RESUMEN
The specific traffic of the membrane components in neurons is a major requirement to establish and maintain neuronal domains-the axonal and the somatodendritic domains-and their polarized morphology. Unlike axons, dendrites contain membranous organelles, which are involved in the secretory pathway, including the endoplasmic reticulum, the Golgi apparatus and post-Golgi apparatus carriers, the cytoskeleton, and plasma membrane. A variety of molecules and factors are also involved in this process. Previous studies have shown that chronic alcohol exposure negatively affects several of these cell components, such as the Golgi apparatus or cytoskeleton in neurons. Yet very little information is available on the possible effects of this exposure on the remaining cell elements involved in intracellular trafficking in neurons, particularly in dendrites. By qualitative and quantitative electron microscopy, immunofluorescence and immunoblotting, we herein show that chronic exposure to moderate levels (30 mM) of ethanol in cultured neurons reduces the volume and surface density of the rough endoplasmic reticulum, and increases the levels of GRP78, a chaperone involved in endoplasmic reticulum stress. Ethanol also significantly diminishes the proportion of neurons that show an extension of Golgi into dendrites and dendritic Golgi outposts, a structure present exclusively in longer, thicker apical dendrites. Both Golgi apparatus types were also fragmented into a large number of cells. We also investigated the effect of alcohol on the levels of microtubule-based motor proteins KIF5, KIF17, KIFC2, dynein, and myosin IIb, responsible for transporting different cargoes in dendrites. Of these, alcohol differently affects several of them by lowering dynein and raising KIF5, KIFC2, and myosin IIb. These results, together with other previously published ones, suggest that practically all the protein trafficking steps in dendrites are altered to a greater or lesser extent by chronic alcohol exposure in neuronal cells, which may have negative repercussions for the development and maintenance of their polarized morphology and function.
Asunto(s)
Dendritas/efectos de los fármacos , Dendritas/ultraestructura , Etanol/farmacología , Transporte de Proteínas/efectos de los fármacos , Animales , Estrés del Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico Rugoso/efectos de los fármacos , Retículo Endoplásmico Rugoso/ultraestructura , Etanol/administración & dosificación , Femenino , Aparato de Golgi/efectos de los fármacos , Aparato de Golgi/ultraestructura , Proteínas de Choque Térmico/metabolismo , Proteínas Motoras Moleculares/efectos de los fármacos , Proteínas Motoras Moleculares/metabolismo , Neuronas/efectos de los fármacos , Neuronas/ultraestructura , Ratas WistarRESUMEN
Vitamin A or retinol which is the natural precursor of several biologically active metabolites can be considered the most multifunctional vitamin in mammals. Its deficiency is currently, along with protein malnutrition, the most serious and common nutritional disorder worldwide. It is necessary for normal embryonic development and postnatal tissue homeostasis, and exerts important effects on cell proliferation, differentiation and apoptosis. These actions are produced mainly by regulating the expression of a variety of proteins through transcriptional and non-transcriptional mechanisms. Extracellular matrix proteins are among those whose synthesis is known to be modulated by vitamin A. Retinoic acid, the main biologically active form of vitamin A, influences the expression of collagens, laminins, entactin, fibronectin, elastin and proteoglycans, which are the major components of the extracellular matrix. Consequently, the structure and macromolecular composition of this extracellular compartment is profoundly altered as a result of vitamin A deficiency. As cell behavior, differentiation and apoptosis, and tissue mechanics are influenced by the extracellular matrix, its modifications potentially compromise organ function and may lead to disease. This review focuses on the effects of lack of vitamin A in the extracellular matrix of several organs and discusses possible molecular mechanisms and pathologic implications.
