RESUMEN
Endocytosis is required for many cellular pivotal processes, including membrane recycling, nutrient uptake, and signal transduction. This complex process is particularly relevant in polarized cells, such as neurons. Previous studies have demonstrated that alcohol alters intracellular traffic, including endocytosis, in several cell types. However, information on the effect of chronic alcohol exposure on this process in neurons is scarce. As an approach, we investigated the effect of alcohol exposure on the internalization of two widely used endocytic markers, albumin and transferrin, in developing hippocampal neurons in primary culture. The effect of this treatment on the levels of several representative proteins involved in the endocytic process was also analyzed. Some of these proteins are also involved in the organization of the actin cytoskeleton. Pretreatment of cells with inhibitors chlorpromazine or nystatin indicates that albumin is internalized mainly by caveolin-dependent endocytosis. On the other hand, alcohol decreases the endocytosis of both markers, although no qualitative changes in the distribution of either of these molecules were observed. Finally, the effect of ethanol on the proteins analyzed was heterogeneous. Alcohol decreases the levels of clathrin, AP-2, SNX9, Rab5, Rab11, EEA1, Cdc42, or RhoA but increases the amount of Arf6. Moreover, alcohol does not affect the levels of caveolin1, dynamin1, Rab7, and LAMP2. This toxic effect of alcohol on endocytosis could affect some of the important neuronal activities, which depend on this process, including cell signaling. Our results in neurons also stress the notion that one of the main targets of ethanol is intracellular transport.
Asunto(s)
Depresores del Sistema Nervioso Central/toxicidad , Endocitosis/efectos de los fármacos , Endosomas/efectos de los fármacos , Etanol/toxicidad , Neuronas/efectos de los fármacos , Albúminas/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Biomarcadores/metabolismo , Células Cultivadas , Depresores del Sistema Nervioso Central/metabolismo , Clatrina/metabolismo , Endocitosis/fisiología , Endosomas/metabolismo , Etanol/metabolismo , Femenino , Hipocampo/citología , Hipocampo/efectos de los fármacos , Hipocampo/embriología , Proteínas del Tejido Nervioso/metabolismo , Neuronas/patología , Neuronas/fisiología , Ratas , Transferrina/metabolismoRESUMEN
Nucleocytoplasmic transport is a crucial process for cell function. We assessed the general effect of chronic alcohol exposure on this transport in growing astrocytes for the first time. Import and export of proteins to the nucleus were examined by pulse-chase experiments using (3)H-methionine, and we showed that ethanol induces a delay in both processes. Furthermore, we took an approach to evaluate the mechanisms involved in this effect. Whereas alcohol did not affect the amount and the distribution of several representative proteins that participate in nuclear import, such as RanBP1, RanGAP1 and the importins alpha2 and beta3, it decreased the amount of Exp1/CRM1, which is a general export receptor involved in the nuclear export. In addition, the density and distribution of nuclear pore complexes, which contribute to nucleocytoplasmic transport, were also affected by ethanol. These effects can be related with changes found in the content of several proteins associated with the nuclear envelope and the nuclear pore complex structure such as lamins A/C, and nucleoporins p62 and RanBP2, respectively. These results suggest that ethanol could interfere with some of the important processes regulated by nucleocytoplasmic transport in astrocytes and support the idea that one of the main ethanol targets is intracellular transport.
Asunto(s)
Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Etanol/toxicidad , Transporte Activo de Núcleo Celular/efectos de los fármacos , Transporte Activo de Núcleo Celular/fisiología , Animales , Astrocitos/ultraestructura , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Núcleo Celular/ultraestructura , Células Cultivadas , RatasRESUMEN
AIMS: Glycoproteins, such as adhesion molecules and growth factors, participate in the regulation of nervous system development. Ethanol affects the synthesis, intracellular transport, distribution, and secretion of N-glycoproteins in different cell types, including astrocytes and hepatocytes, suggesting alterations in the glycosylation process. We analysed the effect of exposure to low doses of ethanol (30 mm, 7 days) on glycosylation in cultured hippocampal neurons. METHODS: Neurons were incubated for short (5 min) and long (90 min) periods with the radioactively labelled carbohydrate precursors 2-deoxy-glucose, N-acetyl-D-mannosamine and mannose. The uptake and metabolism of these precursors, as well as the radioactivity distribution in protein gels, were analysed. The levels of the glucose transporters GLUT1 and GLUT3 were also determined. RESULTS: Ethanol exposure reduces the synthesis of proteins, DNA and RNA and decreased the uptake of mannose, but not of 2-deoxy-glucose and N-acetyl-D-mannosamine, and it increased the protein levels of both glucose transporters. Moreover, it altered the carbohydrate moiety of several proteins. Finally, alcohol treatment results in an increment of cell surface glycoconjugates containing terminal non-reduced mannose. CONCLUSIONS: Alcohol-induced alterations in glycosylation of proteins in neurons could be a key mechanism involved in the teratogenic effects of alcohol exposure on brain development.
