EAATs can transport glutamate aspartate cysteic

EAATs can transport glutamate, aspartate, cysteic acid, and serine-o-sulfate (Bjorn-Yoshimoto and Underhill, 2016). All EAATs work as symporters co-transporting 2 or 3 molecules of Na+ and a proton with each molecule of glutamate (or aspartate). In addition, a K+ ion is counter-transported ending in reorientation of the transporter. In this sense, glutamate-binding site is once again accessible to the extracellular environment (Sheldon and Robinson, 2007). It’s conceived that the natural direction of glutamate is inward under normal conditions, but EAA could also be carried in the outward direction when extracellular sodium/ intracellular potassium decrease and/or intracellular sodium / extracellular potassium increase, therefore glutamate transport is reversible (Grewer et al., 2008). In the rat retina we observed that the primary mechanism to promote glutamate/aspartate efflux is mediated by EAATs. Our results show a sodium-dependent mechanism on [3H]d-aspartate release and an inhibition of l-glutamate-induced [3H]d-aspartate release and [3H]d-aspartate uptake by dl-TBOA. These results highlighted the role of EAATs in regulating extracellular glutamate levels in rat retina development. Another possibility to explain the increased levels of [3H]-d-Aspartate release, observed in immature retina cells, is that NMDAR levels are modified during development. NMDA receptor expression decreases with the retina differentiation (Brandstatter et al., 1998). The NMDAR subunits expression is different among the retina Cy7 NHS ester (non-sulfonated) from different species during development (Shen et al., 2006). NMDAR has been related to programed cell death on developing retinal neurons in vivo (Linden et al., 2005), and glutamate is able to stimulate a transient increase in BDNF (mRNA and protein) in retinal ganglion cells and also, stimulate an early release of BDNF into culture media (Fan et al., 2006). Here, we demonstrated that [3H]d-aspartate release stimulated by l-glutamate depends on NMDAR activation at PN7. Several data in the literature point to an important relationship between the adenosinergic and glutamatergic systems during development of the immature vertebrate retina, as reviewed by (Martins and Pearson, 2008). For instance, adenosine is concentrated in glutamatergic terminals and regulate EAA release (Cunha, 2016). Thus it is likely that blockade of adenosine receptors can modify EAA availability. In fact, one hour treatment with caffeine decreased [3H]d-aspartate uptake at PN3 and PN7 retinas, but it was ineffective at PN14. Different factors can be underlying the caffeine effect, as expression of adenosine receptors during development, downstream signaling pathways associated to them, or developmental changes in NMDAR expression/function (Ferreira et al., 2014). Caffeine is known as an inhibitor of adenosine receptors, and at 200μM increased up to 180% the expression of EAAT3, but not of EAAT1 or EAAT2, compared to basal levels at PN7. This effect might be related to an increase of cAMP and an anticipation of the differentiation process in the postnatal retina. Our data indicate that caffeine decreased EAAT1 and EAAT2 expression, and increased EAAT3 expression in total cellular membranes (P2 fraction). It has been described that caffeine has an effect on GluN2B-containing NMDARs (Ferreira et al., 2014) and its stimulation regulates EAAT3 expression in hippocampal area CA1 (Levenson et al., 2002). EAAT3 is known to be functionally regulated at the level of transcription, trafficking and direct pharmacological modulation (Bjorn-Yoshimoto and Underhill, 2016, Danbolt et al., 2016). Our data provide evidence that EAAT3 total expression is increased after caffeine treatment, as well as in P2 fraction. We suggest that upon caffeine treatment, EAAT1 and EAAT2 are taken out of cellular membranes after 60min, thus reducing aspartate uptake. In addition, EAAT3 is not involved in caffeine’s effect on [3H]d-aspartate uptake, but its increment in total protein levels is a consequence of NMDAR stimulation. We cannot distinguish which transporter, between EAAT1 or EAAT2, is responsible for caffeine’s effect. Expression of EAAT1/GLAST was found in a rat retinal Müller cell line, but EAAT2/GLT-1 or EAAT5 were not detected (Tomi et al., 2003). Therefore, EAAT1 from Muller glia might be a potential candidate for regulating extracellular EAA levels following caffeine treatment.