Caracterización de las alteraciones en la membrana basal pulmonar de ratas deficientes en vitamina A y su reversibilidad por ácido retinoico.

RESUMEN La deficiencia de vitamina A (VAD) es un problema importante de salud pública, que afecta clínicamente a más de 5 millones de personas al año. En países en vías de desarrollo afecta gravemente a niños aumentando la morbilidad y mortalidad de diferentes infecciones y enfermedades del tracto respiratorio. Los retinoides, metabolitos activos de la vitamina A, son necesarios en mamíferos para el desarrollo pulmonar y la diferenciación de células pulmonares, y su deficiencia altera la estructura y función de los pulmones. Las membranas basales (MBs), que están asociadas al epitelio y endotelio alveolar y forman parte de la pared alveolar, también están implicadas en estos procesos, y el ácido retinoico, principal forma biológicamente activa de la vitamina A, modula la expresión de macromoléculas de la matriz extracelular. Por este motivo, el propósito de nuestro trabajo ha sido analizar los efectos de la deficiencia crónica de vitamina A en la estructura y composición de la MB pulmonar durante el desarrollo postnatal de los pulmones, y el del tratamiento posterior con ácido all-trans-retinoico sobre las alteraciones encontradas. Crías macho, recién destetadas, fueron alimentadas con dieta completa o deficiente en vitamina A hasta que cumplieron 60 días de vida. Y un grupo de crías VAD fueron recuperadas con inyecciones intraperitoneales de ácido all-trans-retinoico durante 10 días. El análisis concreto de la MB alveolar, cuya integridad es de gran importancia para el intercambio de gases durante la función respiratoria, mostró en la deficiencia un engrosamiento de la misma, deposiciones ectópicas de fibrillas de colágeno I en su interior, un aumento del contenido total de los colágenos I y IV, y cambios en su composición en cadenas α. Además, la deficiencia de vitamina A produjo una disminución en el contenido de las cadenas de laminina mayoritarias en las MBs pulmonares, y modificó la actividad proteolítica de la MMP2 y MMP9, encargadas de la degradación y renovación de las MBs, disminuyéndola. También observamos que la privación de vitamina A durante este periodo del desarrollo provocó un aumento del factor fibrogénico TGF- β1, un incremento del estrés oxidativo y una respuesta inflamatoria leve en los pulmones. Por otro lado, también demostramos que la mayoría de estas alteraciones, al menos parcialmente, revirtieron al estado control tras tratar con ácido all-trans-retinoico. Interesantemente, la capacidad de recuperación por parte del ácido retinoico ocurre aún existiendo un estrés oxidativo aumentado. En conclusión, la deficiencia de vitamina A ocasiona alteraciones en la estructura y composición de la MB alveolar que probablemente estén mediadas por el TGF-β1 y el ácido retinoico es capaz de revertirlas. Estas alteraciones podrían contribuir a un mal funcionamiento pulmonar y predisponer a diferentes enfermedades pulmonares. __________________________________________________________________________________________________Vitamin A deficiency (VAD) is an important public health problem, affecting clinically more than 5 millions of persons per year. Particularly, VAD affects children of developing countries, increasing the morbidity and mortality of several respiratory tract infections and illnesses. Retinoids, active metabolites of Vitamin A, are essential for lung development and pulmonary cell differentiation and its deficiency results in alterations of lung structure and function. Basement membranes (BMs), that are associated with the alveolar epithelium and endothelium and constitutes part of the air-blood barrier, 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, the aims of our work have been to analyze 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. Alveolar BM, which is very important for the gas exchange and lung function, was enlarged in VAD 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. VAD altered the α chain composition of collagen I and IV, and also reduced the content of the main laminin chains of lung BMs. Moreover proteolitic activity of MMP2 and MMP9 were decreased in VAD animals. Proinflammatory cytokines, IL-1α, IL-1β and TNF-α, did not change, but myeloperoxidase and TGF-β1 were increased. Treatment of VAD rats with retinoic acid reversed to the control state nearly all the alterations, at least partially. Interestingly, 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-β1 and reverted by retinoic acid. These alterations could contribute to the impairment of lung function and predispose to pulmonary disease

