Exogenous Glutamate-Induced Modulation of Neurosecretory Process in Nerve Terminals Obtained from the Rat Brain

Abstract

We studied intracellular processes in nerve terminals of neurons of the rat brain in response to application of exogenous glutamate. Using a рН-sensitive fluorescence probe, acridine orange (AO), and labeled gammaaminobutyric acid ([3Н]GABA), we estimated the effect of application of glutamate on the level of acidification of synaptic vesicles and also on the release of GABA from nerve terminals (synaptosomes) obtained from hippocampal tissue. Our experiments showed that glutamate in a dose-dependent manner stimulated the [3Н]GABA release from nerve terminals, and then we observed re-uptake of this neurotransmitter. A selective blocker of GABA transporters, NO-711, completely blocked the uptake of neurotransmitter but did not influence its release; this observation indicates that the glutamate-induced GABA release was from the vesicular, not cytosolic, pool. We confirmed that glutamate stimulates the process of exocytosis in experiments using AO, where we obtained data indicating that this process is two-phase. The first phase, which reflects probably calcium-induced exocytosis, looked like a “burst” of fluorescent signal typical of the response of synaptosomes to the action of KCl applied in depolarization concentration. Both phases of the response were completely blocked or significantly suppressed in calcium-free medium or in the presence of 25 μM Cd2+. The second (slow) phase of the response developed after a certain lag period and was characterized by a gradual increase in the intensity of fluorescent signal. This phase was completely dependent on the presence of sodium in the extracellular medium and completely blocked when sodium was replaced by choline or N-methyl-D-glucamine. We hypothesize that the second phase of the response can reflect either spontaneous unstimulated exocytosis or dissipation of the proton gradient in synaptic vesicles induced by the entry of Na+ into the nerve terminal.