RESUMEN
BACKGROUND AND AIMS: Oxidized LDL (oxLDL) or pro-inflammatory stimuli lead to increased oxidative stress linked to endothelial dysfunction and atherosclerosis. The oxLDL receptor-1 (LOX1) is elevated within atheromas and cholesterol-lowering statins inhibit LOX1 expression. Berberine (BBR), an alkaloid extracted from plants of gender Berberis, has lipid-lowering and anti-inflammatory activity. However, its role in regulating LOX1-mediated signaling is still unknown. The aim of this study was to investigate the effect of BBR on oxLDL- and TNFα-induced endothelial dysfunction in human umbilical vein endothelial cells (HUVECs) and to compare it with that of lovastatin (LOVA). METHODS AND RESULTS: Cytotoxicity was determined by lactate dehydrogenase assay. Antioxidant capacity was measured with chemiluminescent and fluorescent method and intracellular ROS levels through a fluorescent dye. Gene and protein expression levels were assayed by qRT-PCR and western blot, respectively. HUVECs exposure to oxLDL (30 µg/ml) or TNFα (10 ng/ml) for 24 h led to a significant increase in LOX1 expression, effect abrogated by BBR (5 µM) and LOVA (5 µM). BBR but not LOVA treatment abolished the TNFα-induced cytotoxicity and restored the activation of Akt signaling. In spite of a low direct antioxidant capacity, both compounds reduced intracellular ROS levels generated by treatment of TNFα but only BBR inhibited NOX2 expression, MAPK/Erk1/2 signaling and subsequent NF-κB target genes VCAM and ICAM expression, induced by TNFα. CONCLUSIONS: These findings demonstrated for the first time that BBR could prevent the oxLDL and TNFα - induced LOX1 expression and oxidative stress, key events that lead to NOX, MAPK/Erk1/2 and NF-κB activation linked to endothelial dysfunction. CHEMICAL COMPOUNDS STUDIED IN THIS ARTICLE: Berberine (PubChem CID: 2353); Lovastatin (PubChem CID: 53232).
Asunto(s)
Antiinflamatorios/farmacología , Antioxidantes/farmacología , Berberina/farmacología , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Lipoproteínas LDL/farmacología , Lovastatina/farmacología , Receptores Depuradores de Clase E/agonistas , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Citoprotección , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/patología , Humanos , Glicoproteínas de Membrana/metabolismo , NADPH Oxidasa 2 , NADPH Oxidasas/metabolismo , FN-kappa B/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Receptores Depuradores de Clase E/metabolismo , Transducción de Señal/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Alzheimer's disease (AD) is the most common cause of dementia, clinically characterized by loss of memory and progressive deficits in different cognitive domains. An emerging disease-modifying approach to face the multifactorial nature of AD may be represented by the development of Multi-Target Directed Ligands (MTDLs), i.e., single compounds which may simultaneously modulate different targets involved in the neurodegenerative AD cascade. The structure of tacrine, an acetylcholinesterase (AChE) inhibitor (AChEI), has been widely used as scaffold to provide new MTDLs. In particular, its homodimer bis(7)tacrine represents an interesting lead compound to design novel MTDLs. Thus, in the search of new rationally designed MTDLs against AD, we replaced the heptamethylene linker of bis(7)tacrine with the structure of cystamine, leading to cystamine-tacrine dimer. In this study we demonstrated that the cystamine-tacrine dimer is endowed with a lower toxicity in comparison to bis(7)tacrine, it is able to inhibit AChE, butyrylcholinesterase (BChE), self- and AChE-induced beta-amyloid aggregation in the same range of the reference compound and exerts a neuroprotective action on SH-SY5Y cell line against H(2)O(2)-induced oxidative injury. The investigation of the mechanism of neuroprotection showed that the cystamine-tacrine dimer acts by activating kinase 1 and 2 (ERK1/2) and Akt/protein kinase B (PKB) pathways. This article is part of a Special Issue entitled 'Post-Traumatic Stress Disorder'.