Glutamate aspartate transporters or excitatory amino
Glutamate-aspartate transporters or excitatory amino Go 6983 transporters (EAATs) are essential for the maintenance of glutamate homeostasis. EAATs are widely distributed in central neurons and glial cells (Danbolt, 2001, Martinez-Lozada et al., 2011). They are driven by Na + and K+ gradients (Jiang and Amara, 2011) and five different ‘high-affinity’ glutamate transporters have been cloned: GLAST (EAAT1) (Storck et al., 1992), GLT-1 (EAAT2) (Pines et al., 1992), EAAC1 (EAAT3) (Kanai and Hediger, 1992), EAAT4 (Fairman et al., 1995) and EAAT5 (Arriza et al., 1997).
Adenosine is an important neuromodulator present in the CNS (Paes-De-Carvalho, 2002, Sebastiao and Ribeiro, 2015). Adenosine receptors and glutamate transporters are present very early in the vertebrate retina, acting on synaptic activity and cellular differentiation in this tissue (Kubrusly et al., 1998, Paes-De-Carvalho, 2002, Stutz et al., 2011). It has been identified serine residues that can be phosphorylated by protein Kinase A (PKA) on all EAAT subtypes (Gegelashvili and Schousboe, 1997), changing their activity and expression on cell surface (Garcia-Tardon et al., 2012).
Four adenosine receptor (ARs) subtypes have been cloned, namely A1, A2A, A2B and A3 receptors. The subtypes A1R and A3R are usually coupled to inhibitory G-proteins (Gi and Go) while A2AR and A2BR are coupled to stimulatory G-proteins (Gs). A1R activation inhibits adenylyl cyclase (AC), while A2AR activates this enzyme (Ribeiro et al., 2002), leading to a decrease and increase in cyclic adenosine monophosphate (cAMP) levels, respectively. We have recently shown that caffeine, a non-selective adenosine receptor antagonist, potentiates d-aspartate-stimulated GABA release in the chick retina (Ferreira et al., 2014). However, there is little information concerning the participation of adenosine receptors in glutamate homeostasis. More specifically, whether adenosine modulators, as caffeine, could influence glutamate transporters activity.
Therefore, we aimed to investigate the activity of EAATs during post natal development of rat retinas and how this transport can be modulated by a single caffeine exposure. Here we show that EAA uptake and release are developmentally changed and that caffeine modulates [3H]-Aspartate transport by binding to A2AR in “ex-vivo” retinas.
Discussion The main results described here show that immature first week postnatal rat retina challenged by l-glutamate releases [3H]-d-Aspartate linked to the reverse transport, with the participation of NMDA, but not of non-NMDA receptors. It is well documented that depending on extracellular EAA levels, glutamate or aspartate release is increased or reduced in the CNS (Stutz et al., 2011). In addition, it has been reported that NMDA receptor activation increases EAAT1 and EAAT2 expression in primary cultures of retinal glial cells obtained from neonatal rats (Furuya et al., 2012). In our case, all the main EAATs subtypes were expressed during development of the rat retina and the expression of EAAT1, EAAT2 and EAAT3 increased considerably at PN14. Glutamate and aspartate systems play essential roles in many events of CNS differentiation. The rat retina still undergoes considerable development during the first two post-natal weeks, reaching its complete physiological maturation within 3weeks (Kalloniatis and Tomisich, 1999). Glutamate is first identified at PN6, while aspartate immunoreactivity is weakly detected at PN11 (Fletcher and Kalloniatis, 1997, Kalloniatis and Tomisich, 1999). We observed that glutamate/aspartate transport is dependent on retinae maturation, comparing PN3 and PN7 to PN14 retinas. We showed that both aspartate uptake and glutamate-stimulated aspartate release decrease in the post natal retina development. This profile of [3H] d-aspartate release and uptake could be linked to EAATs expression or function in the retina differentiation. Glutamate transporters belong to the solute carrier 1 (SLC1) family of transport proteins. They are high-affinity Na+-coupled transporters expressed in neurons and glial cells. EAAT1 is exclusively expressed in glial cells, while EAAT2 is present in both neuronal and glial cells. EAAT3 and EAAT4 are commonly found in neurons and EAAT5 is mainly found in the retinal tissue (Stutz et al., 2011). EAAT1 and EAAT2 are responsible for global glutamate clearance and are mostly expressed on the cell surface. EAAT3 is present in intracellular pool and seems to have a local role on glutamate uptake and is dynamically regulated by injurious stimuli (Bianchi et al., 2014). According to our results, total cellular protein levels of EAAT1, EAAT2 and EAAT3 tended to increase along the post-natal rat retina development.