Dissociation of [H]L-Glutamate Uptake from L-Glutamate- Induced [H]D-Aspartate release by 3-Hydroxy-4,5,6,6a- tetrahydro-3aH-pyrrolo[3,4-d]isoxazole-4-carboxylic Acid and 3-Hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]isoxazole-6- carboxylic Acid, Two Conformationally Constrained Aspartate and Glutamate Analogs

We characterized the interaction of two conformationally constrained aspartate and glutamate analogs, 3-hydroxy-4,5,6,6atetrahydro-3aH-pyrrolo[3,4-d]isoxazole-4-carboxylic acid (HIP-A) and 3-hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]isoxazole6-carboxylic acid (HIP-B), with excitatory amino acid transporters (EAATs) in rat brain cortex synaptosomes. HIP-A and HIP-B were potent and noncompetitive inhibitors of [H]L-glutamate uptake, with IC50 values (17–18 M) very similar to that of the potent EAAT inhibitor DL-threo-benzyloxyaspartic acid (TBOA). The two compounds had little effect in inducing [H]D-aspartate release from superfused synaptosomes but they potently inhibited L-glutamate–induced [H]D-aspartate release, thus behaving as EAAT blockers, not substrates, in a manner similar to those of TBOA and dihydrokainate (DHK). HIP-A and HIP-B, but not TBOA and DHK, unexpectedly inhibited L-glutamate–induced [H]D-aspartate release with IC50 values (1.2–1.6 M) 10 times lower than those required to inhibit [H]L-glutamate uptake. There is therefore a concentration window (1–3 M) in which the two compounds significantly inhibited L-glutamate–induced release with very little effect on L-glutamate uptake. This selective inhibitory effect required quite long preincubation ( 5 min) of synaptosomes with the drugs. At these low concentrations, however, HIP-A and HIP-B had no effect on the EAAT-mediated [H]D-aspartate release induced by altering the ion gradients, indicating that they specifically affect some L-glutamate-triggered process(es)—different from L-glutamate translocation itself—responsible for the induction of reverse transport. These data are inconsistent with the classic model of facilitated exchange-diffusion and provide the first evidence that EAAT-mediated substrate uptake and substrate-induced EAAT-mediated reverse transport are independent. Compounds such as HIP-A and HIP-B could be useful to further clarify the mechanisms underlying these operating modes of transporters. Glutamate is considered the main excitatory transmitter in the mammalian brain and is involved in various physiological functions, including development and plasticity, learning and memory, cognition, pain, and nociception. Abnormally high extracellular glutamate levels, however, cause excitotoxic neuronal death, which involves activation of ionotropic glutamate receptors and excessive calcium influx in neurons (Choi et al., 1987) and is implicated in numerous neurodegenerative diseases. Glutamate receptor activation is terminated, and extracellular glutamate levels kept below toxic levels, primarily by reuptake into glial cells and neurons through high-affinity Na -dependent excitatory amino acid transporters (EAATs) (Danbolt, 2001). EAATs can also contribute to neurotoxic glutamate release. Thus, in conditions in which energy levels fall and the transmembrane gradients of Na collapse (e.g., during ischemia), the electrogenic Na -coupled transporters reverse and This work was partly supported financially by Ministero dell’Istruzione, dell’Università e della Ricerca (COFIN2001–Rome). ABBREVIATIONS: EAAT, excitatory amino acid transporter; HIP-A, 3-hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]isoxazole-4-carboxylic acid; HIP-B, 3-hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]isoxazole-6-carboxylic acid; AMPA, (S)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl) propionic acid; TBOA, DL-threo-benzyloxyaspartic acid; DHK, dihydrokainate; CI, confidence interval; FRR, fractional release rate. 0026-895X/04/6603-522–529$20.00 MOLECULAR PHARMACOLOGY Vol. 66, No. 3 Copyright © 2004 The American Society for Pharmacology and Experimental Therapeutics 3178/1168509 Mol Pharmacol 66:522–529, 2004 Printed in U.S.A. 522 at A PE T Jornals on Jne 9, 2017 m oharm .aspeurnals.org D ow nladed from release glutamate into the extracellular space (McMahon and Nicholls, 1991; Levi and Raiteri, 1993). Most of the release in early ischemia probably occurs this way (Seki et al., 1999; Phillis et al., 2000; Rossi et al., 2000), and is also referred to as transporter-mediated release (Levi and Raiteri, 1993). Transporter-mediated release of intracellular neurotransmitter can also be elicited by the extracellular presence of transporter substrates (Koch et al., 