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Cistamina/química , Diseño de Fármacos , Tacrina/química , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Inhibidores de la Colinesterasa/química , Inhibidores de la Colinesterasa/farmacología , Inhibidores de la Colinesterasa/uso terapéutico , Cistamina/farmacología , Cistamina/uso terapéutico , Combinación de Medicamentos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/química , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Proteínas Proto-Oncogénicas c-akt/metabolismo , Tacrina/farmacología , Tacrina/uso terapéuticoRESUMEN
Despite extensive studies, the fundamental mechanisms responsible for the development and progression of cardiovascular diseases have not yet been fully elucidated. Recent experimental and clinical studies have suggested that reactive oxygen species play a major pathological role. Oxidative stress reduction induced by flavonoids has been regarded by many as the most likely mechanism in the protective effects of these compounds; however, there is an emerging view that flavonoids may also exert modulatory actions on protein kinase and lipid kinase signaling pathways. Quercetin, a major flavonoid present in the human diet, has been widely studied, and its biological properties are consistent with its protective role in the cardiovascular system. However, it remains unknown whether the cardioprotective effects of quercetin may also occur through the modulation of genes involved in cell survival. The main goal of this study was to examine the gene expression profiling of cultured rat primary cardiomyocytes treated with quercetin using DNA microarrays and to relate these data to functional effects. Results showed distinct temporal changes in gene expression induced by quercetin and a strong upregulation of phase 2 enzymes, highlighting quercetin ability to act also with an indirect antioxidant mechanism.
Asunto(s)
Expresión Génica/fisiología , Miocitos Cardíacos/metabolismo , Quercetina/fisiología , Animales , Supervivencia Celular , Perfilación de la Expresión Génica , Glutatión/metabolismo , Ventrículos Cardíacos/citología , Hemo-Oxigenasa 1/metabolismo , Peróxido de Hidrógeno/farmacología , Análisis de Secuencia por Matrices de Oligonucleótidos , Oxidantes/farmacología , Estrés Oxidativo/fisiología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The aim of this study was to investigate the potential of quercetin and two of its "in vivo" metabolites, 3'-O-methyl quercetin and 4'-O-methyl quercetin, to protect H9c2 cardiomyoblasts against H(2)O(2)-induced oxidative stress. As limited data are available regarding the potential uptake and cellular effects of quercetin and its metabolites in cardiac cells, we have evaluated the cellular association/uptake of the three compounds and their involvement in the modulation of two pro-survival signalling pathways: ERK1/2 signalling cascade and PI3K/Akt pathway. The three flavonols associated with cells to differing extents. Quercetin and its two O-methylated metabolites were able to reduce intracellular ROS production but only quercetin was able to counteract H(2)O(2) cell damage, as measured by MTT reduction assay, caspase-3 activity and DNA fragmentation assays. Furthermore, only quercetin was observed to modulate pro-survival signalling through ERK1/2 and PI3K/Akt pathway. In conclusion we have demonstrated that quercetin, but not its O-methylated metabolites, exerts protective effects against H(2)O(2) cardiotoxicity and that the mechanism of its action involves the modulation of PI3K/Akt and ERK1/2 signalling pathways.