Searching free zinc at the ultrastructural level in cultured astrocytes

Zinc is an element that is necessary for many physiological functions in the body but may play an important role in diseases affecting most systems in the body if its balance is altered by environmental, toxicological or idiosyncrasy of subjects. We have centred our investigation in central nervous system, using cultured astrocytes since they are involved in clearance of zinc exocytated to the extracellular medium during synaptic transmission. In previous works we have used a zinc fluorochrome, i.e., the TSQ (6-Methoxy-(8-p-toluenesulfonamido)quinoline) to in vivo zinc uptake in cultured astrocytes and its accumulation in organelles named zincosomes. However, the precise location of these zinc-enriched structures (zincosomes) at the ultrastructural level is a very hard task. In a previous attempt at the electron microscopy level, only topographical approximation by combining light and electron microscopy allowed us to identify selected zincosomes previously marked with TSQ. Now, our objective is to adapt zinc autometallography (Timm’s method) to TSQ labelled cultured astrocytes. For the electron microscopic detection of zincosomes, the first important step is to achieve a good zinc precipitation during or previous to glutaraldehyde fixation. Surprisingly, neither ditizone nor selenite were successful as zinc precipitating agents; only sodium sulphide gave us good results. We also found that while glutaraldehyde is the best option for animal experimentation, paraformaldehyde prefixation gave us best results. Paraformaldehyde prefixation allowed both ultrastructure preservation as well as zinc-precipitated-detection with Timm autometallography in semithin sections. These semithin sections were included again and zincosomes become clearly visible in ultrathin sections

Ethanol impairs extracellular zinc intake in cultured astrocytes

Zinc (Zn) deficiency is present in many physiological and health problems. Among circumstances involved in Zn deficiency, ethanol consumption appears as a prominent cause. In the CNS substantial amounts of Zn appear accumulated in synaptic vesicles of a particular class of neurons: the Zn enriched neurons very abundant in the telencephalon and cerebral cortex. This is the so called synaptic Zn which is simultaneously released with the neurotransmitter thus exerting a neuromodulator role during synaptic transmission. Neighbour astrocytic processes have to capture the excess of both extracellular Zn and neurotransmitter in order to maintain efficient synaptic transmission between neurons. In this work we analyze the effect of exposure to 30 mM ethanol for 7 days in the ability of cultured rat astrocytes to capture and manage extracellular Zn. Intracellular Zn levels were visualized by using the TSQ Zn fluorochrome, either in normal culture conditions or after supplementary addition of 50 μM ZnSO4 to the culture. Fluorescence was recorded with an Olympus microscope BX50WI, equipped with a Hamamatsu ORCA digital camera controlled with the Aquacosmos software. Basal Zn levels in cultured astrocytes was greatly and significantly lower in ethanol treated cells (about 30% of control cultures). These differences were consistently maintained after addition of extracellular Zn to cell monolayers, resulting in a lower ability to uptake or retain Zn. The Zn was uptaked by the endocytic pathway, as demonstrated by the marker FM1-43 and was mainly confined to bright organelles that were more abundant in control cells. In conclusion, ethanol impairs astrocyte Zn management resulting in a lower capacity for extracellular Zn intake in resting conditions and after extracellular addition. It has been proposed that an efficient method to palliate Zn deficiency it could be a dietary supplement. Nevertheless, this study suggests that a dietary Zn supplementation may not be enough for recovery of cellular normal function in alcoholic cultured astrocytes

