ELSEVIER European Journal of Pharmacology

Molecular Pharmacology Section 289 (1995) 387-390

ejp molecular pharmacology

Short communication

Inhibition by folded isomers of L-2-(carboxycyclopropyl)glycine of glutamate uptake via the human glutamate transporter hGluT-1

Hiroshi Yamashita a, Hideshi Kawakami a, Yu-xiang Zhang a, Tatsuya Hagiwara b, Kohichi Tanaka b, Shigenobu Nakamura a,*

a Third Department of Internal Medicine, Hiroshima University School of Medicine, 1-2-3 Kasumi, Minami-Ku, Hiroshima734, Japan b Department of Degenerative Neurological Diseases, National Institute of Neuroscience, NCNP, Kodaira187, Japan

Received 23 November 1994; revised 17 January 1995; accepted 24 January 1995

Abstract

The effects of isomers of 2-(carboxycyclopropyl)glycine (CCG) on uptake of L-glutamate were investigated in COS-7 cells that expressed a cloned human glutamate transporter (hGluT-1). The (2S, 3S, 4R)-isomer (L-CCG-III) and the (2S, 3R, 4S)-isomer (L-CCG-IV) markedly inhibited glutamate uptake with a 50% inhibitory concentration of 290 nM and 1.1/~M, respectively. The (2S, 3S, 4S)-isomer (L-CCG-I) and the (2S, 3R, 4R)-isomer (L-CCG-II) did not inhibit glutamate uptake at concentrations of < 10/~M. Thus, hGluT-I showed a markedly higher affinity for L-CCG-III and L-CCG-IV with a folded conformation of the glutamate skeleton, than for L-CCG-I or L-CCG-II with an extended conformation.

Keywords: Human glutamate transporter (hGluT-1); CCG (2-(Carboxycyclopropyl)glycine); COS-7 cell; transfection

1. Introduction

Removal of glutamate from the synaptic cleft, an essential step in the glutamatergic neurotransmission process, is mediated by high-affinity glutamate trans- porters. Four groups have independently cloned com- plementary DNA (cDNA) encoding the high-affinity glutamate transporters GLAST (GluT-l) and GLT-1 from rat, and EAAC1 from rabbit (Storck et al., 1992; Tanaka, 1993; Pines et al., 1992; Kanai and Hediger, 1992). Further more, we have cloned and expressed the human GluT-1 (hGluT-1) cDNA (Kawakami et al., 1994). hGluT-1 is widely distributed in the whole brain.

2-(Carboxycyclopropyl)glycine (CCG) isomers are conformationally restricted glutamate analogs in which the cyclopropyl group fixes the glutamate chain. Be- cause the glutamate molecule is relatively flexible, the carbon chain of glutamate can conform completely with that of L-CCG (Shinozaki et al., 1989). The (2S, 3S, 4S)-isomer (L-CCG-I) and the (2S, 3R, 4R)-isomer (L-CCG-II) adopt an extended conformation of the

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L-glutamate skeleton, whereas the (2S, 3S, 4R)-isomer (L-CCG-III) and the (2S, 3R, 4S)-isomer (L-CCG-IV) adopt a folded conformation of the L-glutamate skele- ton. L-CCG-I and L-CCG-II interact with members of the metabolic glutamate receptor (mGluR) family, and L-CCG-I acts as a potent agonist at mGluR2 (Hayashi et al., 1992). L-CCG-III was identified as an inhibitor of transport activity on the basis of the observation that it potentiates the excitatory effect of glutamate (Shinozaki et al., 1989). L-CCG-IV is a potent and selective agonist for N-methyl-D-aspartate (NMDA) re- ceptors (Kawai et al., 1992). These isomers are useful as probes for elucidating conformational requirements of the glutamate molecule. We have now investigated the effects of L-CCG isomers on L-glutamate uptake by hGluT-1 in order to evaluate how the conformation of the glutamate skeleton affect the transport process.

2. Materials and methods

COS-7 cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum at 37 ° C under an atmosphere of 5% CO 2. Cells (1 × 107) were transfected by electroporation (200 V, 1600 /~F)

388 tt. Yamashita et al. / European Journal of Pharmacology Molecular Pharmacology Section 289 (1995) 387 390

with 20 ~g of the eukaryotic expression vector pcDNA3 containing hGluT-1 cDNA (pcDNA3hGIuT-1). After the electrical discharge, cells were incubated for 10 min at 20 ° C, then diluted with culture medium. Trans- fected COS-7 cells were culturedon 24-well, collagen- coated plastic plates for 2 days.

