Control-nontreated situation. vealed that [ 3H]D-aspartate uptake was sig(n 4, p
Control-nontreated situation. vealed that [ 3H]D-aspartate uptake was sig(n four, p 0.05), from mAChR2 list Gfa2-A2AR-KO mice compared with WT nificantly increased (62.0 7.2 , n four, 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 between A2ARs and NKAs to manage glutamate uptake. 9.0 , n 4, 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 between A2ARs and glutamate transporters (Matos et al., 2012b), we subsequent sought to test whether A2ARs and NKA2s could also copurify inside the cerebral cortex or striatum. The pull-down of A2ARs from cortical and striatal homogenates was followed by a Western blot analysis with the A2AR-immunoprecipate with the anti-NKA- two antibody (Fig. 5, IP) or with an anti-IgG antibody as a negative manage (Fig. 5, CTR ), even though confirming the presence of NKA- 2 in the input sample in nonimmunoprecipitated membranes (Fig. five, CTR ) plus the presence of A2ARs in the input and pull-down samples (Fig. five, upper lanes, WB). As depicted in Figure 5, we observed a close association among NKA- 2s and A2ARs inside the brain extracts from Gfa2-A2AR-WT mice (n three; Fig. 5 A, B, reduce lanes, IP), which was highly decreased in both cortical (Fig. 5A) or striatal extracts (Fig. 5B) from Gfa2A2AR-KO mice (n three), in comparison with all the WT littermates. These information proFigure 3. NKA activity and glutamate uptake are LTB4 Purity & Documentation elevated in parallel selectively in gliosomes from the cortex or striatum of vide powerful evidence of a close association Gfa2-A2AR-KO mice. A , Gliosomes and synaptosomes from Gfa2-A2AR-KO mice and from corresponding WT littermates have been involving A2ARs and NKA- 2s in astroprepared before the NKA activity (A, B) along with the [ 3H]D-aspartate uptake (C, D) assays. The enhanced NKA activity was restricted to cytes, which can be absent in Gfa2-A2AR-KO gliosomes from GFAP-A2AR-KO mice (black columns), particularly in the cortex (A) but additionally in the striatum (B), compared with WT mice. mice (white columns). [ 3H]D-aspartate uptake was also selectively improved in gliosomes in the cortex (C) and striatum (D). Subsequent, employing an in situ PLA, we atData are mean SEM of a minimum of 4 independent experiments. Statistical differences were gauged employing the Tukey’s post hoc test tempted to confirm the existence of A R 2A applied right after one-way ANOVA with p 0.05, p 0.01, and p 0.001. and NKA- 2 complexes in cortical and striatal brain slices of Gfa2-A2AR-KO or GLT-I and NKA- 2 immunoreactivities are enhanced in Gfa2-A2AR-WT littermates (Soderberg et al., 2006; Augusto et al., Gfa2-A2AR-KO mice 2013). PLA is definitely an antibody-based approach in which the A2AR and As a very first step toward testing the hypothesis that A2ARs, NKA- 2s, NKA- two proteins had been 1st immunolabeled with main antiand glutamate transporters could be physically associated in astrobodies after which with secondary antibodies conjugated to comcytes, we compared the density and distribution of GLT-Is and plementary oligonucleotides, which can only ligate and be NKA- 2s in the cerebral cortex and striatum from Gfa2amplified when the A2AR and NKA- 2 antibody mo.