Asunto(s)
Matriz Extracelular/patología , Deficiencia de Vitamina A/fisiopatología , Animales , Apoptosis/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Humanos , Vitamina A/administración & dosificaciónRESUMEN
Dendritic spines are specialised membrane protrusions of neuronal dendrites that receive the majority of excitatory synaptic inputs. Abnormal changes in their density, size and morphology have been associated with various neurological and psychiatric disorders, including those deriving from drug addiction. Dendritic spine formation, morphology and synaptic functions are governed by the actin cytoskeleton. Previous in vivo studies have shown that ethanol alters the number and morphology of spines, although the mechanisms underlying these alterations remain unknown. It has also been described how chronic ethanol exposure affects the levels, assembly and cellular organisation of the actin cytoskeleton in hippocampal neurons in primary culture. Therefore, we hypothesised that the ethanol-induced alterations in the number and shape of dendritic spines are due to alterations in the mechanisms regulating actin cytoskeleton integrity. The results presented herein show that chronic exposure to moderate levels of alcohol (30 mM) during the first 2 weeks of culture reduces dendritic spine density and alters the proportion of the different morphologies of these structures in hippocampal neurons, which affects the formation of mature spines. Apparently, these effects are associated with an increase in the G-actin/F-actin ratio due to a reduction of the F-actin fraction, leading to changes in the levels of the different factors regulating the organisation of this cytoskeletal component. The data presented herein indicate that these effects occur between weeks 1 and 2 of culture, an important period in dendritic spines development. These changes may be related to the dysfunction in the memory and learning processes present in children prenatally exposed to ethanol.
Asunto(s)
Citoesqueleto de Actina/efectos de los fármacos , Espinas Dendríticas/efectos de los fármacos , Espinas Dendríticas/ultraestructura , Etanol/toxicidad , Acetilcolinesterasa/metabolismo , Actinas/metabolismo , Animales , Células Cultivadas , Homólogo 4 de la Proteína Discs Large , Femenino , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Hipocampo/patología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Ratas , Ratas Wistar , Receptores Ionotrópicos de Glutamato/metabolismo , Proteínas Activadoras de ras GTPasa/metabolismoRESUMEN
Vitamin A is essential for lung development and pulmonary cell differentiation. Its deficiency leads to altered lung structure and function and to basement membrane architecture and composition disturbances. Previously, we showed that lack of retinoids thickens the alveolar basement membrane and increases collagen IV, which are reversed by retinoic acid, the main biologically active vitamin A form. This study analyzed how vitamin A deficiency affects the subunit composition of collagen IV and laminin of lung basement membranes and pulmonary matrix metalloproteinase content, plus the recovering effect of all-trans-retinoic acid. Male weanling pups were fed a retinol-adequate/-deficient diet until 60 days old. A subgroup of vitamin-A-deficient pups received daily intraperitoneal all-trans-retinoic acid injections for 10 days. Collagen IV and laminin chain composition were modified in vitamin-A-deficient rats. The protein and mRNA contents of chains α1(IV), α3(IV) and α4(IV) increased; those of chains α2(IV) and α5(IV) remained unchanged; and the protein and mRNA contents of laminin chains α5, ß1 and γ1 decreased. The mRNA of laminin chains α2 and α4 also decreased. Matrix metalloproteinases 2 and 9 decreased, but the tissue inhibitors of metalloproteinases 1 and 2 did not change. Treating vitamin-A-deficient rats with retinoic acid reversed all alterations, but laminin chains α2, α4 and α5 and matrix metalloproteinase 2 remained low. In conclusion, vitamin A deficiency alters the subunit composition of collagen IV and laminin and the lung's proteolytic potential, which are partly reverted by retinoic acid. These alterations could contribute to impaired lung function and predispose to pulmonary disease.
