Ohistochemical data show the immunoreactivity of GLT-I and NKA- 2 inside the
Ohistochemical data show the immunoreactivity of GLT-I and NKA- 2 within the cortex (A ) and within the striatum (E ) of Gfa2-A2AR-KO (D, H ) and Gfa2-A2AR-WT (C, G) littermates with corresponding greater amplifications displayed within the upper ideal corner of each image. Data are imply SEM of a minimum of six independent experiments. Statistical differences have been gauged making use of the Tukey’s post hoc test applied right after one-way ANOVA with p 0.05, p 0.01 and p 0.001, comparison with naive WT littermates. Scale bars: C, D, G, H, 25 m; inset, five m.Matos et al. A2A Receptor Controls Na K -ATPaseJ. Neurosci., November 20, 2013 33(47):184928502 two, GLT-I, and GLAST, as evidenced by their colocalization, copurification, and coimmunoprecipitation (Cholet et al., 2002; Rose et al., 2009; Genda et al., 2011; Bauer et al., 2012) and by the reversed capability of glutamate transporters to modulate NKA activity (Gegelashvili et al., 2007). In parallel, we had previously documented the colocalization and functional interaction among A2AR and GLT-I in astrocytes (Matos et al., 2012a, b). The present demonstration that A2ARs physically associate with NKA- 2s suggests the existence of a macromolecular complex encompassing A2ARs, NKA- 2s, and GLT-Is in astrocytic membranes, in accordance with the function of NKAs as a docking station of molecular signaling hubs (Reinhard et al., 2013) along with the versatility of A2ARs to interact with different neurotransmitters receptors, enzymes, and anchoring proteins (Burgueno et al., 2003; Ferre et al., 2007; Zezula and Freissmuth, 2008; Navarro et al., 2009). This capacity of A2ARs to handle NKA- 2s gives a novel mechanism to know how the acute A2AR activation HIV-2 drug decreases glutamate uptake by astrocytes; MAO-A supplier therefore, A2AR activation not simply triggers a cAMPPKA-dependent pathway to reduce the expression of astrocytic glutamate transporters, but additionally triggers a fast inhibition of astrocytic glutamate transport (Matos et al., 2012b). Albeit the modification of glutamate uptake in astrocytes upon selective A2AR elimination from astrocytes may well result from a short-term andor longterm regulation (Matos et al., 2012b), the observed parallel modification of NKA and glutamate uptake activities selectively in gliosomes of Gfa2-A2AR-KO mice additional suggests an astrocyte-selective coupling among A2ARs and NKAs to regulate glutamate uptake. The molecular Figure five. A2ARs are physically linked with NKA- 2s and this coupling is abrogated in Gfa2-A2AR-KO mice. A, B, Immunomechanism operated by A2ARs to handle precipitation of A2ARs from cerebral cortical (A) or striatal (B) total membranes from Gfa2-A2AR-KO mice and Gfa2-A2AR-WT NKAs may involve a direct conformalittermates with anti-A2AR antibody (IP) or lack of A2AR pull-down with IgG (CTR ), followed by Western blot analysis with anti-NKA- two antibody, revealed an association involving NKA- 2s and A2ARs inside the WT immunoprecipitate (IP), which was absent tional control of NKAs (Arystarkhova and in Gfa2-A2AR-KO mice. The presence of NKA- 2s within the input sample was confirmed in noncoimmunoprecipitated membranes Sweadner, 2005) as a result of the ob(CTR ) inside the decrease (IP) lanes. The presence of A2ARs was confirmed by Western blot evaluation inside the upper lanes (WB). C, D, A PLA served physical association between assay further corroborated the close proximity ( 16 nm) between astrocyte A2ARs and NKA- 2s inside the cortex and striatum from A2ARs and NKA- 2s, which would enable Gfa2-A2AR-WT mice, which was blunted i.