Distinct glycolysis inhibitors determine retinal cell sensitivity to glutamate-mediated injury.
In this study, we analyzed how distinct glycolysis inhibitors influenced the redox status of retinal cells, used as a neuronal model. Three different approaches were used to inhibit glycolysis: the cells were submitted to iodoacetic acid (IAA), an inhibitor of glyceraldehyde 3-phosphate dehydrogenase, to 2-deoxy-glucose (DG) in glucose-free medium, which was used as a substitute of glucose, or in the absence of glucose. The redox status of the cells was evaluated by determining the reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide). By the analysis of dose-response curves of MTT reduction, IAA showed values of IC50 = 7.02 x 10(-5) M, whereas DG showed values of IC50 = 7.42 x 10(-4) M. Upon 30 min-incubation, glucose deprivation, per se, did not significantly affect MTT reduction. We also evaluated the reduction of MTT as an indicator of cell injury by exposing the cells to 100 microM glutamate during the decrement of glycolysis function. In the presence of glutamate, for 2 h, there was a decrease in MTT reduction, which was potentiated in the presence of DG (10-20% decrease), in the presence of IAA (about 30% decrease) or in glucose-free medium (about 30% decrease). Major changes observed by the MTT assay, upon exposure to glutamate, indicative of changes in the redox status of retinal cells, were concomitant with variations in intracellular ATP. Under glucose deprivation, endogenous ATP decreased significantly from 38.9+/-4.4 to 13.3+/-0.7 nmol/mg protein after exposure to 100 microM glutamate. The results support a different vulnerability of retinal cells after being exposed to distinct forms of glycolysis inhibition.
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Center for Neurosciences of Coimbra and Faculty of Medicine, University of Coimbra, Portugal.
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Santos
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Oliveira
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D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D002642:Chick Embryo; D004396:Coloring Agents; D003847:Deoxyglucose / Q000008:administration & dosage / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005947:Glucose / Q000008:administration & dosage / Q000378:metabolism; D018698:Glutamic Acid / Q000494:pharmacology / Q000633:toxicity*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000037:antagonists & inhibitors; D006019:Glycolysis / Q000187:drug effects*; D019807:Iodoacetic Acid / Q000008:administration & dosage / Q000494:pharmacology; D007700:Kinetics; D010084:Oxidation-Reduction; D012160:Retina / Q000166:cytology / Q000187:drug effects* / Q000196:embryology; D013778:Tetrazolium Salts; D013844:Thiazoles
D004396:Coloring Agents; D004791:Enzyme Inhibitors; D013778:Tetrazolium Salts; D013844:Thiazoles; D018698:Glutamic Acid; D000255:Adenosine Triphosphate; D003847:Deoxyglucose; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; C022616:thiazolyl blue; D005947:Glucose; D019807:Iodoacetic Acid