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

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