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

Authors


  • The present address of Dr. T. S. Sihra is Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, NY 10021–6399, U.S.A.

  • The present address of Dr. J. Sanchez-Prieto is Department of Biochemistry, Faculty of Pharmacy, Complutense University of Madrid, 28040 Madrid, Spain.

Address correspondence and reprint requests to Dr. D. G. Nicholls at his present address; Department of Biochemistry, University of Dundee, Dundee DD1 4HN, Scotland.

Abstract

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.

Ancillary