1999b), according to the model of “facilitated exchange diffusion” or “transporter-mediated hetero-exchange” (Fischer and Cho, 1979; Levi and Raiteri, 1993). Blockade of EAATs by nontransportable inhibitors (or blockers) affects glutamate uptake and, when operating, also EAAT-mediated release. EAAT blockers with low or no affinity for ionotropic glutamate receptors could therefore be useful to prevent the glutamate release and neuron death after cerebral ischemia but can produce neurotoxicity in physiological conditions. In the present study, we characterized the interaction of two conformationally constrained aspartate and glutamate analogs (Fig. 1), 3-hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]isoxazole-4-carboxylic acid (HIP-A) and 3-hydroxy4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]isoxazole-6-carboxylic acid (HIP-B), with the EAATs expressed in rat brain cortex synaptosomes, a GLT1-like subtype (Tanaka et al., 1997; Koch et al., 1999a; Suchak et al., 2003). A previous study (Conti et al., 1999b) found that HIP-A and HIP-B had low affinity for (S)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl) propionic acid (AMPA) and kainic acid receptors and none for N-methyl-D-aspartate receptors. HIP-A and HIP-B were also inactive at metabotropic glutamate receptors as agonists or antagonists (Conti et al., 1999b). We first evaluated the compounds’ potency in inhibiting [H]L-glutamate uptake and then carried out release experiments to define these compounds as “substrates” or nontransportable “blockers” of EAATs, as described previously (Koch et al., 1999b). We evaluated the compounds’ ability to induce tritium release from superfused synaptosomes preloaded with [H]D-aspartate, which was used because it is an excellent substrate of EAATs, is not metabolized, and is a poor substrate of vesicular glutamate carrier (Naito and Ueda, 1985; Fykse et al., 1992; Bartlett et al., 1998; Koch et al., 1999a). [H]D-aspartate release from synaptosomes is selectively induced only by EAAT “substrates”, according to the proposed process of EAAT-mediated heteroexchange, and no [H]D-aspartate release can be induced by substrates of other transporters or by selective ligands of ionotropic receptors (Koch et al., 1999b). We therefore used L-glutamate as the prototypical EAAT substrate. Compounds acting as EAAT “blockers” can be identified by evaluating their inhibition of L-glutamate–induced, EAAT-mediated, [H]D-aspartate release. DL-threo-benzyloxyaspartic acid (TBOA) was used here as the reference compound because it is a potent nontransportable blocker of EAAT1–3, with no significant effects on either the ionotropic or metabotropic glutamate receptors (Shimamoto et al., 1998). Dihydrokainate (DHK) was used as a selective EAAT2/GLT1 blocker, with no affinity for EAAT1/GLAST and EAAT3/EAAC1 transporters (Arriza et al., 1994). Finally, we evaluated the compounds’ action in inhibiting [H]D-aspartate release induced in superfused synaptosomes by affecting the ion gradients. With the superfusion apparatus we employed (Raiteri et al., 1974), the neurotransmitter released by the drug is immediately removed and cannot interact with presynaptic receptors or be taken up (Gobbi et al., 1992). Materials and Methods Reagents. HIP-A and HIP-B were synthesized as reported previously (Conti et al., 1999a). L-glutamic acid, DL-threo-hydroxyaspartic acid, 2-deoxy-D-glucose, and rotenone were obtained from Sigma (St Louis, MO). TBOA was obtained from Tocris (Ellisville, MO). DHK was purchased both from Tocris and from Sigma. [H]L-glutamate and [H]D-aspartate were purchased from Amersham Biosciences (Buckinghamshire, UK). Animals. Adult male CRL:CD(SD)BR rats (Charles River, Calco, Italy) were used. They were kept in a controlled environment with a 12-h light/dark cycle and 21°C temperature. Food and water were provided ad libitum. Procedures involving animals and their care were conducted in conformity with the institutional guidelines that are in compliance with national (D.L. n.116 G.U., suppl. 40, 1992 Feb 18) and international laws and policies (EEC Council Directive 86/ 609, OJ L 358, 1, 1987 Dec 12; Guide for the Care and Use of Laboratory Animals, US National Research Council, 1996). Preparation of the Synaptosomal Fraction. Rats were killed by decapitation and the cortex was rapidly dissected out and homogenized in 40 volumes of ice-chilled 0.32 M sucrose, pH 7.4, in a glass homogenizer with a Teflon pestle. The homogenates were centrifuged at 1000g for 5 min and the supernatants centrifuged again at 12,000g for 20 min to yield the crude synaptosomal pellet (P2) (Whit-