To assess glutamate uptake, the subconfluent cells were washed three times with 1 ml of modified phos- phate-buffered saline (PBS) that contained 137 mM NaCI, 2.7 mM KCI, 8.1 mM NazHPO4, 1.5 mM KH2PO 4, 1 mM MgCl2, 0.9 mM CaC12 and 5.6 mM D-glucose (pH 7.35). Unless otherwise indicated, cells were incubated with 50 nM L-[3H]glutamate (46 Ci/mmol; Amersham) in 200 /xl of modified PBS for 10 rain at 37°C. Uptake was terminated by three washes with 1 ml of ice-cold modified PBS, and ra- dioactivity was extracted with 1 M NaOH and mea- sured by scintillation counting. NaCl in modified PBS was replaced by LiCI to assess nonspecific uptake.

Data are reported as statistical analyses utilized analysis of variance, and a level of P < 0.05 was ac- cepted as statistically significant.

L-CCG-I, L-CCG-III, and L-CCG-IV were obtained from Tocris Cookson (Langford, Bristol, UK). L-CCG- II was provided by Professor S. Nakanishi.

3. Results

Transfected COS-7 cells accumulated up to five times the amount of L-[3H]glutamate taken up by non- transfected cells. Kinetic studies showed that L- [3H]glutamate uptake in transfected cells was saturable with respect to glutamate concentration, and an Eadie Hofstee plot of the data revealed a Michaelis constant (K m) of 21 /.tM and a maximum uptake velocity (Vm~) of 228 pmol per milligram of protein per minute (Fig. 1A). L-Glutamate, L-aspartate, and D-aspartate, each at 100 /.tM, inhibited 50 nM L-[3H]glutamate uptake by 81, 87, and 77%, respectively (Fig. 1B). D-Glutamate (100 /xM) failed to inhibit L-[3H]glutamate uptake. DL-Threo-/3-hydroxyaspartate, one of the most potent glutamate uptake inhibitors, inhibited L-[3H]glutamate uptake by 88% at 100/zM. However, two region-specific glutamate uptake inhibitors, dihydrokainate and L-a- aminoadipate inhibited L-[3H]glutamate uptake by only 61 and 41%, respectively at a concentration of 300/xM.

L-CCG-III and L-CCG-IV markedly inhibited L- [3H]glutamate uptake in transfected cells in a dose dependent manner (Fig. 2A). L-CCG-III almost totally inhibited L-[3H]glutamate uptake at concentrations of > 10 /zM, with a 50% inhibitory concentration (ICs0) of 290 nM. L-CCG-IV almost totally inhibited L- [3H]glutamate uptake at 100/.tM, with an ICs0 value of 1.1 /.tM. L-glutamate inhibited L-[3H]glutamate uptake at 100 /.tM, with an ICs0 value of 4.2 /xM. L-CCG-I

and L-CCG-II failed to inhibit L-[3H]glutamate uptake at concentrations of_< 10 /.tM. Lineweaver-Burk plots clearly demonstrated the concentration-dependent ef- fects of L-CCG-III and L-CCG-IV on L-[3H]glutamate uptake, reflecting competitive inhibition in both in- stances (Fig. 2B).

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Fig. 1. (A) Effect of glutamate concentration on Na+-dependent L-13Hlglutamate uptake in COS-7 cells transfected with pcDNA3hG- luT-1. Cells were incubated for 15 min with various concentrations of l.-[3H]glutamate in modified PBS. Results are means of triplicate incubations. (Inset) Eadie-Hofstee plot of the concentration-depen- dence of L-[3H]glutamate uptake [v, uptake (pmol/mg protein per min); [S], glutamate (/zM) ]. (B) Pharmacological characterization of L-[3H]~utamate uptake in COS-7 cells transfected with pcDNA3hG- luT-1. Cells were incubated with the indicated concentration (mM) of various glutamate uptake inhibitors for 15 min. Results are means of triplicate incubations. Cont., control; THA, DL-threo-/3-hydroxy- aspartate; DHK, dihydrokainate; AAD, L-a-aminoadipate.