Asunto(s)
Antioxidantes/metabolismo , Antioxidantes/farmacología , Estrés Oxidativo/efectos de los fármacos , Quercetina/metabolismo , Quercetina/farmacología , Transducción de Señal/efectos de los fármacos , Animales , Western Blotting , Caspasa 3/efectos de los fármacos , Caspasa 3/metabolismo , Fragmentación del ADN/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Peróxido de Hidrógeno/toxicidad , Fosfatidilinositol 3-Quinasas/efectos de los fármacos , Fosfatidilinositol 3-Quinasas/metabolismo , Ratas , Especies Reactivas de Oxígeno/metabolismoRESUMEN
BACKGROUND: Consumers consider plant food products from organic origin healthier than the corresponding conventional plant foods. Clear experimental evidence supporting this assumption is still lacking. AIM OF THE STUDY: To determine if the organic red oranges have a higher phyto-chemical content (i. e., phenolics, anthocyanins and ascorbic acid), total antioxidant activity and in vitro bioactivity, in terms of protective effect against oxidative damage at cellular level, than nonorganic red oranges. METHODS: Total phenolics were measured using the Folin Ciocalteau assay, while total anthocyanins and ascorbic acid levels were determined by spectrophotometric and HPLC analysis, respectively. In addition, the total antioxidant activity of red orange extracts was measured by the ABTS(*+) test. The ability of red orange extracts to counteract conjugated diene containing lipids and free radical production in cultured rat cardiomyocytes and differentiated Caco-2 cells, respectively, was assessed. RESULTS: Organic oranges had significantly higher total phenolics, total anthocyanins and ascorbic acid levels than the corresponding non-organic oranges (all p < 0.05). Moreover, the organic orange extracts had a higher total antioxidant activity than non-organic orange extracts (p < 0.05). In addition, our results indicate that red oranges have a strong capacity of inhibiting the production of conjugated diene containing lipids and free radicals in rat cardiomyocytes and differentiated Caco-2 cells, respectively. Statistically higher levels of antioxidant activity in both cell models were found in organically grown oranges as compared to those produced by integrated agriculture practice. CONCLUSIONS: Our results clearly show that organic red oranges have a higher phytochemical content (i. e., phenolics, anthocyanins and ascorbic acid), total antioxidant activity and bioactivity than integrated red oranges. Further studies are needed to confirm whether the organic agriculture practice is likely to increase the antioxidant activity of other varieties of fruits and vegetables.
Asunto(s)
Agricultura/métodos , Antioxidantes/análisis , Citrus sinensis/química , Alimentos Orgánicos , Antocianinas/análisis , Antocianinas/metabolismo , Antioxidantes/metabolismo , Ácido Ascórbico/análisis , Ácido Ascórbico/metabolismo , Células CACO-2/metabolismo , Cromatografía Líquida de Alta Presión/métodos , Alimentos Orgánicos/análisis , Humanos , Hidroxibenzoatos/análisis , Hidroxibenzoatos/metabolismo , Miocitos Cardíacos/metabolismo , Oxidación-ReducciónRESUMEN
Hypoxia/reoxygenation (H/R) is one of the causes of the increased expression of inducible nitric oxide synthase (iNOS) in cardiomyocytes. Since an aberrant NOS induction has detrimental consequences, we evaluated the effect of a green tea extract (GTE) on the NOS induction and activity in H/R-cardiomyocytes to define a nutritional strategy. Cultured rat cardiomyocytes were exposed to H/R in the presence of two concentrations of a green tea extract (GTE), which is reported to inhibit NOS expression and activity in different cells. In cultured cardiomyocytes two NOS isoforms were constitutively expressed, but only iNOS was induced by H/R. GTE supplementation at the lowest concentration, comparable to that in human plasma after dietary consumption, was ineffective, while the highest, comparable to that achievable by dietary supplements, counteracted the effect of H/R on iNOS induction and activity. It is necessary to verify in humans the relationship between the modulation of NO production and green tea dietary consumption.
Asunto(s)
Hipoxia de la Célula , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/enzimología , Óxido Nítrico Sintasa/metabolismo , Oxígeno/metabolismo , Té , Animales , Células Cultivadas , Suplementos Dietéticos , Regulación Enzimológica de la Expresión Génica , Óxido Nítrico Sintasa de Tipo II , Ratas , Ratas WistarRESUMEN
Doxorubicin cardiotoxicity is associated with the generation of free radicals, and involves not only lipid peroxidation but also a decreased biosynthesis of highly unsaturated fatty acids, leading to significant modification in cardiomyocyte fatty acid composition. We have evaluated whether naturally occurring antioxidants could counteract this side-effect. Green tea is an excellent source of catechins; we supplemented cultured rat cardiomyocytes with different green tea extracts to relate their catechin content and composition to their ability in protecting cells against doxorubicin-induced damage. The determination of total lipid fatty acid composition, of conjugated diene production (indicator of lipid peroxidation), and of lactate dehydrogenase release revealed that supplementation with tea extracts could counteract significant modifications in the fatty acyl pattern due to doxorubicin exposure, although to different extents. These differences could be ascribed to the different total catechin content and to qualitative differences among the tea extracts, determined by HPLC analysis.
