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Notes: Abstract: The effects of the enantiomers of (±)-CAMP and(±)-TAMP [(±)-cis- and(±)-trans-2-aminomethylcyclopropanecarboxylic acids,respectively], which are cyclopropane analogues of GABA, were tested onGABAA and GABAC receptors expressed in Xenopuslaevis oocytes using two-electrode voltage clamp methods. (+)-CAMP wasfound to be a potent and full agonist at homooligomeric GABACreceptors (KD∼40 μM andImax∼100% at ρ1;KD∼17 μM and Imax∼100% at ρ2) but a very weak antagonist atα1β2γ2L GABAAreceptors. In contrast, (-)-CAMP was a very weak antagonist at bothα1β2γ2L GABAAreceptors and homooligomeric GABAC receptors (IC50∼900 μM at ρ1 and ∼400 μM atρ2). Furthermore, (+)-CAMP appears to be a superior agonist tothe widely used GABAC receptor partial agonistcis-4-aminocrotonic acid (KD∼74μM and Imax∼78% at ρ1;KD∼70 μM and Imax∼82% at ρ2). (-)-TAMP was the most potent of thecyclopropane analogues on GABAC receptors (KD∼9 μM and Imax∼40% atρ1; KD∼3 μM andImax∼50-60% at ρ2), but it was also amoderately potent GABAA receptor partial agonist(KD∼50-60 μM and Imax∼50% at α1β2γ2LGABAA receptors). (+)-TAMP was a less potent partial agonist atGABAC receptors (KD∼60 μM andImax∼40% at ρ1; KD∼30 μM and Imax∼60% atρ2) and a weak partial agonist atα1β2γ2L GABAAreceptors (KD∼500 μM andImax∼50%). None of the isomers of (±)-CAMP and(±)-TAMP displayed any interaction with GABA transport at theconcentrations tested. Molecular modeling based on the present resultsprovided new insights into the chiral preferences for either agonism orantagonism at GABAC receptors.

Notes: 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 µmol/L), but it also potently inhibited a fraction of GluT (approximately 50% of [3H]-l-glutamate uptake was eliminated; IC50 〈 1 µmol/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 µmol/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.

Notes: The role of glutamine and alanine transport in the recycling of neurotransmitter glutamate was investigated in Guinea pig brain cortical tissue slices and prisms, and in cultured neuroblastoma and astrocyte cell lines. The ability of exogenous (2 mm) glutamine to displace 13C label supplied as [3-13C]pyruvate, [2-13C]acetate, l-[3-13C]lactate, or d-[1-13C]glucose was investigated using NMR spectroscopy. Glutamine transport was inhibited in slices under quiescent or depolarising conditions using histidine, which shares most transport routes with glutamine, or 2-(methylamino)isobutyric acid (MeAIB), a specific inhibitor of the neuronal system A. Glutamine mainly entered a large, slow turnover pool, probably located in neurons, which did not interact with the glutamate/glutamine neurotransmitter cycle. This uptake was inhibited by MeAIB. When [1-13C]glucose was used as substrate, glutamate/glutamine cycle turnover was inhibited by histidine but not MeAIB, suggesting that neuronal system A may not play a prominent role in neurotransmitter cycling. When transport was blocked by histidine under depolarising conditions, neurotransmitter pools were depleted, showing that glutamine transport is essential for maintenance of glutamate, GABA and alanine pools. Alanine labelling and release were decreased by histidine, showing that alanine was released from neurons and returned to astrocytes. The resultant implications for metabolic compartmentation and regulation of metabolism by transport processes are discussed.