Ncreased selectively in astrocytes from Gfa2A2AR-KO mice To superior understand the association among A2ARs and NKAs to handle astrocytic SphK2 Inhibitor drug glutamate uptake, we subsequent made use of Gfa2-A2AR-KO mice (Matos et al., 2012b) to investigate how the selective deletion of A2ARs in astrocytes affects NKA and GLT-I activities in astrocytes and neurons. As portrayed in Figure three, gliosomes collected from the cortex (Fig. 3A) or striatum (Fig. 3B) of Gfa2-A2AR-KO mice Figure 2. The NKA-inhibitor ouabain has a parallel influence on the activities of NKA and of glutamate transport and blunt the displayed a drastically greater NKA ac- effect of A Rs on [ 3H]D-aspartate uptake in cortical gliosomes. A, Concentration-dependent inhibition of NKA activity by ouabain 2A tivity than gliosomes collected from WT in cerebral cortical gliosomes from WT mice. Ouabain at 0.1 M enhanced NKA activity, but at ten M inhibited NKA activity. NKA littermates (58.1 9.0 , n four, p 0.05 activity was expressed as micromole Pi liberated from ATP by 1 g of protein ( mol Pi/ g protein). B, Concentration-dependent in the cortex; 33.1 6.0 , n 4, p 0.05 inhibition of [ 3H]D-aspartate uptake in cerebral cortical gliosomes from WT mice. Ouabain at 0.1 M enhanced [ 3H]D-aspartate inside the striatum). In contrast, NKA activity uptake, but at one hundred M inhibited [ 3H]D-aspartate uptake. The specific uptake of [ 3H]D-aspartate was expressed as nanomoles of was not considerably different in cortical [ 3H]D-aspartate retained per milligram of gliosome protein per minute. C, Acute (30 min) incubation of cerebral cortical gliosomes with all the A2AR-selective agonist CGS 21680 (one hundred nM) decreased [ 3H]D-aspartate uptake, an impact no longer observed upon pertur(n four, p 0.94) or striatal (n 4, p 0.24) synaptosomes from Gfa2-A2AR-KO bation from the activity of NKA by preincubation with either a low (0.1 M) or a higher (1 mM) concentration of ouabain. Information would be the or Gfa2-A2AR-WT mice. A equivalent evaluation mean SEM of five independent experiments carried out in triplicate. Statistical distinction was assessed making use of a two-way ANOVA from the activity of glutamate β-lactam Chemical Purity & Documentation transporters re- analysis. p 0.05, p 0.01, p 0.001, comparison with control-nontreated situation. vealed that [ 3H]D-aspartate uptake was sig(n four, p 0.05), from Gfa2-A2AR-KO mice compared with WT nificantly elevated (62.0 7.two , n 4, p 0.001) in cerebral littermates (Fig. 3D). The observed parallel modification of NKA cortical gliosomes, but not in synaptosomes (n 4, p 0.05), from and glutamate uptake activities selectively in gliosomes of Gfa2Gfa2-A2AR-KO mice compared with WT littermates (Fig. 3C). SimA2AR-KO mice is additional suggestive of an astrocyte-selective couilarly, [ 3H]D-aspartate uptake was also selectively improved (44.0 pling among A2ARs and NKAs to handle glutamate uptake. 9.0 , n four, p 0.01) in striatal gliosomes, but not in synaptosomesMatos et al. A2A Receptor Controls Na /K -ATPaseJ. Neurosci., November 20, 2013 33(47):184928502 ciation among A2ARs and glutamate transporters (Matos et al., 2012b), we subsequent sought to test irrespective of whether A2ARs and NKA2s may possibly also copurify within the cerebral cortex or striatum. The pull-down of A2ARs from cortical and striatal homogenates was followed by a Western blot analysis of the A2AR-immunoprecipate with all the anti-NKA- 2 antibody (Fig. 5, IP) or with an anti-IgG antibody as a damaging control (Fig. five, CTR ), whilst confirming the presence of NKA- 2 inside the input sample in nonimmunoprecipitated membranes (Fig. five, CTR ) along with the presen.