Asunto(s)
Doxorrubicina/toxicidad , Ácidos Grasos/metabolismo , Corazón/efectos de los fármacos , Peroxidación de Lípido/efectos de los fármacos , Extractos Vegetales/farmacología , Té/química , Alcadienos/análisis , Animales , Catequina/química , Células Cultivadas , Hidroliasas/biosíntesis , Peroxidación de Lípido/fisiología , Miocardio/citología , Extractos Vegetales/química , Ratas , Ratas WistarRESUMEN
Some thiazolylbenzimidazole-4,7-diones were synthesized and tested in vitro on two tumor cell lines. Compounds 2d and 2e show a very good activity on K562 cells, whereas compounds 2a and 2b are active on SW620 cells. The importance of the methoxy group on the quinone moiety is confirmed and the function at 4-position of the thiazole ring plays a determining role for the activity.
Asunto(s)
Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Bencimidazoles/síntesis química , Bencimidazoles/farmacología , Tiazoles/química , Replicación del ADN/efectos de los fármacos , Humanos , Células Tumorales CultivadasRESUMEN
The clinical usefulness of doxorubicin is limited by cardiotoxicity. We have demonstrated that doxorubicin has a dual negative effect on myocardial lipids, acting against highly unsaturated fatty acids (HUFAs) directly and desaturating/elongating enzymes required for their biosynthesis, thus decreasing linoleic and alpha -linolenic conversion to higher metabolites. Primary cultures of rat cardiomyocytes were challenged with different doxorubicin concentrations and doxorubicin exposure was followed by a 24-h recovery period in the absence or presence of serum, and of gamma -linolenic acid. Serum in the recovery medium did not appear to be essential for the restoration of the desaturating/elongating activities, and gamma -linolenic acid supplementation influenced only alpha -linolenic acid conversion. Serum, and particularly gamma-linolenic acid, were very important in increasing HUFA levels behind the pure biosynthesis. HUFA biosynthesis plays a role in counteracting doxorubicin toxicity, but it cannot completely overcome the depletion of these fatty acids; serum and exogenous gamma-linolenate are critical in filling the decreased HUFA pool.
Asunto(s)
Doxorrubicina/farmacología , Miocardio/citología , Ácido gammalinolénico/fisiología , Animales , Antineoplásicos/efectos adversos , Antineoplásicos/farmacología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Doxorrubicina/efectos adversos , Ácidos Grasos Insaturados/metabolismo , Miocardio/metabolismo , Ratas , Ratas Wistar , Factores de Tiempo , Ácido alfa-Linolénico/metabolismoRESUMEN
Arachidonic acid (AA) has been related to both stimulation and inhibition of cellular proliferation. During replicative senescence of human fibroblasts, increased levels of AA have been thought to play a causal role in the limited proliferative capacity of the cells. To clarify the role of AA in the proliferation of normal fibroblasts and in cellular senescence, we examined uptake from and release of AA into the culture media and its effects on DNA synthesis. Our results indicate that some aspects of AA metabolism in normal human fibroblasts aged in culture are significantly different in comparison to early passage cells. Particularly, AA release following different mitogenic stimulation is higher in senescent than in young cells. Notwithstanding this significant difference, AA, at the concentration used, has no inhibitory effect on fibroblast DNA synthesis. Moreover AA and prostaglandins are responsible for the proliferative block in neither senescent cells nor mediate ceramide inhibition of DNA synthesis. So our results suggest that the increasing AA release is not causal, but rather the result of in vitro aging.
