RESUMO
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.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Ácido Glutâmico/análise , Ácido Glutâmico/farmacocinética , Retina/química , Retina/metabolismo , Análise de Variância , Animais , Galinhas , Cloretos/química , Homosserina/análise , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Espectrometria de Fluorescência , Temperatura , Compostos de Zinco/químicaRESUMO
Quorum sensing is a widespread regulatory mechanism among Gram-negative bacteria. In this study, Acinetobacter strains were assayed for the presence of quorum sensing signal molecules capable of activating N-acylhomoserine lactone biosensors. By using an Agrobacterium tumefaciens reporter strain it was shown that all the cultures produced two to four detectable signal molecules with different chromatographic patterns. In A. calcoaceticus BD413 supernatants four compounds were detected in a time-dependent manner, and maximal activity was reached at stationary phase. The number of signal molecules was dependent on medium composition; typically, cultures in minimal medium displayed one or two more signals, as compared to complex medium. None of the Acinetobacter supematants showed autoinduction activity with an Chromobacterium violaceum reporter strain, neither in direct or competition assays.