepatectomy, the amount of active tRNA (called 'total acceptor capacity') is significantly higher in regenerating liver than in control livers, owing to a general, possibly not uniform, increase in the various tRNA families, which suggests that it may contribute to the increased protein synthesis and to decreased protein degradation as well. The activities of most, but not of all, aminoacyl-tRNA synthetases in cell sap of regenerating liver tend to be greater than normal. Increased activity of histidyl-tRNA synthetase fits in with the possibility that the mechanisms that control the rate of protein degradation through aminoacylation of tRNAHis in cultured cells [Scornik (1983) J. Biol. Chem. 258, 882-886] also operate in the liver and play a role in regeneration. Sedimentation analysis of cell sap in sucrose density gradients shows a shift of prolyl-tRNA synthetase activity toward the high-Mr form in regenerating liver. This change might be related to the positive protein balance and to growth in vivo, since it is also observed in the anaplastic Yoshida ascites hepatoma AH 130.
Asunto(s)
Aminoacil-ARNt Sintetasas/metabolismo , Regeneración Hepática , Hígado/metabolismo , ARN de Transferencia/metabolismo , Aminoácidos/metabolismo , Animales , Centrifugación por Gradiente de Densidad , Hepatectomía , Hígado/enzimología , Neoplasias Hepáticas Experimentales/metabolismo , Masculino , Nitrógeno/metabolismo , Ratas , Ratas EndogámicasRESUMEN
In this paper we describe a procedure to determine glycosaminoglycan and oligosaccharide composition of biological samples such as cell cultures or tissue explants. We demonstrate that heparin species of different molecular mass can be easily fractionated by sequential ethanol precipitation in 4.0 M guanidine hydrochloride. We studied by gradient polyacrylamide gel electrophoresis fractionation of standard heparin and heparin-derived oligosaccharides by anion-exchange chromatography on DEAE-Sephacel resin eluted by increasing concentration of guanidine hydrochloride. The use of guanidine salts followed by sequential precipitation by increasing ethanol concentration allowed recovery of heparin and heparin-derived oligosaccharides.
Asunto(s)
Precipitación Química , Etanol , Guanidina , Heparina/aislamiento & purificación , Animales , Aniones , Bovinos , Células Cultivadas , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Técnicas de Cultivo , Electroforesis en Gel de Poliacrilamida , Glicosaminoglicanos/aislamiento & purificación , Heparina/análisis , Peso Molecular , Oligosacáridos/aislamiento & purificación , SolubilidadRESUMEN
The aim of this study was to examine how macrophages could act on ethanol-induced oxidative stress in rat hepatocytes during inflammatory conditions, well-known to induce nitric oxide (NO) synthase. For this purpose, RAW 264.7 macrophages were added to primary rat hepatocyte cultures. Co-cultures were then supplemented with lipopolysaccharide (LPS) and interferon gamma (IFN) for 18 h, in order to induce NO synthase before the addition of 50 mM ethanol. In cultures of hepatocytes alone, the addition of LPS and IFN protected from ethanol-induced oxidative stress. It has been shown previously that NO generated in hepatocytes was responsible for this effect. When macrophages were added to primary rat hepatocyte cultures supplemented with LPS and IFN, protection provided by NO against ethanol-induced oxidative stress in hepatocytes ceased. Using a pretreatment of macrophages with N(g)-monomethyl-l-arginine, a NO synthase inhibitor, it was concluded that NO generated by macrophages was responsible for macrophage toxicity. Taken together, our observations suggest that NO biosynthesis in hepatocytes protects them from ethanol-induced oxidative stress, whereas NO production in macrophages deprives hepatocytes of this NO protection.
