Glutationa modula a liberação de adenosina em cultura primária de astrócitos corticais de camundongo

Abstract

Glutathione (GSH) is one of the main antioxidants in the Central Nervous System (CNS) and a potential gliotransmitter, inducing calcium waves in the cytosol of glial cells. Adenosine (Adn) is a neuromodulator widely expressed in the CNS and its extracellular levels are a critical factor in determining its effect on nervous tissue. It is known that Ca2+- dependent pathways regulate Adn release. Since GSH has the ability to induce Ca2+ waves in the cytosol of glial cells, the present work aims to investigate whether this molecule can regulate extracellular DNA levels. To assess this, we used primary cultures of cortical astrocytes maintained in DMEM+10% SBF in a CO2 oven (37oC, 95% O2/5% CO2) for 12-15 days, when they reached confluence. The cells were incubated with Hank buffer for different time intervals, after which this solution was collected and the neurotransmitters present there were quantified by High Performance Liquid Chromatography. Our data show that GSH induces an 80% increase in extracellular Adn levels at two analyzed times: 5 and 20 minutes. Removal of GSH from the incubation medium returns the Adn concentration to baseline levels. Removal of Na+ or Ca2+ from the medium did not affect the effect of GSH. Blockade of nucleoside equilibrative transporters by dipyridamole (10 µM) significantly decreased the levels of Adn in the medium, but did not interfere with the action of GSH. In order to assess whether the effect of GSH derives from an indirect modulation on the release of glutamate or GABA (two agents described as regulators of Adn release), the quantification of these transmitters was performed. Both were significantly increased in the presence of GSH. However, unlike what was observed with Adn, the removal of Na+ from the incubation medium mitigated the effect of GSH on glutamate release. The incubation of astrocytes with GABA (50 and 100 µM) did not influence the extracellular Adn concentration in our experimental model, ruling out a GABAergic modulation behind the effect of GSH. The evaluation of redox agents showed that thiol compounds reproduce the effect of GSH, while the non thiol antioxidant alpha-tocopherol did not regulate extracellular Adn levels. Thus, the present work concludes that astrocytes express a GSH-sensitive component that can be modulated by its sulfhydryl group.