Modulation of Aspartate Release by Ascorbic Acid and Endobain E, an Endogenous Na+, K+-ATPase Inhibitor

Abstract

The isolation of a soluble brain fraction which behaves as an endogenous ouabain-like substance, termed endobain E, has been described. Endobain E contains two Na+, K+ -ATPase inhibitors, one of them identical to ascorbic acid. Neurotransmitter release in the presence of endobain E and ascorbic acid was studied in non-depolarizing (0 mM KCl) and depolarizing (40 mM KCl) conditions. Synaptosomes were isolated from cerebral cortex of male Wistar rats by differential centrifugation and Percoll gradient. Synaptosomes were preincubated in HEPES-saline buffer with 1 mM D-[3H]aspartate (15 min at 37 degrees C), centrifuged, washed, incubated in the presence of additions (60 s at 37 degrees C) and spun down; radioactivity in the supernatants was quantified. In the presence of 0.5-5.0 mM ascorbic acid, D-[3H]aspartate release was roughly 135-215% or 110-150%, with or without 40 mM KCI, respectively. The endogenous Na+, K+ -ATPase inhibitor endobain E dose-dependently increased neurotransmitter release, with values even higher in the presence of KCl, reaching 11-times control values. In the absence of KCl, addition of 0.5-10.0 mM commercial ouabain enhanced roughly 100% D-[3H]aspartate release; with 40 mM KCl a trend to increase was recorded with the lowest ouabain concentrations to achieve statistically significant difference vs. KCl above 4 mM ouabain. Experiments were performed in the presence of glutamate receptor antagonists. It was observed that MPEP (selective for mGluR5 subtype), failed to decrease endobain E response but reduced 50-60% ouabain effect; LY-367385 (selective for mGluR1 subtype) and dizocilpine (for ionotropic NMDA glutamate receptor) did not reduce endobain E or ouabain effects. These findings lead to suggest that endobain E effect on release is independent of metabotropic or ionotropic glutamate receptors, whereas that of ouabain involves mGluR5 but not mGluR1 receptor subtype. Assays performed at different temperatures indicated that in endobain E effect both exocytosis and transporter reversion are involved. It is concluded that endobain E and ascorbic acid, one of its components, due to their ability to inhibit Na+, K+ -ATPase, may well modulate neurotransmitter release at synapses.