Asunto(s)
Depresores del Sistema Nervioso Central/farmacología , Etanol/farmacología , Hígado/efectos de los fármacos , Activación de Macrófagos/fisiología , Macrófagos/fisiología , Óxido Nítrico/biosíntesis , Estrés Oxidativo/efectos de los fármacos , Animales , Antineoplásicos/farmacología , Células Cultivadas , Interferón gamma/farmacología , Lipopolisacáridos/farmacología , Hígado/citología , Hígado/fisiología , Ratones , Estrés Oxidativo/fisiología , Ratas , Ratas Sprague-DawleyRESUMEN
Many controversies still exist with regard to the relationship between alcoholic intoxication and the occurrence of an oxidative stress. To attempt to resolve this question, first we investigated the induction by acute ethanol intoxication of lipid peroxidation in primary rat hepatocyte cultures using simultaneously two indices for each sample. When considering conjugated-diene indice, any lipid peroxidation elevation could be observed, whereas a net increase of extracellular free malondialdehyde was noted at 5 hours of incubation. These results led us to estimate the intracellular pool of low molecular weight iron which is known to be the iron species catalytically active in hydroperoxide degradation. An early enhancement of +20-30% of cellular low molecular weight iron was observed. Thus the discrepancy between conjugated dienes and malondialdehyde could be ascribed to an increase of hydroperoxide degradation into malondialdehyde by the transient cellular pool of low molecular weight iron. Lipid peroxidation and low molecular weight iron augmentation were linked to ethanol metabolism, since both were suppressed by the addition of 4-methylpyrazole, an alcohol dehydrogenase inhibitor. Superoxide dismutase activity was increased in the early incubation time (1 hour) and then markedly reduced. We conclude that ethanol metabolism can induce a lipid peroxidation accompanied by an elevation of intracellular pool of low molecular weight iron and a decrease of superoxide dismutase activity.
Asunto(s)
Etanol/farmacología , Hígado/efectos de los fármacos , Estrés Oxidativo , Alcohol Deshidrogenasa/metabolismo , Animales , Células Cultivadas , Etanol/metabolismo , Fomepizol , Hierro/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Hígado/metabolismo , Malondialdehído/metabolismo , Peso Molecular , NADPH-Ferrihemoproteína Reductasa/metabolismo , Pirazoles/farmacología , Ratas , Superóxido Dismutasa/metabolismo , Vitamina E/farmacologíaRESUMEN
Several studies are reviewed dealing with the mechanisms which regulate the cell cycle progression in normal and cancer cells. Using Yoshida AH 130 ascites tumor cells, it has been found that the G1-S transition of these cells is impaired by specific inhibitors of the electron flow through the respiratory chain (antimycin A), although respiratory ATP can be replaced by glycolytic ATP. The above transition can be also inhibited by the addition of physiologic substrates, mainly pyruvate, by a mechanism which appears linked to a modification of the cellular redox state and can be totally reversed by adding adenine to the culture medium. Adenine equally removes the block produced by antimycin A, pointing out a respiration-linked step of purine metabolism restricting the cell recruitment into S. A substantial protection of this step against the inhibitory effects of pyruvate and antimycin A has been obtained by the addition of folate and tetrahydrofolate, suggesting that the respiration-linked limiting step of tumor cell cycling involves folate metabolism and its connection to purine synthesis. The biologic relevance of these findings is stressed by the fact that pyruvate addition also inhibits the proliferation of concanavalin A-stimulated lymphocytes as well as of bone marrow hemopoietic cells in the presence of colony-stimulating factors. On the other hand, pyruvate only slightly affects the growth kinetics of malignant lymphoblasts and of Friend erythroleukemia cells either in the absence or in the presence of the differentiation inducer dimethylsulfoxide.