Asunto(s)
Colágeno Tipo IV/metabolismo , Laminina/metabolismo , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Metaloproteinasas de la Matriz/metabolismo , Tretinoina/farmacología , Deficiencia de Vitamina A/metabolismo , Animales , Membrana Basal/metabolismo , Colágeno Tipo IV/genética , Femenino , Expresión Génica , Laminina/genética , Masculino , Ratas , Ratas Wistar , Inhibidores Tisulares de Metaloproteinasas/metabolismo , Vitamina A/sangre , Deficiencia de Vitamina A/tratamiento farmacológicoRESUMEN
AIMS: Ethanol affects not only the cytoskeletal organization and activity, but also intracellular trafficking in neurons in the primary culture. Polyphosphoinositide (PPIn) are essential regulators of many important cell functions, including those mentioned, cytoskeleton integrity and intracellular vesicle trafficking. Since information about the effect of chronic ethanol exposure on PPIn metabolism in neurons is scarce, this study analysed the effect of this treatment on three of these phospholipids. METHODS: Phosphatidylinositol (PtdIns) levels as well as the activity and/or levels of enzymes involved in their metabolism were analysed in neurons chronically exposed to ethanol. The levels of phospholipases C and D, and phosphatidylethanol formation were also assessed. The consequence of the possible alterations in the levels of PtdIns on the Golgi complex (GC) was also analysed. RESULTS: We show that phosphatidylinositol (4,5)-bisphosphate and phosphatidylinositol (3,4,5)-trisphosphate levels, both involved in the control of intracellular trafficking and cytoskeleton organization, decrease in ethanol-exposed hippocampal neurons. In contrast, several kinases that participate in the metabolism of these phospholipids, and the level and/or activity of phospholipases C and D, increase in cells after ethanol exposure. Ethanol also promotes phosphatidylethanol formation in neurons, which can result in the suppression of phosphatidic acid synthesis and, therefore, in PPIn biosynthesis. This treatment also lowers the phosphatidylinositol 4-phosphate levels, the main PPIn in the GC, with alterations in their morphology and in the levels of some of the proteins involved in structure maintenance. CONCLUSIONS: The deregulation of the metabolism of PtdIns may underlie the ethanol-induced alterations on different neuronal processes, including intracellular trafficking and cytoskeletal integrity.
Asunto(s)
Etanol/toxicidad , Aparato de Golgi/efectos de los fármacos , Hipocampo/efectos de los fármacos , Neuronas/efectos de los fármacos , Fosfatos de Fosfatidilinositol/metabolismo , Animales , Células Cultivadas , Etanol/administración & dosificación , Femenino , Aparato de Golgi/metabolismo , Aparato de Golgi/ultraestructura , Hipocampo/metabolismo , Hipocampo/ultraestructura , Neuronas/metabolismo , Neuronas/ultraestructura , Ratas , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
In the present study, we analyze the effects of ethanol on the Golgi structure and membrane transport in differentiated PC12 cells, which are used as a model of neurons. Chronic exposure to moderate doses of ethanol induces Golgi fragmentation, a common characteristic of many neurodegenerative diseases. Alcohol impaired the lateral linking of stacks without causing microtubule damage. Extensive immunocytochemical and western blot analyses of representative Golgi proteins showed that few, but important, proteins are significantly affected. Thus, alcohol exposure induced a significant ER-to-Golgi transport delay, the retention of the GTPase Rab1 in the Golgi membranes and the accumulation of tethering factor p115 in the cytosol. These modifications would explain the observed fragmentation. The amount of p115 and the stacking protein GRASP65 increased in alcohol-treated cells, which might be a mechanism to reverse Golgi damage. Importantly, the overexpression of GTP-tagged Rab1 but not of a dominant-negative Rab1 mutant, restored the Golgi morphology, suggesting that this protein is the main target of alcohol. Taken together, our results support the view that alcohol and neurodegenerative diseases such as Parkinson have similar effects on intracellular trafficking and provide new clues on the neuropathology of alcoholism.
