Ethanol stimulates ROS generation by mitochondria through Ca 2+ mobilization and increases GFAP content in rat hippocampal astrocytes

Abstract

We have employed rat hippocampal astrocytes in culture to investigate the effect of ethanol on reactive oxygen species (ROS) production as well as its effect on [Ca 2+] c and GFAP expression. Cells were loaded with the fluorescent probes fura-2 and H 2DCFDA for the determination of changes in [Ca 2+] c and ROS production respectively, employing spectrofluorimetry. GFAP content was determined by immunocytochemistry and confocal scanning microscopy. Our results show ROS production in response to 50 mM ethanol, that was reduced in Ca 2+-free medium (containing 0.5 mM EGTA) and in the presence of the intracellular Ca 2+ chelator BAPTA (10 μM). The effect of ethanol on ROS production was significantly reduced in the presence of the alcohol dehydrogenase inhibitor 4-methylpyrazole (1 mM), and the antioxidants resveratrol (100 μM) or catalase (300 U/ml). Preincubation of astrocytes in the presence of 10 μM antimycin plus 10 μM oligomycin to inhibit mitochondria completely blocked ethanol-evoked ROS production. In addition, ethanol led to a sustained increase in [Ca 2+] c that reached a constant level over the prestimulation values. Finally, incubation of astrocytes in the presence of ethanol increased the content of GFAP that was significantly reduced in the absence of extracellular Ca 2+ and by resveratrol and catalase pretreatment. The data obtained in the present study suggest that astrocytes are able to metabolize ethanol, which induces two effects on intracellular homeostasis: an immediate response (Ca 2+ release and ROS generation) and later changes involving GFAP expression. Both effects may underline various signaling pathways which are important for cell proliferation, differentiation and function.