Asunto(s)
Ciclo Celular , Neoplasias Experimentales/metabolismo , Adenosina Trifosfato/biosíntesis , Aerobiosis , Animales , Transporte de Electrón , Glucólisis , Humanos , Neoplasias Experimentales/patología , Fosforilación Oxidativa , Consumo de Oxígeno , Purinas/metabolismo , Piruvatos/metabolismoRESUMEN
Kupffer cells and other macrophages play an important role in pathogenesis of toxicants in the liver. The aim of this study was to evaluate the effect of macrophages on hepatocyte production of nitric oxide (NO), which has been previously reported to be protective toward oxidative stress induced in primary rat hepatocytes. For this purpose, RAW 264.7 macrophages were added to primary rat hepatocytes at various ratios between macrophages and hepatocytes. These cocultures were supplemented with lipopolysaccharide (LPS) and interferon gamma (IFN-gamma) for 23 hours to induce NO synthase and trigger NO production. NO production was followed by quantification of nitrites in culture medium and dinitrosyl iron complexes (DNIC) in intact hepatocytes after separation from macrophages. In cocultured hepatocytes incubated with LPS and IFN-gamma, DNIC and nitrite levels decreased compared with those observed in hepatocytes cultured without macrophages in the same conditions. Moreover, inhibition of NO production in hepatocyte cocultures was macrophage-number-dependent. Macrophage-conditioned medium also inhibited NO production in hepatocytes, suggesting that the effect of macrophages was mediated by soluble factors. Among the soluble factors known to decrease NO levels are some cytokines, growth factors, reactive oxygen species, and prostaglandins. Ultrafiltration of macrophage-conditioned medium through a 500-d membrane to rule out higher-molecular-weight molecules, such as anti-inflammatory cytokines and growth factors, failed to restore NO production. In the same way, the use of superoxide dismutase (SOD) and catalase (CAT) to eliminate reactive oxygen species produced by macrophages did not lead to recovery of NO levels in hepatocytes. However, when NO synthesis was inhibited in macrophages by NG-monomethyl-L-arginine (L-NMMA), hepatocytes recovered the capacity to produce NO. A net decrease of prostaglandin E2 (PGE2) release by macrophages was concomitantly observed. Moreover, inhibition of PGE2 production in macrophages by indomethacin led to restoration of NO levels. Taken together, our observations suggest that NO synthesized by macrophages can decrease NO production in hepatocytes via PGE2 release. Because of the protective role of NO toward many liver injuries, it may be postulated that macrophages contribute through this mechanism to liver damage.
Asunto(s)
Dinoprostona/metabolismo , Hígado/metabolismo , Macrófagos/fisiología , Óxido Nítrico/biosíntesis , Animales , Línea Celular , Células Cultivadas , Técnicas de Cocultivo , Medios de Cultivo Condicionados , Inducción Enzimática , Inhibidores Enzimáticos/farmacología , Interferón gamma/farmacología , Lipopolisacáridos/farmacología , Ratones , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico Sintasa/biosíntesis , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , omega-N-Metilarginina/farmacologíaRESUMEN
The interaction of urokinase-type plasminogen activator (u-PA) or of u-PA amino-terminal fragment (u-PA-ATF) with the cell surface receptor (u-PAR) was found to stimulate an increase of glucose uptake in many cell lines, ranging from normal and transformed human fibroblasts, mouse fibroblasts transfected with human u-PAR, and cells of epidermal origin. Such increase of glucose uptake reached a peak within 5-10 min, depending on the cell line, and occurred through the facilitative glucose transporters (GLUTs), since it was inhibited by cytochalasin B. Each cell line showed a specific mosaic of glucose transporter isoforms, GLUT2 being the most widespread and GLUT1 the most abundant, when present. u-PAR stimulation was followed by translocation of GLUT1 from the microsomal to the membrane compartment, as shown by both immunoblotting and immunofluorescence of sonicated plasma membrane sheets and by activation of GLUT2 on the cell surface. Both translocation and activation resulted inhibitable by protein-tyrosine kinase inhibitors and independent of downregulation of protein kinase C (PKC). The increase of intracellular glucose was followed by neosynthesis of diacylglycerol (DAG) from glucose, as previously shown. Such neosynthesis was completely inhibited by impairment of facilitative GLUT transport by cytochalasin B. DAG neosynthesis was followed by activation of PKC, whose activity translocated into the intracellular compartment (PKM), where it probably phosphorylates substrates required for u-PAR-dependent chemotaxis. Our data show that u-PAR-mediated signal transduction, related with u-PA-induced chemotaxis, involves activation of tyrosine kinase-dependent glucose transporters, leading to increased de novo DAG synthesis from glucose, eventually resulting in activation of PKC.