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  • We confirmed a decrease in extracellular glutamate


    We confirmed a decrease in extracellular glutamate uptake and the presence of efflux in an endothelial cell model of oxygen-glucose deprivation (OGD), which effectively simulates the inefficient energy supply after brain injury [15], and analysed the function of endothelial EAATs and explored the role and regulatory mechanisms of A2AR to confirm this hypothesis. Finally, the key mechanisms by which A2AR regulates endothelial EAATs were verified in wild-type (WT) and A2AR knock-out (KO) mice after traumatic brain injury (TBI).
    Materials and methods
    Discussion Endothelial EAATs have important and unique functions in maintaining glutamate homeostasis in the brain [8,28]. However, after brain injury, changes in the EAAT functions and their roles in rapidly increasing glutamate levels in the brain are not clear. In this study, the Maprotiline HCl synthesis endothelial cell model of OGD was constructed to show that the transport efficiency of EAATs was significantly reduced and even reversed. OGD depleted ATP from endothelial cells, resulting in abnormal intra- and extracellular Na+/K+ distributions. Since EAATs transport glutamate against its concentration gradient primarily through secondary Maprotiline HCl synthesis coupled to the Na+ gradient [6], the abnormal distribution of Na+ leads to the dysfunction of or even reverse transport by Na+-dependent EAATs [7]. This reversal of the EAAT transport function thus represents an important mechanism underlying the elevated brain glutamate levels observed after brain injury. Interactions among the major subunits of NKAs are required for ion transport [29]. In this study, the specific NKA inhibitor ouabain decreased glutamate uptake in cultured endothelial cells under OGD conditions and even triggered reverse transfer, with the concomitant destruction of the Na+/K+ gradient inside and outside of the cells, suggesting a key role for NKAs in controlling glutamate transport [30]. Furthermore, conserved phosphorylation sites provide additional targets for the regulation of NKA activity [31]. After brain injury, a significant increase in adenosine levels initially activates A1R, one of the adenosine receptors (A1R, A2AR, A2BR and A3R, all of which are G-protein-coupled receptors), although it is rapidly downregulated, and thus A2AR activation becomes more prominent [32]. Although the activation of A2ARs after brain injury did not significantly alter the expression of FXYD1 or NKA-α1 in endothelial cells, activated A2AR decreased NKA activity in response to high glutamate concentrations by inhibiting the classical downstream PKA pathway [33] to promote the interaction of two PKA subunits, leading to significant increases in [Na+]i and [K+]o and thus rapidly reversing glutamate transport. Additionally, based on the relatively low glutamate conditions, the mechanism by which A2AR reduces glutamate transporter activity involves its enhanced interaction with GLAST/GLT-1, which is mediated by PKA. Because the direction of glutamate transport was reversed, the increased interaction of EAATs induced by A2AR activation subsequently decreased glutamate uptake and exacerbated glutamate release. Consistent with these findings, the in vitro and in vivo results together suggest the existence of a macromolecular complex in endothelial membranes comprising A2AR, GLAST, GLT-1, FXYD1 and NKA-α1 [34]. On the one hand, A2AR activation decreases NKA activity by mediating the interaction between NKA-α1 and FXYD1 and then indirectly but rapidly reversing the transport efficiency of EAATs. On the other hand, A2ARs also enhance this reverse transport function by promoting GLAST/GLT-1 phosphorylation. This dual regulatory mechanism provides a new explanation for the regulation of intraparenchymal glutamate homeostasis in the presence of different glutamate levels induced by endothelial A2ARs. Notably, the interaction between NKA-α1 and FXYD1 was only decreased by A2AR activation under high glutamate conditions. Although the specific mechanism underlying this observation has not been identified, we speculate that it may be related to the allosteric regulation of both PKA subunits induced by over-activated A2ARs in response to high glutamate concentrations [35].