Asunto(s)
Envejecimiento/metabolismo , Ácido Araquidónico/metabolismo , Ácido Araquidónico/farmacología , Ácido Araquidónico/fisiología , Línea Celular , Senescencia Celular/fisiología , Ceramidas/farmacología , ADN/antagonistas & inhibidores , ADN/biosíntesis , Ácidos Grasos/metabolismo , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Mitógenos/farmacología , Prostaglandinas/fisiologíaRESUMEN
The antioxidant activities of three different green tea extracts were investigated and compared by two different methods. By the first method, which evaluated the direct protective effect of the green tea extracts on lipid peroxidation, the extracts were added, at different concentrations, to a lipid model system, made by refined peanut oil, freshly submitted to a further bleaching and subjected to forced oxidation at 98 degrees C, by an oxidative stability instrument. By the second method, the effectiveness of the same extracts was checked in cultures of neonatal rat cardiomyocytes exposed to a free radical-generating system by evaluating conjugated diene production and lactate dehydrogenase release. All of the extracts revealed a strong antioxidant activity by both the methods, and a particular effectiveness was demonstrated by the extracts having higher amounts of (-)-epigallocathechin-3-gallate and (-)-epigallocathechin, as analyzed by reverse-phase HPLC analysis.
Asunto(s)
Catequina/química , Extractos Vegetales/farmacología , Té/química , Animales , Células Cultivadas , Estrés Oxidativo , Extractos Vegetales/química , Ratas , Ratas WistarRESUMEN
Sphingosine-1-phosphate (SPP) acts as a first messenger in immortalized human airway epithelial cells (CFNPE9o(-)), possibly interacting with an Edg family receptor. Expression of the SPP receptors Edg-1 and Edg-3, as well as a low level of Edg-5/H218, was detected in these cells, in agreement with their ability to specifically bind SPP. The related lipids, lysophosphatidic acid and sphingosylphosphorylcholine, were unable to displace SPP from its high affinity binding sites, suggesting that the biological responses to these different lysolipids are mediated by distinct receptors. SPP markedly inhibited forskolin-stimulated cAMP accumulation in a dose-dependent manner and caused a remarkable elevation of intracellular calcium, both effects being sensitive to pertussis toxin treatment. Most importantly, SPP stimulated phosphatidic acid formation, which was maximal after 2 min and decreased within 8-10 min. In the presence of butan-1-ol, suppression of SPP-induced phosphatidic acid formation and production of phosphatidylbutanol were found, clearly indicating activation of phospholipase D (PLD). This finding was also confirmed by analysis of the fatty acid composition of phosphatidic acid, showing an increase in the monounsaturated oleic acid only. The decrease of phosphatidic acid level after 8-10 min incubation with SPP was accompanied by a parallel increase of diacylglycerol production, which was abolished in the presence of butan-1-ol. This result indicates that activation of phospholipase D is followed by stimulation of phosphatidate phosphohydrolase activity. Phosphatidic acid formation was insensitive to protein kinase C inhibitors and almost completely inhibited by pertussis toxin treatment, suggesting that SPP activates phospholipase D via a G(i/o) protein-coupled receptor.