Asunto(s)
Diferenciación Celular , Retículo Endoplásmico/metabolismo , Etanol/farmacología , Aparato de Golgi/metabolismo , Proteínas de Unión al GTP rab1/genética , Animales , Proteínas de la Matriz de Golgi , Proteínas de la Membrana/metabolismo , Células PC12 , Transporte de Proteínas , Ratas , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Unión al GTP rab1/metabolismoRESUMEN
AIMS: Zinc is an ion that participates in basic cellular and tissular functions. Zinc deficiency is present in many physiological and health problems affecting most body organs, including the brain. Among the circumstances involved in zinc deficiency, ethanol consumption is probably one of the most frequent. A dietary zinc supplement has been proposed as possibly being an efficient method to palliate zinc deficiency. Astrocytes form part of the hematoencephalic barrier, and they are apparently implicated in the homeostasis of the neuronal medium. In this work, we analyze the effect of ethanol on extracellular zinc management by rat astrocytes in culture. METHODS: Intracellular levels of 'free zinc ions', in controls and 30 mM ethanol-treated astrocytes, were visualized by using the zinc fluorochrome TSQ. Cytoplasmic fluorescence and zincosome formation were measured after adding extracellular 50 µM ZnSO(4) to cell monolayers. Zincosomes were also observed at the electron microscopy level. RESULTS: Exposure to ethanol for 7 days lowered the basal zinc levels of astrocytes by â¼30%. This difference was consistently maintained after the zinc pulse. Zinc ions were confined to bright fluorescent particles, the 'zincosomes', which appeared to be formed by the endocytic pathway. Zincosomes were less abundant in alcohol-treated cells, indicating a deficit in endocytoses as the origin of low zinc intake in astrocytes after ethanol treatment. CONCLUSIONS: Ethanol reduces both intracellular ionic zinc levels and extracellular zinc uptake, resulting in poorer zincosome formation. Given the endocytic nature of zincosomes, the effect of ethanol on membrane trafficking is apparently the origin of this deficit.
Asunto(s)
Astrocitos/metabolismo , Vesículas Citoplasmáticas/metabolismo , Etanol/farmacología , Zinc/deficiencia , Zinc/metabolismo , Animales , Astrocitos/efectos de los fármacos , Astrocitos/ultraestructura , Barrera Hematoencefálica/metabolismo , Células Cultivadas , Vesículas Citoplasmáticas/ultraestructura , Endocitosis/efectos de los fármacos , Homeostasis , Ratas , Zinc/química , Zinc/farmacologíaRESUMEN
The organization and dynamics of microtubules (MTs) and the actin cytoskeleton are critical for the correct development and functions of neurons, including intracellular traffic and signaling. In vitro ethanol exposure impairs endocytosis, exocytosis, and nucleocytoplasmic traffic in astrocytes and alters endocytosis in cultured neurons. In astrocytes, these effects relate to changes in the organization and/or function of MTs and the actin cytoskeleton. To evaluate this possibility in hippocampal cultured neurons, we analyzed if chronic ethanol exposure affects the levels, assembly, and cellular organization of both cytoskeleton elements and the possible underlying mechanisms of these effects by morphological and biochemical methods. In the experiments described below, we provide the first evidence that chronic alcohol exposure decreases the amount of both filamentous actin and polymerized tubulin in neurons and that the number of MTs in dendrites lowers in treated cells. Alcohol also diminishes the MT-associated protein-2 levels, which mainly localizes in the somatodendritic compartment in neurons. Ethanol decreases the levels of total Rac, Cdc42, and RhoA, three small guanosine triphosphatases (GTPases) involved in the organization and dynamics of the actin cytoskeleton and MTs. Yet when alcohol decreases the levels of the active forms (GTP bound) of Rac1 and Cdc42, it does not affect the active form of RhoA. We also investigated the levels of several effector and regulator molecules of these GTPases to find that alcohol induces heterogeneous results. In conclusion, our results show that MT, actin cytoskeleton organization, and Rho GTPase signaling pathways are targets for the toxic effects of ethanol in neurons.
