Método baseado me cromatografia líquida de alta eficiência para analisar a atividade dos transportadores de GABA no sistema nervoso central

Abstract

The GATs are the membrane proteins responsible for the uptake of GABA in the central nervous system. Alterations in GAT activity are implicated in several neurological diseases, including retinopathies. The present study describes an alternative method to determine GAT activity in tissue preparations of the central nervous system, using high performance liquid chromatography (HPLC) with fluorescence detection. The GABA concentration in the medium was determined using the o-phthaldehyde (OPA)-derivation protocol validated by the Brazilian Health Regulatory Agency (ANVISA) and the United States Food and Drug Administration (US-FDA). The GAT activity in the retinal preparations was determined through the evaluation of the GABA uptake, which was measured by assessing the difference between the initial and final concentrations of GABA in the incubation médium (Δ𝐺𝑎𝑏𝑎 = [𝐺𝑎𝑏𝑎] 𝑡_0 − [𝐺𝑎𝑏𝑎] 𝑡_𝑒𝑛𝑑). The evaluation of the GAT kinetics returned values of Km = 382.5 ± 32.2 μM and Vmax = 34 nmol/mg of protein. The data also demonstrated that the GABA uptake was predominantly Na+ and temperature-dependent, and was also inhibited by incubation with nipecotic acid, a substrate of GABA transporters. Taken together, these findings confirm that our approach provided a specific measure of GAT activity in retinal tissue. The data presented here thus validate, for the first time, an alternative, simple and sensitive method for the evaluation of GAT activity using high performance chromatography on preparations of the central nervous system.