Asunto(s)
Células Epiteliales/enzimología , Proteínas I-kappa B , Lisofosfolípidos , Fosfolipasa D/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores Acoplados a Proteínas G , Sistema Respiratorio/enzimología , Esfingosina/análogos & derivados , Células 3T3 , Animales , Unión Competitiva/efectos de los fármacos , Calcio/metabolismo , Línea Celular Transformada , Colforsina/farmacología , AMP Cíclico/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Diglicéridos/biosíntesis , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Células Epiteliales/citología , Proteínas de Unión al GTP/metabolismo , Humanos , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Ratones , Inhibidor NF-kappaB alfa , Toxina del Pertussis , Ácidos Fosfatidicos/biosíntesis , ARN Mensajero/biosíntesis , Receptores Lisofosfolípidos , Sistema Respiratorio/citología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Esfingosina/metabolismo , Esfingosina/farmacología , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
An anomalous n-6 polyunsaturated fatty acid composition in plasma and erythrocyte membrane phospholipids, namely increased levels of arachidonic acid (AA), has been reported in calcium nephrolithiasis and has been proposed to play an important role in its pathogenesis. To confirm this, in rats we modified phospholipid AA levels by dietary manipulation of the delta-6-desaturase, the rate-limiting enzyme of the fatty acid biosynthetic pathway, and evaluated the effect on cellular and renal functions predisposing to lithogenesis. Increased AA levels led to conditions at risk for nephrolithiasis: higher oxalate flux and lower sodium cotransport in erythrocytes and a rise in urinary prostaglandin E2, calcium, sodium, and oxalate levels; reduced AA levels reversed these changes. In vitro, in human erythrocytes the incorporation of exogenous AA into membranes increased band 3 protein phosphorylation directly activating the Ser/Thr protein kinase CK1 and induced a parallel raise in band 3-mediated oxalate transport. These findings demonstrate the pivotal role of phospholipid AA in modulating erythrocyte and renal transport of calcium and oxalate.
Asunto(s)
Ácido Araquidónico/sangre , Calcio/orina , Dieta , Ácido Graso Desaturasas/metabolismo , Nefrocalcinosis/metabolismo , Oxalatos/orina , Fosfolípidos/sangre , Animales , Proteína 1 de Intercambio de Anión de Eritrocito/metabolismo , Ácido Araquidónico/farmacología , Quinasa de la Caseína II , Caseína Quinasas , Dinoprostona/orina , Membrana Eritrocítica/efectos de los fármacos , Membrana Eritrocítica/enzimología , Eritrocitos/efectos de los fármacos , Eritrocitos/metabolismo , Humanos , Linoleoil-CoA Desaturasa , Hígado/enzimología , Masculino , Nefrocalcinosis/etiología , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Ratas Sprague-Dawley , Sodio/orinaRESUMEN
The clinical use of the antitumoral doxorubicin (DOX) is limited by its cardiotoxicity, which is mediated through different mechanisms. The membrane lipid peroxidation induced by DOX may cause disruption of the unsaturated fatty acyl chains; in the endoplasmic reticulum, containing the system catalyzing the desaturation/elongation of fatty acids, DOX could interfere with the metabolism of linoleic and alpha-linolenic acids. Using primary cultures of neonatal rat cardiomyocytes we demonstrated that the exposure to different concentrations of DOX (10(-5) and 10(-7) M) for 24 h caused an increase in the production of conjugated dienes, an impairment in the desaturation/elongation of essential fatty acids, and a reduction in the cellular content of highly unsaturated fatty acids. Conversely, 1 h exposure to 10(-5) M DOX was sufficient to induce alterations in the desaturation/elongation of linoleic and alpha-linolenic acids, but did not cause either formation of conjugated dienes or modification of the fatty acyl pattern. Therefore, DOX has a dual negative effect, depending on its concentration and on the time of exposure, one directed against the membrane highly unsaturated fatty acids, the other against the system which is required for the synthesis of these fatty acids themselves. These two effects synergically act in causing heart cell damage.
Asunto(s)
Doxorrubicina/toxicidad , Ácidos Grasos Esenciales/metabolismo , Corazón/efectos de los fármacos , Animales , Células Cultivadas , Peroxidación de Lípido/efectos de los fármacos , Ratas , Ratas WistarRESUMEN
Gamma-linolenic acid (GLA) supplemented to neuroblastoma SK-N-BE, tubal carcinoma TG and colon carcinoma SW-620 cells was incorporated into phospholipids in all the cell lines (although to different extents), in a concentration- and time-dependent manner. All the cell lines were able to metabolize GLA to arachidonic acid, SK-N-BE being the most active. Supplementation with low GLA concentrations for short periods was not sufficient to impair cell proliferation; only higher amounts of GLA had an anti-proliferative effect also in short times. In these conditions, the antiproliferative effect of GLA is probably due to cellular dysfunction caused by fatty acid modifications.
