Comparison of [3H]-(2S,4R)-4-methylglutamate and [3H]d-aspartate as ligands for binding and autoradiographic analyses of glutamate transporters

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Abstract

While studies with [3H]d-aspartate ([3H]d-Asp) illustrate specific interactions with excitatory amino acid transporters (EAATs), new insights into the pharmacological characteristics and localization of specific EAAT subtypes depend upon the availability of novel ligands. One such ligand is [3H]-(2S,4R)-4-methylglutamate ([3H]4MG) which labels astrocytic EAATs in homogenate binding studies. This study examined the utility of [3H]4MG for binding and autoradiography in coronal sections of rat brain. Binding of [3H]4MG was optimal in 5 mM HEPES buffer containing 96 mM NaCl, pH 7.5. Specific binding of [3H]4MG exhibited two components, but was to a single site when glutamate receptor (GluR) sites were masked with kainate (KA; 1 μM): t1/2  5 min, Kd 250 nM and Bmax 5.4 pmol/mg protein. Pharmacological studies revealed that [3H]4MG, unlike [3H]d-Asp, labeled both EAAT and ionotropic GluR sites. Further studies employed 6-cyano-7-nitroquinoxaline (30 μM) to block GluR sites, but selective EAAT ligands displayed lower potency than expected for binding to transporters relative to drugs possessing mixed transporter/receptor activities. Autoradiography in conjunction with densitometry with [3H]4MG and [3H]d-Asp revealed wide, but discrete distributions in forebrain; significant differences in binding levels were found in hippocampus, nucleus accumbens and cortical sub-areas. Although EAAT1 and EAAT2 components were detectable using 3-methylglutamate and serine-O-sulphate, respectively, the majority of [3H]4MG binding was to KA-related sites. Overall, in tissue sections [3H]4MG proved unsuitable for studying the autoradiographic localization of EAATs apparently due to its inability to selectively discriminate Na+-dependent binding to Glu transporters.

Introduction

Excitatory amino acid transporters (EAATs) play a fundamental role in l-glutamate (Glu) homeostasis at the synapse (Danbolt, 2001, O'Shea, 2002, Beart and O'Shea, 2007). Altered EAAT function has been implicated in the potentiation of excitotoxicity and in a number of neuropathologies, such as stroke and amyotrophic lateral sclerosis (Heath and Shaw, 2002, Allen et al., 2004). Of the five subtypes of EAATs (EAAT1–5) described, EAAT1–3 have been the most intensely studied with a considerable body of evidence revealing that their characteristics and localization are altered by the manipulations likely to model physiological changes and pathological insults (Gegelashvili and Schousboe, 1998, Butchbach et al., 2004, Maragakis and Rothstein, 2004, Rothstein et al., 2005, O'Shea et al., 2006, Beart and O'Shea, 2007). Such changes can involve EAATs found in both neurons and astrocytes, thus methods allowing the analyses of the localization and distribution of the different subtypes of EAATs in the brain aid greatly in understanding the regulation of EAAT function. Whilst in this context immunocytochemistry has proved invaluable (Danbolt, 2001), autoradiography with selective radioligands represents a powerful technique for determining the localization and pharmacological properties of binding sites in discrete regions of complex tissues (Wharton et al., 1993). Studies using this technique have contributed fundamental knowledge concerning the location and characteristics of EAAT binding sites with the use of [3H]d-aspartate ([3H]d-Asp), a non-metabolised substrate of the EAATs, in binding studies illustrating specific interactions with EAATs (Davies and Johnston, 1976, Balcar and Li, 1992). Autoradiographic experiments with this ligand in rat and mouse tissue have demonstrated the heterogeneity of EAATs, and that the majority of sites are located throughout the grey matter of the brain (Anderson et al., 1990, Greenamyre et al., 1990, Li and Balcar, 1994, Killinger et al., 1996). Studies of the pharmacological characteristics of these binding sites reveal that the binding of [3H]d-Asp is Na+-dependent, saturable, specific and of high affinity (Parsons and Rainbow, 1983, Anderson et al., 1990, Anderson and Vickroy, 1990, Li and Balcar, 1994), and are consistent with binding to Glu transporters. Moreover, when the binding of [3H]d-Asp was tested in the presence of Glu receptor (GluR) agonists, such as kainic acid (KA) and AMPA, no effect on [3H]d-Asp binding was noted, demonstrating selective interaction of [3H]d-Asp with EAAT binding sites and not GluRs (Anderson and Vickroy, 1990). [3H]d-Asp does not, however, distinguish between the binding sites for the different EAAT subtypes (Anderson et al., 1990).

As more pharmacological agents targeted at the various subtypes of EAATs become available, the data arising from their use expands existent knowledge concerning EAAT binding sites (for review see Bridges and Esslinger, 2005). For example, the use of the differing isomers of threo-β-hydroxyaspartic acid (THA) by Anderson and Vickroy (1990) distinguished between two anatomically distinct d-Asp binding sites, with the d-isomer of THA being a less potent inhibitor of [3H]d-Asp binding than l-THA in rat cerebellum compared with forebrain, indicating a pharmacological difference between the EAATs located in these brain regions. This regional variation in transporter binding was also revealed through the use of weak transporter inhibitors (IC50  100 μM) such as dl-2-aminoadipic acid, 3-aminoadipic acid, KA and dihydrokainate (DHK) (Killinger et al., 1996). These studies also showed that KA and DHK were more effective inhibitors of EAAT-related binding in the forebrain of rat tissue than in the cerebellum. Higher-affinity inhibitors (e.g. l-trans-pyrrolidine-2,4-dicarboxylate; PDC) also possessed similar inhibitory profiles, however, their lower IC50 values would indicate that ligands with two negative and one positive charge have increased affinity for the EAAT binding site (Killinger et al., 1996).

