EFFECTS OF GLUTAMATE TRANSPORT SUBSTRATES AND GLUTAMATE RECEPTOR LIGANDS ON THE ACTIVITY OF Na⁺/K⁺‐ATPase IN BRAIN TISSUE IN VITRO

Abstract

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.

Distribution of Glutamate Transporter GLAST in Membranes of Cultured Astrocytes in the Presence of Glutamate Transport Substrates and ATP

“…, K ? -dependent ATPase, the variations in the synaptic activity into changes in the astrocytic metabolism and eventually regulate glucose uptake from blood [51][52][53]; see also [54][55][56]). However, as indicated by the sensitivity to inhibition by suramin, the ATP effect on GLAST distribution seems to be mediated by P2 receptors.…”

Rottlerin Inhibits (Na+, K+)-ATPase Activity in Brain Tissue and Alters d-Aspartate Dependent Redistribution of Glutamate Transporter GLAST in Cultured Astrocytes

“…acts only as a ''marker'' of K ? in medium [24]. Incubation was terminated 10 min later by rapid filtration (assisted by negative-pressure at *20 psi) through Whatman No.…”

“…)-dependent ATPase in rat brain tissue. The methodology has been modified from that which has been previously used to study uptake of radiolabelled amino acids [11,24]. Prisms of adult (3-6 months) rat cerebral cortex (0.1 9 0.1 9 thickness of cortex) were prepared using the McIlwain tissue chopper, suspended at 25 mg/10 ml of incubation medium (phosphate buffered Krebs-Ringer [16]) and allowed 15 min for recovery in a shaking water bath at 37°C.…”

“…by AAS while the rest, including the pellet, were mixed with 200 mM NaOH, left at room temperature for 24 h and used to estimate the protein by Lowry technique. AAS was carried out as described previously [24] using SpectrAA-20plus (Varian Aust P/L, Melbourne, Victoria, Australia) and Photron Rb ? lamp (k = 780.2 mm, slit width 0.2 mm).…”

Altered electrolyte handling of the choroid plexus in rats with glycerol‐induced acute renal failure

“…The inhibitor solution was the DMSO solution containing bumetanide (200 mg/ml), amiloride (8 mg/ml) or ouabain (7.5 mg/ml). These concentrations were selected by considering their half-maximal inhibitory concentration (IC 50 ) for their target transporter and the volume of CSF in rats [20][21][22][23]. Following introduction of inhibitor into the cerebroventricle, the marker solution was administered intravenously, and the disposition profile from the plasma to CSF was evaluated as described above.…”