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  • The existence of EAAT subtypes raises obvious

    2022-01-11

    The existence of 5 EAAT subtypes raises obvious questions as to the cellular and anatomical distribution of the various transporters (for review, see Gegelashvili & Schousboe, 1998, Danbolt, 2001). While difference in techniques (i.e., protein vs. mRNA), reagents, and preparations (i.e., in vivo vs. in vitro, developmental age) can lead to conflicting results and make definitive conclusions difficult (see, for example, Danbolt, 2001), a number of generalizations can be made regarding the localizations of the subtypes. EAATs 1–3 are present in the highest levels in the CNS, although all 3 appear to have been detected in peripheral tissues such as heart, liver, kidney, and intestine (Gegelashvili & Schousboe, 1998). Within the CNS, EAAT3, and EAAT4 are considered to be neuronal transporters (Furuta et al., 1997). EAAT3 is generally distributed throughout the forebrain and spinal cord, with enriched levels found in the hippocampus, basal ganglia, and cortical structures. Colocalization studies further suggest that EAAT3 may act not only to recapture the transmitter into excitatory glutamatergic neurons but also serve to provide glutamate as a precursor to GABAergic neurons (Sepkuty et al., 2002). EAAT4, on the other hand, is expressed at the highest levels in the cerebellum, where it is localized to Purkinje neurons. In contrast to EAAT3 and 4, EAAT1 and EAAT2 are typically considered to be glial transporters, with EAAT1 being distributed predominantly in the cerebellum and EAAT2 in the forebrain. However, the recent identification of an EAAT2 splice variant that appears to be expressed in both astrocytes and neurons calls the cellular specificity of EAAT2 distribution into question (Chen et al., 2002, Schmitt et al., 2002). This is a particularly relevant and potentially controversial issue because EAAT2 is considered to mediate the bulk of glutamate uptake in the adult Norfloxacin sale and many previous studies with synaptosomes (presumed isolated presynaptic terminals) indicate that uptake into this compartment is most consistent with an EAAT2 pharmacology (Koch et al., 1999a, Danbolt, 2001). Among all the subtypes the distribution of EAAT5 may be the most restricted, as its expression appears to be limited to the retina, where it is found on neurons and Muller cells (Eliasof et al., 1998).
    Excitatory amino acid transporter pharmacology Early attempts to delineate the pharmacological specificity of the EAATs utilized a variety of CNS tissue preparations (e.g., synaptosomes, brain slices, primary cultures of neurons and glia) and typically quantified the uptake of radiolabeled substrates, such as l-glutamate, l-aspartate, or d-aspartate, in the presence and absence of EAA analogues. The collections of compounds used in these first structure activity relationship (SAR) experiments were typically designed around varying the most basic aspects of the acidic amino acid structure, such as enantioselectivity (e.g., d- vs. l-glutamate or aspartate), chain length (e.g., aspartate, glutamate, α-aminoadipate), and distal acidic group composition (e.g., COOH, SO2H, SO3H, PO3H2, CONHOH; Balcar & Johnston, 1972, Logan & Snyder, 1972, Roberts & Watkins, 1975). While these studies were somewhat constrained by (i) the use of heterogeneous preparations, (ii) the availability of compounds, and (iii) a bias toward characterizing binding (i.e., competitive inhibition) more so than actual translocation, the experiments nonetheless provided a good fundamental composite profile of glutamate transport. Both d- and l-aspartate were identified as substrates of the system, although d-glutamate was inactive as an inhibitor. The recognition that d-aspartate was both a transporter substrate and metabolically inert led to it being used in many later studies as a substrate of choice. Inhibitors of l-glutamate uptake generally shared the properties of being α-amino acids that possessed a second carboxylate mimic 2–4 carbon atoms from the α-carboxyl group. Longer-length dicarboxylic amino acids, such as l-α-aminoadipate, exhibited little or no activity as inhibitors. Substitutions of either sulfinic or sulfonic acid groups for the distal carboxylate (e.g., l-cysteine sulfinic and l-cysteic acid) were tolerated and retained inhibitory activity, while phosphate derivatives did not. In general, modification to the acid groups, such as esterification, resulted in loss of activity. An interesting exception may be found among a number of aspartate derivatives where the distal COOH groups has been converted to a hydroxamate or N-nitroso hydroxylamine (Roberts & Watkins, 1975, Palos et al., 1996).