Centrifuge-induced hypergravity and glutamate efflux by reversal of high-affinity, sodium-dependent transporters from rat brain synaptosomes.

Glutamate uptake by high affinity sodium-dependent glutamate transporters is essential for termination of the synaptic transmission. Glutamate transporters may also contribute to an increase in extracellular glutamate. Glutamate efflux can occur by reversal of the sodium-dependent glutamate transporters during ATP depletion and dissipation of the sodium gradient across the cell membrane. Depolarization-induced calcium independent release of neurotransmitter from synaptosomal cytosolic pool is Na⁺-dependent and due to reverse of the neurotransmitter transporters also. We used monovalent organic cations N-methyl-D-glucamine (NMDG) to replace extracellular sodium, suggesting that the reducing of Na⁺ elucidate further the mechanism underlying Ca²⁺-independent glutamate release. A reduction in extracellular sodium would facilitate reversal of sodium-dependent transporters with extrusion of glutamate. We have compared the basal release of glutamate in Ca²⁺-free Na⁺-supplemented and NMDG-supplemented medium in control and after exposure of animals to long-arm centrifuge-induced hypergravity (ten G, during one hour). Replacement of sodium by NMDG enhanced basal level of neurotransmitter. The value of basal level increased to 110± 4% and 140± 2% in the medium with NMDG in comparison with Na⁺ under the control and hypergravity conditions, respectively. It is likely to reflect the enhancement of the neurotransmitter level in cytosolic pool. Thermodynamic considerations show that the extracellular level of a amino acid neurotransmitter, such as glutamate, that can be generated by transporter reversal are directly proportional to the intracellular concentration of the intracellular concentration of amino acid. KCl-stimulated glutamate release from cytosolic pool increased not statistically after hypergravity loading. We examined the effects of transporter inhibitors DL-threo-beta-benzyloxyaspartate ( DL-TBOA) on the release to elucidate whether reverse transport via the glutamate transporter is involved in the KCl-stimulated glutamate release in sodium - supplemented Ca²⁺-free medium. DL-TBOA is a newly developed competitive inhibitor of the high-affinity, Na⁺-dependent glutamate transporters. Application of 10 μ M DL-TBOA caused a decrease of 20 % of initial glutamate efflux, 100 μ M DL-TBOA -50 %, respectiely as in control as after centrifuge-induced hypergravity. The data are interpreted as indicating that glutamate release was reduced as a consequence of DL-TBOA inhibition of reversed transport by high-affinity, Na-dependent carriers.