Asunto(s)
Ácido gammalinolénico/farmacología , Ácido Araquidónico/metabolismo , División Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ácidos Grasos/análisis , Humanos , Fosfolípidos/química , Fosfolípidos/metabolismo , Timidina/metabolismo , Factores de Tiempo , Células Tumorales CultivadasRESUMEN
In long term (21 days) primary cultures of neonatal rat cardiomyocytes, utilized as a model of in vitro senescence, we investigated the dual effect of the time length in culture and of the supplementation with n-6:n-3 fatty acid mixtures on linoleic (LA) and alpha-linolenic acid (ALA) metabolism. Cardiomyocytes were divided into groups receiving: (1) control medium; (2) control medium plus n-3 fatty acids; (3) and (4) control medium plus n-6 and n-3 fatty acids in the ratio 1:2 or 2:1, respectively. In control cells. senescence caused a reduction in the conversion of LA and ALA, and the decrease in their metabolites was bypassed by the different supplementations. The fatty acid composition of cardiomyocyte lipids was therefore affected by both senescence and supplementation, as evidenced by the n-6:n-3 fatty acid ratio and the unsaturation index (U.I.) in cellular lipids. The final result of ageing in culture and of fatty acid supplementations was in all the groups of cells but one (n-6:n-3, 2:1) an unbalance in the n-6:n-3 fatty acid ratio. All the supplementations were able to counteract the decrease in the U.I. observed with senescence, but only the n-6:n-3 (2:1) was able to do so by increasing the cellular content of the fatty acids which are precursors of anti-aggregation eicosanoids without altering the n-6:n-3 fatty acid ratio.
Asunto(s)
Grasas de la Dieta/metabolismo , Ácidos Grasos Esenciales/metabolismo , Miocardio/metabolismo , Animales , Células Cultivadas , Senescencia Celular , Suplementos Dietéticos , Ácidos Grasos Omega-3/metabolismo , Ácidos Grasos Omega-6 , Ácidos Grasos Insaturados/metabolismo , Miocardio/citología , Ratas , Factores de TiempoRESUMEN
Extracellular sphingosylphosphorylcholine (SPC) caused a remarkable elevation in the intracellular Ca2+ concentration ([Ca2+]i) in immortalized human airway epithelial cells (CFNP9o-). An increase in total inositol phosphates formation was determined; however, the dose responses for [Ca2+]i elevation and inositol phosphates production were slightly different and, furthermore, PMA and pertussis toxin almost completely inhibited [Ca2+]i mobilization by SPC, whereas inositol phosphates production was only partially reduced. The possible direct interaction of SPC with Ca2+ channels of intracellular stores was determined by experiments with permeabilized cells, where SPC failed to evoke Ca2+ release, whereas lysophosphatidic acid was shown to be effective. The level of phosphatidic acid was increased by SPC only in the presence of AACOCF3, a specific inhibitor of phospholipase A2 (PLA2) and blocked by both pertussis toxin and R59022, an inhibitor of diacylglycerol kinase. R59022 enhanced diacylglycerol production by SPC and also significantly reduced [Ca2+]i mobilization. Only polyunsaturated diacylglycerol and phosphatidic acid were generated by SPC. Lastly, SPC caused stimulation of arachidonic acid release, indicating the involvement of PLA2. Taken together, these data suggest that, after SPC stimulation, phospholipase C-derived diacylglycerol is phosphorylated by a diacylglycerol kinase to phosphatidic acid, which is further hydrolysed by PLA2 activity to arachidonic and lysophosphatidic acids. We propose that lysophosphatidic acid might be the intracellular messenger able to release Ca2+ from internal stores.