H. Yamashita et aL / European Journal of Pharmacology - Molecular Pharmacology Section 289 (1995) 387-390 389

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Fig. 2. (A) Effect of L-CCG isomers on L-[3H]glutamate uptake in COS-7 cells transfected with pcDNA3hGIuT-1. Values are the means of six independent experiments, each performed in triplicate, and are expressed as a percentage of the control (Cont.) uptake. SEM values were < 4.7% of the mean. (B) Lineweaver-Burk plots [v, uptake (pmol/mg protein per min; [S], glutamate (~M)] demonstrating competitive inhibition of glutamate uptake by L-CCG-III and L-CC-G-IV. Transfected COS-7 cells were incubated with 50 nM L-[3H]glutamate. Glutamate uptake was measured in triplicate in the linear range in the absence (0) or presence of 1 /zM (~), 3 #M (©), or 5 /~M (zx) L-CCG-III or L-CCG-IV.

4. Discussion

Glutamate uptake by COS-7 cells that expressed hGluT-1 was specific and saturable, with an estimated K m of 21 ~M, consistent with the properties of a high-affinity glutamate transport system. The affinity for glutamate of hGluT-1 in transfected COS-7 cells (Km = 21 ~M) was a slightly higher than that observed in Xenopus oocytes injected with hGluT-1 synthetic RNA ( K m = 78 ~ M ) (Kawakami et al., 1994), which is probably attributable to differences in the properties of the two membranes. Glutamate uptake in transfected COS-7 cells was effectively inhibited by L-glutamate, L-aspartate, D-aspartate or DL-threo-/3-hydroxyaspar- tate, whereas D-glutamate failed to inhibit glutamate uptake and dihydrokainate or L-a-aminoadipate only weakly inhibited this process; similar pharmacological properties were observed in Xenopus oocytes express- ing hGluT-1 and rat GluT-1 (Kawakami et al., 1994; Tanaka, 1994).

Chemical modification of substrate and charac- terization of the actions of various substrate analogs is an important approach to analyze the active conforma- tions of the glutamate molecule. We therefore investi- gated the effects of L-CCG isomers on glutamate up- take in COS-7 cells expressing hGluT-1, which is thought to be expressed predominantly in glial cells in vivo. L-CCG-I I I and L-CCG-IV inhibited glutamate uptake competitively with ICs0 values of 0.29 and 1.1 /zM, respectively, although it is unknown whether L- CCG is taken up into the cells through hGluT-1. L- CCG-I and L-CCG-II did not inhibit glutamate uptake

at concentrations of_< 10/xM. Thus, the rank order of potency for inhibition of glutamate uptake was L-CCG- II I > L-CCG-IV = L-glutamate >> L-CCG-I = L-CCG-II. L-CCG-I I I has been shown to inhibit D-aspartate up- take in synaptosomes of rat cerebral cortex (Kawai et al., 1992). Nakamura et al. (1993) showed that L-CCG- I l l potently inhibited glutamate uptake in glial plas- malemmal vesicles and synaptosomes from rat hip- pocumpus with IC50 values of 2.0 and 1.6/~M, respec- tively. L-CCG-I I I also showed a potent inhibitory ef- fect on glutamate uptake in crude synaptosomes from rat cortex and cerebellum, with ICs0 values of 1.6 and 1.0 /zM, respectively. L-CCG-II also inhibited gluta- mate uptake in crude synaptosomes from rat cerebel- lum with an ICs0 of 5.5 /xM (Robinson et al., 1993). The effects of L-CCG isomers on glutamate uptake observed in these studies may reflect compound ac- tions at more than one transporter.

In summary, we have demonstrated that hGluT-1 shows a higher affinity for L-CCG-I I I or L-CCG-IV, which adopt a folded conformation of the glutamate skeleton, than for L-CCG-I or L-CCG-II , which adopt an extended conformation. The physiological relevance of the active conformations of the glutamate molecule for glutamate transporters remain to be elucidated.

Acknowledgements

We thank Dr. S. Nakanishi for supplying L-CCG-II , and Dr. S. Kitayama for valuable advice and discus- sion. Supported by a grand-in-aid from the Research

390 H. Yamashita et al. / European Journal o f Pharmacology - Molecular Pharmacology Section 289 (1995) 387-390

C o m m i t t e e o f C N S D e g e n e r a t i v e D i s e a s e s o f t h e M i n -

is t ry o f H e a l t h a n d W e l f a r e o f J a p a n .

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