Calcium‐Dependent and‐Independent Release of Glutamate from Synaptosomes Monitored by Continuous Fluorometry

Abstract

An enzyme-linked fluorometric assay is described for the continuous monitoring of the unidirectional efflux of glutamate from guinea-pig synaptosomes. Glutamate efflux from freshly suspended, polarized synaptosomes occurs at 0.35–0.39 nmol min⁻¹ mg of protein⁻¹ and is not significantly affected by external Ca²⁺. KC1 depolarization (30 mM KCI) in the absence of Ca²⁺ doubles this rate, whereas in the presence of Ca²⁺, the initial kinetics of the assay are consistent with the release in the first 5 s of 0.6 nmol mg of protein⁻¹. The final extent of Ca²⁺-dependent release amounts to 1.9 nmol mg of protein⁻¹, or 8.5% of the total intrasynaptosomal glutamate content. Preincubation of synaptosomes at 30°C for 2 h before depolarization leads to a decrease in Ca²⁺-independent release and an increase in Ca²⁺-dependent release, consistent with an intrasynaptosomal relocation of the amino acid.