Asunto(s)
Calcio/metabolismo , Fosfolípidos/metabolismo , Fosforilcolina/análogos & derivados , Sistema Respiratorio/efectos de los fármacos , Sistema Respiratorio/metabolismo , Esfingosina/análogos & derivados , Ácido Araquidónico/metabolismo , Bradiquinina/farmacología , Canales de Calcio/efectos de los fármacos , Canales de Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Línea Celular , Permeabilidad de la Membrana Celular , Diglicéridos/biosíntesis , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Humanos , Fosfatos de Inositol/biosíntesis , Líquido Intracelular/metabolismo , Ácidos Fosfatidicos/biosíntesis , Fosforilcolina/farmacología , Esfingosina/farmacología , Tapsigargina/farmacologíaRESUMEN
The sphingosine derivatives sphingosylphosphorylcholine (SPC) and sphingosine-1-phosphate (S1P) caused a similar elevation of the intracellular Ca2+ concentration ([Ca2+]i) in an immortalized airway epithelial cell line (CFNP9o-) incubated in Ca(2+)-free medium. The maximal effect was obtained with 2 microM SPC and 0.1 microM S1P and was sensitive to pre-incubation with pertussis toxin, indicating the involvement of a Gi/G(o) type of G protein. In Ca2+ containing medium, [Ca2+]i elevation by SPC was significantly higher than that by S1P, due to the fact that SPC was able to stimulate Mn2+ entry, whereas S1P was ineffective. SPC, but not S1P, caused a dose-dependent production of total inositol phosphates. Conversely, S1P, but not SPC, increased the level of phosphatidic acid. These findings suggest the presence of two distinct receptors, specific for SPC and S1P, respectively. Depletion of intracellular Ca2+ stores by SPC makes cells unable to respond to a subsequent addition of S1P. Conversely, cells do respond to SPC after a challenge with S1P, suggesting that the two receptors likely share one or more intracellular signalling component(s).
Asunto(s)
Calcio/metabolismo , Citosol/metabolismo , Lisofosfolípidos , Cavidad Nasal/metabolismo , Fosforilcolina/análogos & derivados , Esfingosina/análogos & derivados , Células Cultivadas , Relación Dosis-Respuesta a Droga , Células Epiteliales/metabolismo , Proteínas de Unión al GTP/metabolismo , Histamina/farmacología , Humanos , Fosfatos de Inositol/metabolismo , Magnesio/metabolismo , Ácido Palmítico/farmacología , Toxina del Pertussis , Ácidos Fosfatidicos/metabolismo , Fosforilcolina/farmacología , Esfingosina/farmacología , Estaurosporina/farmacología , Tapsigargina/farmacología , Factores de Tiempo , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
Delta-6-desaturase (D6D) activity is influenced by many nutritional and non-nutritional factors, among which one of the most important is aging. D6D activity could be susceptible to the dual influence of aging itself and of nutritional deficiencies, due to the reduced intake and/or absorption of essential nutrients. Particularly, vitamin B6 deficiency might be a crucial factor for D6D activity in aged people. Using 20 month old Sprague-Dawley rats fed a diet with a subnormal level of vitamin B6, we evaluated D6D activity for linoleic acid (LA) and alpha-linolenic acid (ALA) in liver microsomes, and the fatty acid composition of microsomal total lipids. We observed a diminished D6D activity for LA and also for ALA in vitamin B6-deficient animals, being approximately 63% and 81% respectively of the corresponding activity in control rats. As a consequence, significant modifications in the relative molar content of microsomal fatty acids were observed. The content of arachidonic and docosahexaenoic acid, the main products of the conversion of LA and ALA respectively, decreased, LA content increased and a decrease in the unsaturation index was observed in liver microsomes of B6-deficient rats. The foregoing results suggest that the impairment of D6D activity by vitamin B6 deficiency might be an important factor in decreasing the synthesis of n-6 and n-3 PUFAs. This may be particularly important in aging, where D6D activity is already impaired.