Effects of glutamate transport substrates and glutamate receptors ligands on the activity of (Na+,K+)-ATPase in brain tissue in vitro

E NANITSOS, GB ACOSTA, Y SAIHARA, D TANTON, LP LIAO, JW SHIN, C RAE, AND VJ BALCAR

CLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY

Blackwell Science Asia

Lugar: Australia; Año: 2004 vol. 31 p. 762 - 769

0305-1870

1. It has been suggested that Na+/K(+)-ATPase and Na(+)-dependent glutamate transport (GluT) are tightly linked in brain tissue. In the present study, we have investigated Na+/K(+)-ATPase activity using Rb+ uptake by ´minislices´ (prisms) of the cerebral cortex. This preparation preserves the morphology of neurons, synapses and astrocytes and is known to possess potent GluT that has been well characterized. Uptake of Rb+ was determined by estimating Rb+ in aqueous extracts of the minislices, using atomic absorption spectroscopy. 2. We determined the potencies of several known substrates/inhibitors of GluT, such as L-trans-pyrrolidine-2,4-dicarboxylate (LtPDC), DL-threo-3-benzyloxyaspartic acid, (2S,3S,4R)-2-(carboxycyclopropyl)-glycine (L-CCG III) and L-anti,endo-3,4-methanopyrrolidine dicarboxylic acid, as inhibitors of [3H]-L-glutamate uptake by cortical prisms. In addition, we established the susceptibility of GluT, measured as [3H]-L-glutamate uptake in brain cortical prisms, to the inhibition of Na+/K(+)-ATPase by ouabain. Then, we tested the hypothesis that the Na+/K(+)-ATPase (measured as Rb+ uptake) can respond to changes in the activity of GluT produced by using GluT substrates as GluT-specific pharmacological tools. 3. The Na+/K(+)-ATPase inhibitor ouabain completely blocked Rb+ uptake (IC50 = 17 micromol/L), but it also potently inhibited a fraction of GluT (approximately 50% of [3H]-L-glutamate uptake was eliminated; IC50 < 1 micromol/L). 4. None of the most commonly used GluT substrates and inhibitors, such as L-aspartate, D-aspartate, L-CCG III and LtPDC (all at 500 micromol/L), produced any significant changes in Rb+ uptake. 5. The N-methyl-D-aspartate (NMDA) receptor agonists (R,S)-(tetrazol-5-yl)-glycine and NMDA decreased Rb+ uptake in a manner compatible with their known neurotoxic actions. 6. None of the agonists or antagonists for any of the other major classes of glutamate receptors caused significant changes in Rb+ uptake. 7. We conclude that, even if a subpopulation of glutamate transporters in the rat cerebral cortex may be intimately linked to a fraction of Na+/K(+)-ATPase, it is not possible, under the present experimental conditions, to detect regulation of Na+/K(+)-ATPase by GluT.