Substituted quinolines as inhibitors of l-glutamate transport into synaptic vesicles
Introduction
As the primary excitatory neurotransmitter in the mammalian CNS, l-glutamate participates in both fast synaptic communication and the higher order signal processing required in learning, memory, development, and plasticity (for review see Cotman et al., 1995). Consistent with classical mechanisms of neurotransmission, l-glutamate has been shown to be sequestered in synaptic vesicles (Storm-Mathisen et al., 1983) and upon depolarization, to be released from neurons in a calcium-dependent manner (Cotman et al., 1981, Nicholls and Sihra, 1986). l-Glutamate is concentrated in these synaptic vesicles in an ATP-dependent manner by a specific uptake system (Naito and Ueda, 1985). This transporter has also been shown to correlate anatomically and temporally with the presence of glutamatergic synapses (Ueda, 1986, Fischer-Bovenkerk et al., 1988, Kish and Ueda, 1989).
Pharmacological and kinetic studies of the vesicular uptake system demonstrate that it is distinct from the well characterized cellular glutamate transporters present on neurons and glia (e.g. EAAC-1, GLT-1, and GLAST; human counterparts, EAAT 1–3 (Arriza et al., 1994, Gegelashvili and Schousboe, 1997)). In contrast to the sodium-dependency exhibited by the cellular systems, vesicular uptake is sodium-independent, coupled to an electrochemical proton gradient generated by a vacuolar type Mg++-ATPase, and requires physiologically relevant concentrations of chloride ion (e.g. 1–4 mM) (Naito and Ueda, 1985, Maycox et al., 1988). The potential regulatory role of a chloride binding site was suggested by studies demonstrating that the anion channel blocker, 4,4′diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) inhibits vesicular glutamate transport and that this inhibition could be prevented by excess levels of chloride (Hartinger and Jahn, 1993). Kinetic studies with isolated vesicles also indicate significant differences in the Km values determined for glutamate uptake: 1–2 mM (Naito and Ueda 1985), versus 5–50 μM for the high-affinity cellular systems (Bridges et al., 1994, Garlin et al., 1995). The specificities of the respective systems are also distinct, as a number of well known substrates and inhibitors of cellular transporters exhibit little or no activity at the vesicular system (e.g. d- and l-aspartate, l-cysteate) (Dunlop et al., 1991, Fyske and Fonnum, 1991, Tabb and Ueda, 1991). While large numbers of excitatory amino acid (EAA) analogues have been tested as potential blockers of vesicular glutamate uptake, only a few potent competitive inhibitors have been identified; e.g. trans-1-aminocyclopentane-1,3-dicarboxylic acid (trans-ACPD) (Winter and Ueda, 1993), bromocriptine (Carlson et al., 1989), certain naphthalene disulfonic acids (Roseth et al., 1995), and kynurenate (Fyske et al., 1992). This last compound is of particular interest because it is not only present in the CNS (for review see Stone, 1993) but also because it exhibits activity at EAA ionotropic receptors. Specifically, kynurenate acts as a competitive inhibitor at the glutamate sites on the NMDA, KA and AMPA receptors (Ganong et al., 1983) and at the glycine site on the NMDA receptor (Watson et al., 1988, Kessler et al., 1989).
In the present work we analyze the structure–activity relationships and kinetic properties of a library of kynurenate analogues as inhibitors of the vesicular glutamate transporter and identify two closely related derivatives, 7-chloro-kynurenate and xanthurenate, that are markedly more potent as uptake inhibitors than the parent compound. Parallel studies assessed the activity of these inhibitors at the sodium-dependent synaptosomal transporter and at the EAA ionotropic receptors. Computer-based molecular modeling was used to analyze the structure–activity data in an attempt to explain the inhibitory activities of these compounds and to postulate an active conformation of glutamate that binds to the vesicular transporter. The findings will help to further define the pharmacological specificity of the glutamate binding site on the vesicular uptake system and identify novel pharmacological probes with which to investigate its function.
Section snippets
Isolation of synaptic vesicles
Synaptic vesicles were isolated from the forebrains of male Sprague–Dawley rats (200–300 g) essentially as described by Kish and Ueda (1989). Briefly, rats were rapidly decapitated and their cerebrums were removed and minced with scissors in an ice-cold solution consisting of 0.32 M sucrose, 1.0 mM sodium bicarbonate, 1.0 mM magnesium acetate, and 0.5 mM calcium acetate (pH 7.2). The finely minced cerebrums were homogenized (motorized Potter-Elvejhem, Teflon/glass (Wheaton) and centrifuged
Materials
l-Glutamic acid, kynurenic acid, 7-chloro-kynurenic acid, picolinic acid, 4-hydroxy-pyridine, quinaldic acid, and xanthurenic acid were purchased from Sigma (St. Louis, MO.). Pyridine, 3-hydroxy-picolinic acid, quinaldic acid, quinoline, 4-hydroxy-quinoline, and 2-pyrazine carboxylic acid were purchased from Aldrich (Milwaukee, WI). 4-Hydroxy-picolinic acid was synthesized as previously described by Clark-Lewis and Mortimer (1961). l-[3,4-3H]-Glutamic acid, [vinylidine-3H] -kainic acid, D,l-α
Pharmacology of the vesicular glutamate transporter
A series of quinoline and pyridine analogues of kynurenate were tested for their ability to inhibit the uptake of 3H-l-glutamate into synaptic vesicles prepared from rat forebrain (Table 1). Consistent with previous studies (Fyske et al., 1992), kynurenate markedly reduced the uptake of 3H-l-glutamate (0.25 mM) to 11% of control values (1532±111 pmol/min per mg protein) when included in the assay at 5.0 mM. Analogues of kynurenate were selected in an effort to examine the contribution of the
Discussion
The identification of kynurenate as an inhibitor of glutamate uptake into synaptic vesicles prompted our investigation into the structure–function relationships governing this activity (Fyske et al., 1992). The demonstration that several of the substituted quinolines are competitive inhibitors and exhibit Ki values lower than l-glutamate itself is consistent with their binding to the substrate site on the transporter and raises intriguing questions as to the chemical/conformational basis of
Acknowledgements
This research was supported in part by NIH grants: NS30570 (RJB) and NSRA NS10156 (CSE). The authors would like to thank Hans Koch and Danielle Dauenhauer for their technical assistance with the biochemical assays.
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