D by A2ARs (Fig. 1, compare A, D). Ouabain triggered a
D by A2ARs (Fig. 1, evaluate A, D). Ouabain brought on a bimodal but parallel impact on the activities of each NKA (Fig. 2A) and of glutamate transporters (Fig. 2B) in cortical gliosomes. As a result, a low ouabain cIAP review concentration (0.1 M) induced a 40.0 five.0 increase (n 4, p 0.05) of NKA activityResultsActivation of A2ARs decreases NKA activity in gliosomes Since A2ARs control the uptake of glutamate by the astrocytic glutamate transporters GLT-I (Matos et al., 2012b) and the efficiency of glutamate transporters rely on the sodium gradientMatos et al. A2A Receptor Controls Na K -ATPaseJ. Neurosci., November 20, 2013 33(47):184928502 Figure 1. Activation of A2ARs leads to a selective decrease in the activities of each NKA and glutamate transporters in gliosomes but not in synaptosomes from either the cerebral cortex or striatum. Gliosomes and synaptosomes from brain cortex or striatum have been incubated with out or with the A2AR-selective agonist CGS 21680 (30 00 nM) andor antagonist SCH 58261 (50 nM). A, The activation of A2ARs by CGS 21680 in cortical gliosomes (open symbols) reduces NKA activity, whereas it increases NKA activity in synaptosomes (closed symbols). B, C, These opposite effects of CGS 21680 (100 nM) on NKA activity have been prevented by SCH 58261 in cortical gliosomes and synaptosomes (B) and in striatal gliosomes (C). D, E, The activation of A2ARs with CGS 21680 (30 00 nM) inhibited [ 3H]COX-1 drug D-aspartate uptake both in cortical gliosomes and in synaptosomes (D) and SCH 58261 prevented this effect of CGS 21680 (one hundred nM; E). F, A2AR activation by CGS 21680 (one hundred nM) also inhibited [ 3H]D-aspartate uptake in striatal gliosomes, whereas no significant effects were observed in striatal synaptosomes. NKA activity was determined by subtracting the total ATPase activity from the ATPase activity in the presence of membrane ATPase inhibitor ouabain and was expressed as micromole Pi liberated from ATP by 1 g of protein ( mol Pi g protein), whereas the specific uptake of [ 3H]D-aspartate was calculated by subtracting the uptake activity from the uptake activity within the presence of Na -free buffer NMG and was expressed as nanomoles of [ 3H]D-aspartate retained per milligrams of gliosome protein per minute. Data are mean SEM of no less than 3 independent experiments completed in triplicate. Statistical variations have been gauged making use of the Tukey’s post hoc test applied immediately after one-way ANOVA with p 0.05 and p 0.01, when compared with nontreated conditionspared with nontreated gliosomes, in agreement with prior reports (Lichtstein et al., 1985; Gao et al., 2002; Antolovic, 2006) in addition to a lowmoderate concentration of ouabain (1 M) had no effect on NKA activity. Meanwhile, moderatehigher concentrations (10 00 M) inhibited NKA activity (n 4, p 0.05), and a higher concentration (2 mM) of ouabain caused a 73.0 11.two inhibition (n 4, p 0.01) of NKA activity (Fig. 2A). In accordance with all the essential NKA-mediated handle of GLT-I activ-ity, a low ouabain concentration (0.1 M) enhanced [ 3H]Daspartate uptake by 26.1 4.1 (n 4, p 0.05), a low moderate concentration (1 M) had no impact on [ 3H]D-aspartate uptake, a moderatehigher concentration (ten M) inhibited (n four, p 0.05) [ 3H]D-aspartate uptake, along with a larger concentration (two mM) inhibited [ 3H]D-aspartate uptake by 75.0 9.0 (n four, p 0.001; Fig. 2B), as previously observed (Pellerin and Magistretti, 1997; Rose et al., 2009).18496 J. Neurosci., November 20, 2013 33(47):18492Matos et al. A2A Receptor Controls Na K -ATPaseWe next analyzed.