Asunto(s)
Etanol/farmacología , Hipocampo/citología , Monosacáridos/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Western Blotting , División Celular , Células Cultivadas , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Técnica del Anticuerpo Fluorescente Indirecta , Transportador de Glucosa de Tipo 1 , Glicosilación/efectos de los fármacos , Microscopía Electrónica , RatasRESUMEN
Ethanol exposure induces retention of glycoproteins in growing astrocytes. We examined the intracellular sites at which this retention occurs and investigated whether this effect is accompanied by alterations in the Golgi complex and microtubular system. We studied the effects of ethanol on the Golgi complex structure, as well as on the secretory pathway functionality by monitoring both the transport of the VSV-G protein and the protein levels of several molecules involved in the regulation of this pathway. Ethanol was found to delay VSV-G transport, modify Golgi complex morphology, and reduce the number of secretory vesicles. Moreover, ethanol affected the levels of mannosidase II, p58, betaCOP, rbet1, and several Rab GTPases. It also affected microtubule organization and polymerization and the levels of the motor proteins kinesin and dynein. Most of these effects were dose-dependent. These alterations, together with those previously reported concerning biosynthesis of glycoconjugates, provide novel insights into how ethanol impairs brain development.
Asunto(s)
Astrocitos/efectos de los fármacos , Encéfalo/efectos de los fármacos , Encéfalo/crecimiento & desarrollo , Etanol/toxicidad , Aparato de Golgi/efectos de los fármacos , Trastornos del Sistema Nervioso Inducidos por Alcohol/metabolismo , Trastornos del Sistema Nervioso Inducidos por Alcohol/fisiopatología , Animales , Astrocitos/metabolismo , Encéfalo/fisiopatología , Células Cultivadas , Proteína Coatómero/efectos de los fármacos , Proteína Coatómero/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Trastornos del Espectro Alcohólico Fetal/metabolismo , Trastornos del Espectro Alcohólico Fetal/fisiopatología , Aparato de Golgi/metabolismo , Lectinas de Unión a Manosa/efectos de los fármacos , Lectinas de Unión a Manosa/metabolismo , Manosidasas/efectos de los fármacos , Manosidasas/metabolismo , Proteínas de la Membrana/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Proteínas Motoras Moleculares/efectos de los fármacos , Proteínas Motoras Moleculares/metabolismo , Embarazo , Efectos Tardíos de la Exposición Prenatal/metabolismo , Efectos Tardíos de la Exposición Prenatal/fisiopatología , Proteínas Qc-SNARE/efectos de los fármacos , Proteínas Qc-SNARE/metabolismo , Ratas , Vesículas Secretoras/efectos de los fármacos , Vesículas Secretoras/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Proteínas de Transporte Vesicular/efectos de los fármacos , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Unión al GTP rab/efectos de los fármacos , Proteínas de Unión al GTP rab/metabolismoRESUMEN
AIMS: Prenatal ethanol exposure (PEA) increases both liver weight and total protein content in the Golgi complex and alters its morphological and functional properties. As PEA-induced protein retention could be the synergetic consequence of alterations in the cytoskeleton and in the glycan biosynthesis, and there are no data that in liver PEA perturbs the cytoskeleton, we examined in hepatocytes whether PEA affects the main cytoskeleton elements. We also analysed whether ethanol induces glycoprotein microheterogeneity by altering the sugar composition of glycoproteins. METHODS: Livers from 0-day newborn control and PEA rats were used. The carbohydrate moiety of glycoproteins was determined by lectin blotting. The content and intracellular distribution of cytoskeleton proteins was analysed using immunoblotting, immunofluorescence and immunogold. RESULTS: PEA delayed the post-Golgi transport of albumin but not of transferrin. PEA also increased the levels of cytokeratin and tubulin, but it decreased the amount of tubulin capable of assembling into functional microtubules. PEA perturbed the distribution of cytokeratin and tubulin and induced microheterogeneity in several glycoproteins. CONCLUSIONS: PEA-induced retention of proteins in fetal hepatocytes could be the result of an alteration of glycoprotein biosynthesis and cytoskeleton-mediated transport.