期刊
NEUROCHEMISTRY INTERNATIONAL
卷 158, 期 -, 页码 -出版社
PERGAMON-ELSEVIER SCIENCE LTD
DOI: 10.1016/j.neuint.2022.105359
关键词
GAT; GABAuptake; HPLC; Retina; Transport
资金
- Conselho Nacional de Pesquisa e Desenvolvimento (CNPq, Brazil)
- Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES, Brazil)
This study presents an alternative method for determining GAT activity in tissue preparations of the central nervous system using high performance liquid chromatography (HPLC) with fluorescence detection. The results demonstrate that this method provides a specific measure of GAT activity and validates the Na+- and temperature-dependent uptake of GABA.
The GATs are the membrane proteins responsible for the uptake of GABA in the central nervous system. Al -terations 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 con-centration 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 medium. The evaluation of the GAT kinetics returned values of Km = 382.5 +/- 32.2 mu M 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.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据