Asunto(s)
Depresores del Sistema Nervioso Central/toxicidad , Citoesqueleto/efectos de los fármacos , Etanol/toxicidad , Hipocampo/efectos de los fármacos , Microtúbulos/efectos de los fármacos , Neuronas/efectos de los fármacos , Actinas/metabolismo , Animales , Células Cultivadas , Citoesqueleto/metabolismo , Citoesqueleto/ultraestructura , Femenino , Hipocampo/metabolismo , Hipocampo/patología , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Neuronas/metabolismo , Neuronas/ultraestructura , Ratas , Transducción de Señal/efectos de los fármacos , Proteínas de Unión al GTP rho/metabolismoRESUMEN
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
Chronic vitamin A deficiency induces a substantial delay in the rates of weight and height gain in both humans and experimental animals. This effect has been associated with an impaired nutrient metabolism and loss of body protein. Therefore, we analyzed the effect of vitamin A deficiency on endogenous proteolysis and nitrogen metabolism and its reversibility with all-trans retinoic acid (RA). Male weanling rats, housed in pairs, were pair-fed a vitamin A-deficient (VAD) or control diet until they were 60 d old. A group of deficient rats were further treated with daily intraperitoneal injections of all-trans RA for 10 d. Final body and tissue (i.e. liver and heart) weights were significantly lower and tissue:body weight ratios were similar in VAD rats and in controls. Conversely, the epididymal white fat:body weight ratio and the plasma concentrations of alanine aminotransferase and adiponectin were significantly higher in VAD rats, which also had hepatic macrovesicular lipid accumulations. Plasma and gastrocnemius muscle 3-methylhistidine, urine nitrogen, and plasma and urine urea concentrations were all significantly higher in the VAD group. The expression of the genes encoding urea cycle enzymes and their activities increased in VAD livers. These changes were partially reverted by all-trans RA. We propose that fuel partitioning in vitamin A deficiency may shift from fatty acids to protein catabolism as an energy source. Our results emphasize the importance of vitamin A on the energy balance control system and they provide an explanation for the role of vitamin A in protein turnover, development, and growth.
Asunto(s)
Antioxidantes/uso terapéutico , Hígado/metabolismo , Tretinoina/uso terapéutico , Urea/metabolismo , Deficiencia de Vitamina A/metabolismo , Animales , Antioxidantes/farmacología , Inducción Enzimática , Peroxidación de Lípido/efectos de los fármacos , Hígado/enzimología , Hígado/ultraestructura , Masculino , Metilhistidinas/sangre , Metilhistidinas/metabolismo , Músculo Esquelético/metabolismo , Nitrógeno/metabolismo , Ratas , Retinoides/sangre , Retinoides/metabolismo , Tretinoina/farmacología , Triglicéridos , Deficiencia de Vitamina A/tratamiento farmacológico , Deficiencia de Vitamina A/enzimologíaRESUMEN
Vitamin A is essential for lung development and pulmonary cell differentiation and its deficiency results in alterations of lung structure and function. Basement membranes (BMs) are also involved in those processes, and retinoic acid, the main biologically active form of vitamin A, influences the expression of extracellular matrix macromolecules. Therefore, we have analyzed the ultrastructure and collagen content of lung alveolar BM in growing rats deficient in vitamin A and the recovering effect of all-trans retinoic acid. Male weanling pups were fed a retinol-adequate or -deficient diet until they were 60 days old. A group of vitamin A-deficient pups were recovered by daily intraperitoneal injections of all-trans retinoic acid for 10 days. Alveolar BM in vitamin A-deficient rats doubled its thickness and contained irregularly scattered collagen fibrils. Immunocytochemistry revealed that these fibrils were composed of collagen I. Total content of both collagen I protein and its mRNA was greater in vitamin-deficient lungs. In agreement with the greater size of the BM the amount of collagen IV was also increased. Proinflammatory cytokines, IL-1alpha, IL-1beta and TNF-alpha, did not change, but myeloperoxidase and TGF-beta1 were increased. Treatment of vitamin A-deficient rats with retinoic acid reversed all the alterations, but the BM thickness recovered only partially. Retinoic acid recovering activity occurred in the presence of increasing oxidative stress. In conclusion, vitamin A deficiency results in alterations of the structure and composition of the alveolar BM which are probably mediated by TGF-beta1 and reverted by retinoic acid. These alterations could contribute to the impairment of lung function and predispose to pulmonary disease.
