AI Article Synopsis

  • - The study presents a method using high performance liquid chromatography (HPLC) with fluorescence detection to measure how nerve tissue takes up glutamate, a key neurotransmitter.
  • - Retinal tissue from 7-day-old chicks was tested with different glutamate concentrations, revealing a saturable uptake mechanism characterized by specific kinetic parameters (K(m)=8.2 and V(max)=9.8 nmol/mg protein/min) that is influenced by sodium and temperature.
  • - The research also found that zinc chloride inhibits glutamate uptake, confirming the effectiveness of the method, which could aid in studying changes in glutamate transport linked to central nervous system injuries.

Article 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.

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Source
http://dx.doi.org/10.1016/j.jchromb.2012.07.027DOI Listing

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