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.

Intracerebroventricular Administration of Ouabain to Rats Changes the Expression of NMDA Receptor Subunits in Cerebral Cortex and Hippocampus

“…-ATPase inhibitor, termed endobain E, which shares several biological properties with ouabain. They include the blockade of high affinity [ 3 H]ouabain binding [42], norepinephrine release enhancement [43] and uptake decrease [44] from rat hypothalamus slices, excitatory amino acid release enhancement from synaptosomes [19], as well as phosphoinositide turnover increase [45].…”

“…Ouabain evokes the release of neurotransmitters in several experimental models, including that of excitatory amino acids from cerebral cortex synaptosomes [18,19], and from cerebellar granule cells in culture [4]. Besides, ouabain is responsible for an increase in intracellular sodium concentration and depolarization of the plasma membrane, as well as for calcium entry to the cell through several calcium channels, including those related to NMDA receptor.…”

Repeated Electroconvulsive Shock Induces Changes in High-affinity [3H]-ouabain Binding to Rat Striatal Membranes

“…Ouabain induces several effects in brain neurotransmission: it increases the release of aspartate and other amino acids [14,15]; decreases norepinephrine uptake [16] and increases its release [17]; blocks the uptake of dopamine [18] and increases its release [14]; decreases the binding of muscarinic antagonist [19] and it induces the release of acetylcholine [14,20]. Some of these effects of ouabain are also induced by the endogenous ouabain-like substance [15][16][17].…”

“…Some of these effects of ouabain are also induced by the endogenous ouabain-like substance [15][16][17]. Recently, other effects of the interaction of ouabain with the sodium-potassium/ATPase were demonstrated and involve the induction of intracellular signaling molecules as a result of the binding of ouabain to the sodium-potassium/ATPase and promoting the interaction between sodium-potassium/ATPase and other proteins in the cell membrane, such as tyrosine kinase Src [21,22].…”

Co-administration effects of α-lipoic acid and nucleo CMP on arousal and sensory cortical activity

“…18 Vitamin C modulates brain neurotransmitter system mainly glutaminergic,dopaminergic, cholinergic and serotonergic that are linked in memory, learning and cognitive function, moreover ,vitamin C regarded as important cofactor in the synthesis and releasing of noradrenaline and cerebral acetylcholine 19 ,this may explained the cognitive enhancing effects of lipoic acid that augments endogenous cerebral ascorbate level in aged and healthy subjects also , neurobehavioral effect of different herbs was explained depending on their antioxidant properties . 20 Animal model study showed that mice deficient in gene encoding for vitamin C lactone oxidase developed a sever cognitive impairment due to oxidative stress that measured by F4-neuroprostans, also, vitamin C reverses and attenuate scopolamine induced cognitive impairment in rats due to stimulation of central acetylcholineastrase enzyme at median forebrain bundle or through activation of cholinergic neurons, and in spite of vitamin C does not cross blood brain barrier, 21,22 therefore ,oral and intraperitoneal administration of vitamin C will not elevates cerebral ascorbate level ,but lipoic acid elevate brain ascorbate concentration that be able for central arousal effects which improve attention and learning due to modulation of hippocampal and forebrain glutamate concentration. Moreover, lipoic acid may improve psychomotor performance through its modulator effects on glutamate receptor which per see improve a brain nitric oxide.…”