Protein traffic is an intracellular target in alcohol toxicity

Eukaryotic cells comprise a set of organelles, surrounded by membranes with a unique composition, which is maintained by a complex synthesis and transport system. Cells also synthesize the proteins destined for secretion. Together, these processes are known as the secretory pathway or exocytosis. In addition, many molecules can be internalized by cells through a process called endocytosis. Chronic and acute alcohol (ethanol) exposure alters the secretion of different essential products, such as hormones, neurotransmitters and others in a variety of cells, including central nervous system cells. This effect could be due to a range of mechanisms, including alcohol-induced alterations in the different steps involved in intracellular transport, such as glycosylation and vesicular transport along cytoskeleton elements. Moreover, alcohol consumption during pregnancy disrupts developmental processes in the central nervous system. No single mechanism has proved sufficient to account for these effects, and multiple factors are likely involved. One such mechanism indicates that ethanol also perturbs protein trafficking. The purpose of this review is to summarize our understanding of how ethanol exposure alters the trafficking of proteins in different cell systems, especially in central nervous system cells (neurons and astrocytes) in adult and developing brains

Ethanol impairs extracellular zinc intake in cultured astrocytes

Zinc is an ion that participates in increasing described cellular and tissular functions. On the other hand, zinc deficiency is also present in many physiological and health problems affecting most organs along the body, including teratological problems. Among circumstances involved in zinc deficiency, ethanol consumption is probably one of the most frequent. In the central nervous system zinc is also present and it is especially important in neurons that include zinc in the transmission synaptic vesicles. This zinc is delivered as the neurotransmitter exerting a neuromodulator role in the synaptic transmission. Neighbour astrocytes have to maintain the extracellular zinc homeostasis in order to maintain neurons continuously able to perform synaptic transmission. It has been proposed that an efficient method to palliate zinc deficiency it could be a dietary supplement. In this work we analyze the ability of cultured astrocytes in the management of extracellular zinc. Cultured rat astrocytes were used in this work, incubated in presence or not of 30 mM Ethanol for 7 days. Intracellular zinc levels were visualized by using the TSQ zinc fluorochrome, after addition or not of 50 µM ZnSO4. Fluorescence was recorded with an Olympus microscope BX50WI, equipped with a Hamamatsu ORCA digital camera controlled with the Aquacosmos software. Basal zinc levels in cultured astrocytes was greatly and significantly lower in ethanol treated cells (about 30% of control cultures). These differences were consistently maintained after addition of extracellular zinc to cell monolayers, resulting in a lower ability to uptake or retain zinc. The zinc was uptaked by the endocitic pathway, as demonstrated with the endosome marker FM 1-43 and was mainly confined to bright organelles that were more abundant in control cells. In conclusion, ethanol impairs astrocyte zinc management resulting in a lower capacity for extracellular zinc intake in resting conditions and after extracellular addtion. Consequently the efficiency of a dietary zinc supplementation may not be enough for recovery of cellular normal function

Extracellular zinc intake in cultured astrocytes is altered by ethanol exposure

Ethanol reduces the amount of intracellular zinc detectable with TSQ. Ethanol impairs astrocyte Zn management. It results in a lower capacity for exogenous Zn intake and delivering to zincosomes. Thus, Zn supplementation (dietary?) may not be enough for recovery of cellular normal function; as it happens in alcohol treated astrocytes. Zincosomes are a kind of low density primary endosomes

Endocytosis is altered by chronic alcohol exposure in cultured neurons

Endocytosis is required for many cellular pivotal processes including membrane recycling, nutrient uptake and signal transduction. This process is particularly relevant in polarized cells such as neurons. Exposure to ethanol causes a variety of anomalies in the developing brain and 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 (BSA) and transferrin (Tf) 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. The present study shows that the exposure of neurons in primary culture to chronic ethanol (0 to 14 DIV, 30 mM) affects both clathrindependent and clathrinindependent endocytosis by probably acting not only on the several proteins which are directly involved in the different steps of these processes, but also on the proteins that participate in the organization and dynamics of the actin cytoskeleton as well as on cholesterol cellular levels. Therefore, a toxic effect of alcohol on endocytosis could affect some of the important neuronal activities which depend on the endocytic process, including synaptic vesicle recycling, trafficking of postsynaptic receptors, polarized axon elongation, growth cone navigation and neuronal migration