title: Determination of glutamate uptake by high performance liquid chromatography (HPLC) in preparations of retinal tissue.
abstract: The present study describes a simple and efficient method utilizing high performance liquid chromatography (HPLC) coupled to fluorescence detection for the determination of kinetic parameters of glutamate uptake in nervous tissue. Retinal tissue obtained from 7-day-old chicks was incubated with known concentrations of glutamate (50-2000 μM) for 10 min, and the levels of the o-phtaldehyde (OPA)-derivatized neurotransmitter in the incubation medium were measured. By assessing the difference between initial and final concentrations of glutamate in the medium, a saturable uptake mechanism was characterized (K(m)=8.2 and V(max)=9.8 nmol/mg protein/min). This measure was largely sodium- and temperature-dependent, strongly supporting that the mechanism for concentration decrements is indeed uptake by high-affinity transporters. Added to this, our results also demonstrated that zinc chloride (an inhibitor of glutamate/aspartate transporters) evoked a concentration-dependent decrease in glutamate uptake, demonstrating the specificity of our methodology. Overall, the present work characterizes an alternative methodology to evaluate glutamate uptake in nervous tissue using HPLC. This approach could be an important tool for studies associated to the characterization of minute alterations in glutamate transport related with central nervous system injury.
authors.affiliation: Universidade Federal do Pará, Instituto de Ciências Biológicas, Laboratório de Neuroendocrinologia, Belém, Pará State, Brazil.
authors.forename: Edinaldo Rogério da Silva
authors.initials: ER
authors.lastname: Moraes
authors.forename: Alan Barroso Araújo
authors.initials: AB
authors.lastname: Grisolia
authors.forename: Karen Renata Matos
authors.initials: KR
authors.lastname: Oliveira
authors.forename: Domingos Luiz Wanderley
authors.initials: DL
authors.lastname: Picanço-Diniz
authors.forename: Maria Elena
authors.initials: ME
authors.lastname: Crespo-López
authors.forename: Caio
authors.initials: C
authors.lastname: Maximino
authors.forename: Evander de Jesus Oliveira
authors.initials: Ede J
authors.lastname: Batista
authors.forename: Anderson Manoel
authors.initials: AM
authors.lastname: Herculano
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D002645:Chickens; D002712:Chlorides / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D018698:Glutamic Acid / Q000032:analysis* / Q000493:pharmacokinetics*; D006714:Homoserine / Q000032:analysis; D015203:Reproducibility of Results; D012160:Retina / Q000737:chemistry* / Q000378:metabolism*; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D013696:Temperature; D017967:Zinc Compounds / Q000737:chemistry
chemical_list: D002712:Chlorides; D017967:Zinc Compounds; D018698:Glutamic Acid; D006714:Homoserine; C016837:zinc chloride