Glutamate is the main excitatory neurotransmitter in the central nervous system , and the overstimulation of glutamatergic receptors, including the ionotropic receptors of N-methyl-d-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA), seems to be related to neuronal death caused by excitotoxicity [1, 2]. Glutamate-induced neurotoxicity apparently underlies a variety of neurologic disorders, including epilepsy, Huntington’s disease, Parkinson’s disease and Alzheimer’s disease . Excessive accumulation of glutamate in the synaptic cleft can be due to higher glutamate release, lower uptake by pre-synaptic terminals and/or reverse transportation of glutamate from the pre-synaptic terminal towards the synaptic cleft [2, 3]. These processes can result from alterations in the Na+ and K+ concentration gradients between the intracellular and extracellular environments, which depend on the activity of the transmembrane enzyme Na+,K+-ATPase [4–6].
Na+,K+-ATPase is the enzyme responsible for the maintenance of low concentrations of Na+ and high concentrations of K+ in the intracellular environment, maintaining the resting potential and aiding in the reestablishment of this potential after neuronal depolarization . Structurally, Na+,K+-ATPase is a heterotrimer formed by three subunits (α1-4, β1-3 and γ), and the kinetic properties of the isozymes are mainly determined by the α subunit . In the central nervous system, α1 and α2 function as the “housekeeping” isoforms, while the α3 isoform is predominantly activated in situations involving high neuronal activity [8, 9]. The inhibition of Na+, K+-ATPase activity by the glycoside ouabain elicits an excitatory effect leading to convulsions [10, 11] and neuronal death through both apoptosis and necrosis . Additionally, a deficiency in Na+,K+-ATPase activity has been linked to central nervous system disorders, including epilepsy and neurodegenerative diseases [4, 6].
Several in vitro studies have been performed in an attempt to understand whether and how glutamatergic activation influences the kinetics of Na+,K+-ATPase in nervous tissue. The results, however, have not been consistent. Although some researchers have reported a stimulation of Na+,K+-ATPase enzymatic activity by glutamatergic agonists [12–14], others have demonstrated an inhibition of its activity [15–19] or neither stimulation nor inhibition [20, 21]. It is worthwhile to mention that such contradictions may be due to some technical factors, including differences in brain tissues, tissue preparation and the biochemical assays performed; for example, 42 K+ uptake has been analyzed using scintillation counting , Rb+ uptake has been analyzed using atomic absorption spectroscopy  or scintillation counting  and inorganic phosphate release has been analyzed using spectrophotometry [15–17, 20, 21] and scintillation counting . The results can also be discrepant depending on the family of glutamatergic receptor that is being stimulated [16, 18, 19].
Therefore, the present study sought to verify whether different concentrations of glutamate and the ionotropic glutamatergic agonists NMDA, AMPA and kainate would alter the kinetic behavior of Na+,K+-ATPase in the crude synaptosomal fraction of the hippocampus and the frontal cortex. These studies were performed using two different biochemical methods. The first method was a Na+,K+-ATPase assay involving the complete enzymatic reaction, including both Na+-dependent phosphorylation and K+-dependent dephosphorylation. The second method was a K+
p-NPPase) assay involving only K+-dependent dephosphorylation. In the Na+,K+-ATPase assay, we performed studies with both saturating and sub-saturating concentrations of ATP. Under higher ATP concentrations, the activities of the isozymes containing α1, α2 and α3 are indistinguishable. At lower ATP concentrations, isozymes containing α3 can be preferentially assayed because they have a higher affinity for ATP . Concomitant with these assays, we also investigated a putative influence of the glutamatergic agonists on the activities of ouabain-insensitive Mg2+-ATPase and Mg2+