(2S,4R)-4-Methylglutamate (4MG) is a Glu analogue displaying affinity for EAATs, and has been reported to possess greater affinity for EAAT2 relative to EAAT1 (Vandenberg et al., 1997). We found that the radiolabeled form of this compound ([3H]4MG) allowed the development of robust binding assays in brain homogenates and cultured astrocytes, with data revealing a preference of this ligand for astrocytic EAATs (Apricò et al., 2001, Apricò et al., 2004). Our use of this radiolabeled synthetic analogue of Glu to examine the characteristics of EAATs represented the first introduction of a novel radioligand in this area of study since the synthesis of [3H]d-Asp (Davies and Johnston, 1976). Further investigation of this ligand was, therefore, undertaken to determine its potential for use in autoradiographic studies. Given the differing affinities of 4MG at the astrocytic transporters (EAAT1 and 2; Vandenberg et al., 1997), further studies using preferential inhibitors for each EAAT subtype sought to explore the differential localization of these two EAAT subtypes.

Section snippets

Materials

[3H]4MG (27 Ci/mmol), 6-cyano-7-nitroquinoxaline (CNQX), l-anti-endo-3,4-methanopyrrolidine dicarboxylate (MPDC), PDC, l-(2S,3S,4R)-2-(carboxycyclopropyl)-glycine (l-CCG-III), l-threo-3-methylglutamate (3MG), dl-threo-β-benyloxyaspartate (TBOA) and 4MG were all obtained from Tocris Cookson (Bristol, UK). l-Glu, l-Asp, d-Asp, dihydrokainate (DHK), KA, l-serine-O-sulphate (SOS) and gelatine were purchased from Sigma Chemical Co. (St. Louis, USA). Ecolite® scintillant and Hyperfilm™ were purchased

Preliminary studies

Numerous experiments were performed to determine the optimal conditions needed to establish how [3H]4MG might be employed as a radioligand in autoradiographic studies for EAATs. Buffers, differing in ionic composition, were initially examined to determine if this variable would influence the amount of binding observed in coronal sections (data not shown). In the presence of a buffer lacking K+ (“Binding Buffer”), binding was inhibited by 3MG, TBOA and PDC (100, 10 and 10 μM respectively, 25–45%

Discussion

The labeled forms of d/l-Asp have been used in numerous autoradiographic studies to visualise the distribution of EAATs (Anderson et al., 1990, Anderson and Vickroy, 1990, Balcar and Li, 1992, Li and Balcar, 1994, Lieb et al., 2000). More recently, the concept of using labeled synthetic compounds acting at EAATs has been proposed (Balcar et al., 1995). Following our observation that [3H]4MG could be used as a radioligand for EAATs in binding assays (Apricò et al., 2001, Apricò et al., 2004),

Acknowledgement

Supported in part by Monash University and a Program Grant (#236805) from the NH&MRC (Australia), of which PMB is a Research Fellow.

References (44)

  • J.A. Larm et al.

    Neurotoxin domoic acid produces cytotoxicity via kainate- and AMPA-sensitive receptors in cultured cortical neurones

    Neurochem. Int.

    (1997)
  • I. Lieb et al.

    Quantitative autoradiography of Na+-dependent [3H]l-aspartate binding to l-glutamate transporters in rat brain: structure-activity studies using l-trans-pyrrolidine-2,4-dicarboxylate (l-t-PDC) and 2-(carboxycyclopropyl)-glycine (CCG)

    Neurochem. Int.

    (2000)
  • H. Lomeli et al.

    High-affinity kainate and domoate receptors in rat brain

    FEBS Lett.

    (1992)
  • O.H. Lowry et al.

    Protein measurement with the Folin phenol reagent

    J. Biol. Chem.

    (1951)
  • N.J. Maragakis et al.

    Glutamate transporters: animal models to neurologic disease

    Neurobiol. Dis.

    (2004)
  • R.D. O'Shea et al.

    Effects of lipopolysaccharide on glial phenotype and activity of glutamate transporters: evidence for delayed up-regulation and redistribution of GLT-1

    Neurochem. Int.

    (2006)
  • B. Parsons et al.

    Quantitative autoradiography of sodium-dependent [3H]d-aspartate binding sites in rat brain

    Neurosci. Lett.

    (1983)
  • N.J. Toms et al.

    A novel kainate receptor ligand [3H]-(2S,4R)-4-methylglutamate: pharmacological characterization in rabbit brain membranes

    Neuropharmacology

    (1997)
  • J.R. Unnerstall et al.

    Autoradiographic localization of high-affinity [3H]kainic acid binding sites in the rat forebrain

    Eur. J. Pharmacol.

    (1983)
  • A.B. Young et al.

    Excitatory amino acid receptors in the brain: membrane binding and receptor autoradiographic approaches

    Trends Pharmacol. Sci.

    (1990)
  • N.J. Allen et al.

    Reversal or reduction of glutamate and GABA transport in CNS pathology and therapy

    Pflügers Arch.

    (2004)
  • K. Apricò et al.

    Binding and transport of [3H](2S,4R)-4-methylglutamate, a new ligand for glutamate transporters, demonstrate labeling of EAAT1 in cultured murine astrocytes

    J. Neurosci. Res.

    (2004)
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    These authors contributed equally to this work.

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