Asunto(s)
Membrana Basal/efectos de los fármacos , Alveolos Pulmonares/efectos de los fármacos , Tretinoina/uso terapéutico , Deficiencia de Vitamina A/patología , Deficiencia de Vitamina A/fisiopatología , Animales , Membrana Basal/ultraestructura , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Colágeno Tipo IV/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Inmunohistoquímica , Interleucinas/metabolismo , Pulmón/metabolismo , Pulmón/patología , Masculino , Malondialdehído/metabolismo , Estrés Oxidativo/efectos de los fármacos , Peroxidasa/metabolismo , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Alveolos Pulmonares/ultraestructura , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Factor de Crecimiento Transformador beta1/metabolismo , Tretinoina/efectos adversos , Tretinoina/sangre , Tretinoina/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Deficiencia de Vitamina A/tratamiento farmacológicoRESUMEN
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
This work proposes a practical experimental approach that allows the rapid in situ generation of a wide range of intracellular GSH concentrations in the intact hepatocyte under highly reproducible conditions. The strategy involves the use of diethyl maleate, a thiol-reactive electrophile that causes rapid and extensive GSH depletion, as well as GSH monoethylester, a GSH analogue that is readily taken up by cells and deesterified intracellularly to render GSH. For both agents, we have analyzed (i) the minimal exposure time required to produce a maximal and dose-related effect on intracellular GSH without altering hepatocyte viability or subsequent survival in culture, and (ii) the relative stability of the GSH levels achieved after removal of both modulators from the culture medium. Results show that with the appropriate timing of exposure, hepatocytes from the same preparation can be quickly synchronized to provide an extreme intracellular GSH concentration gradient (from 0.1- to 4-fold the normal liver content), which simulates an in vitro GSH dose dependency curve and remains stable for at least the subsequent 4 h of culture. The experimental design, which can be easily adapted to different cell types, provides an improved testing protocol to evaluate under reliable conditions the dose-response relationships of the thiol-redox state in a variety of biological processes. It has been applied here to investigate the role of endogenous GSH content in the covalent binding of N-acetyl-p-benzoquinoneimine, the reactive species formed during CYP-dependent bioactivation of acetaminophen, to hepatic proteins.
Asunto(s)
Glutatión/metabolismo , Hepatocitos/metabolismo , Acetaminofén/farmacocinética , Animales , Biotransformación , Células Cultivadas , Sistema Enzimático del Citocromo P-450/metabolismo , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Retinoids can modulate the expression of extracellular matrix (ECM) proteins with variable results depending on other contributing factors. Because changes in these proteins may alter the composition and impair the function of specialized ECM structures such as basement membranes (BMs), we studied the effects of vitamin A deficiency on renal BMs during the growing period. Newborn male rats were fed a vitamin A-deficient (VAD) diet for 50 d. The ultrastructure of renal BMs was analyzed by electron microscopy. Total collagen IV, the different alpha(IV) chains, matrix degrading metalloproteinases (MMP), and tissue inhibitors of metalloproteinases (TIMP) were quantified by immunocytochemistry and/or Western blotting. Tumor necrosis factor-alpha and interleukin-1beta were measured by ELISA. Semiquantitative RT-PCR was used for determining the steady-state levels for each alpha(IV) chain mRNA. VAD renal BMs showed an irregular thickening, particularly tubular BM. The total collagen IV content was increased, but there was a differential expression of the collagen IV chains. The protein amounts for alpha1(IV), alpha4(IV), and alpha5(IV) were similarly increased, whereas alpha2(IV) and alpha3(IV) were decreased. The levels of mRNA for each collagen IV chain changed in parallel with those of the corresponding protein. Both MMP2 and MMP9 were diminished, but no change was detected in TIMP1 or TIMP2. Our data indicate that nutritional VAD leads to alterations in the structure of renal BMs and to quantitative and qualitative variations in its collagen IV composition. These changes may be a factor predisposing to or resulting in kidney malfunction and renal disease.