Keiko Shimamoto

A novel metabotropic glutamate receptor agonist: marked depression of monosynaptic excitation in the newborn rat isolated spinal cord.

1 Neuropharmacological actions of a novel metabotropic glutamate receptor agonist, (2S,1′R,2′R,3′R)‐2‐(2,3‐dicarboxycyclopropyl)glycine (DCG‐IV), were examined in the isolated spinal cord of the newborn rat, and compared with those of the established agonists of (2S,1′S,2′S)‐2‐(carboxycyclopropyl)glycine (l‐CCG‐I) or (1S,3R)‐1‐aminocyclopentane‐1,3‐dicarboxylic acid ((1S,3R)‐ACPD). 2 At concentrations higher than 10 μm, DCG‐IV caused a depolarization which was completely blocked by selective N‐methyl‐d‐aspartate (NMDA) antagonists. The depolarization was pharmacologically quite different from that caused by l‐CCG‐I and (1S,3R)‐ACPD. 3 DCG‐IV reduced the monosynaptic excitation of motoneurones rather than polysynaptic discharges in the nanomolar range without causing postsynaptic depolarization of motoneurones. DCG‐IV was more effective than l‐CCG‐I, (1S,3R)‐ACPD or l‐2‐amino‐4‐phosphonobutanoic acid (l‐AP4) in reducing the monosynaptic excitation of motoneurones. 4 DCG‐IV (30 nm–1 μm) did not depress the depolarization induced by known excitatory amino acids in the newborn rat motoneurones, but depressed the baseline fluctuation of the potential derived from ventral roots. Therefore, DCG‐IV seems to reduce preferentially transmitter release from primary afferent nerve terminals. 5 Depression of monosynaptic excitation caused by DCG‐IV was not affected by any known pharmacological agents, including 2‐amino‐3‐phosphonopropanoic acid (AP3), diazepam, 2‐hydroxysaclofen, picrotoxin and strychnine. 6 DCG‐IV has the potential of providing further useful information on the physiological function of metabotropic glutamate receptors.

Agonist analysis of 2-(carboxycyclopropyl)glycine isomers for cloned metabotropic glutamate receptor subtypes expressed in Chinese hamster ovary cells

1 2‐(Carboxycyclopropyl)glycines (CCGs) are conformationally restricted glutamate analogues and consist of eight isomers including l‐ and d‐forms. The agonist potencies and selectivities of these compounds for metabotropic glutamate receptors (mGluRs) were studied by examining their effects on the signal transduction of representative mGluR1, mGluR2 and mGluR4 subtypes in Chinese hamster ovary cells expressing the individual cloned receptors. 2 Two extended isomers of l‐CCG, l‐CCG‐I and l‐CCG‐II, effectively stimulated phosphatidylinositol hydrolysis in mGluR1‐expressing cells. The rank order of potencies of these compounds was l‐glutamate > l‐CCG‐I > l‐CCG‐II. 3 l‐CCG‐I and l‐CCG‐II were effective in inhibiting the forskolin‐stimulated adenosine 3′:5′‐cyclic monophosphate (cyclic AMP) accumulation in mGluR2‐expressing cells. Particularly, l‐CCG‐I was a potent agonist for mGluR2 with an EC50 value of 3 × 10−7 m, which was more than an order of potency greater than that of l‐glutamate. 4 l‐CCG‐I evoked an inhibition of the forskolin‐stimulated cyclic AMP production characteristic of mGluR4 with a potency comparable to l‐glutamate. 5 In contrast to the above compounds, the other CCG isomers showed no appreciable effects on the signal transduction involved in the three mGluR subtypes. 6 This investigation demonstrates not only the importance of a particular isomeric structure of CCGs in the interaction with the mGluRs but also a clear receptor subtype specificity for the CCG‐receptor interaction, and indicates that the CCG isomers would serve as useful agonists for investigation of functions of the mGluR family.

Syntheses of optically pure β-hydroxyaspartate derivatives as glutamate transporter blockers

DL-threo-beta-benzyloxyaspartate (DL-TBOA) is a non-transportable blocker of the glutamate transporters that serves as an indispensable tool for the investigation of the physiological roles of the transporters. To examine the precise interaction between a blocker and the transporters, we synthesized the optically pure isomers (L- and D-TBOA) and its erythro-isomers. L-TBOA is the most potent blocker for the human excitatory amino acid transporters (EAAT1-3), while D-TBOA revealed a difference in the pharmacophores between EAAT1 and EAAT3. We also synthesized the substituent variants (methyl or naphthylmethyl derivatives) of L-TBOA. The results obtained here suggest that bulky substituents are crucial for non-transportable blockers.

Effects of threo-β-hydroxyaspartate derivatives on excitatory amino acid transporters (EAAT4 and EAAT5)

d,lthreo‐β‐Benzyloxyaspartate (d,l‐TBOA), an analog of threo‐β‐hydroxyaspartate (THA) possessing a bulky substituent, is a potent non‐transportable blocker for the excitatory amino acid transporters, EAAT1, 2 and 3, while lthreo‐β‐methoxyaspartate (l‐TMOA) is a blocker for EAAT2, but a substrate for EAAT1 and EAAT3. To characterize the actions of these THA analogs and the function of EAAT4 and EAAT5, we performed electrophysiological analyses in EAAT4 or EAAT5 expressed on Xenopus oocytes. In EAAT4‐expressing oocytes, d,l‐TBOA acted as a non‐transportable blocker, while l‐TMOA like d,l‐THA was a competitive substrate. In contrast, d,l‐THA, d,l‐TBOA and l‐TMOA all strongly attenuated the glutamate‐induced currents generated by EAAT5. Among them, l‐TMOA showed the most potent inhibitory action. Moreover, d,l‐THA, d,l‐TBOA and l‐TMOA themselves elicited outward currents at negative potentials and remained inward at positive potentials suggesting that d,l‐TBOA and l‐TMOA, as well as d,l‐THA, not only act as non‐transportable blockers, but also block the EAAT5 leak currents. These results indicate that EAATs 4 and 5 show different sensitivities to THA analogs although they share properties of a glutamate‐gated chloride channel.

Role of glutamine and neuronal glutamate uptake in glutamate homeostasis and synthesis during vesicular release in cultured glutamatergic neurons

Glutamate exists in a vesicular as well as a cytoplasmic pool and is metabolically closely related to the tricarboxylic acid (TCA) cycle. Glutamate released during neuronal activity is most likely to a large extent accumulated by astrocytes surrounding the synapse. A compensatory flux from astrocytes to neurons of suitable precursors is obligatory as neurons are incapable of performing a net synthesis of glutamate from glucose. Glutamine appears to play a major role in this context. Employing cultured cerebellar granule cells, as a model system for glutamatergic neurons, details of the biosynthetic machinery have been investigated during depolarizing conditions inducing vesicular release. [U-13C]Glucose and [U-13C]glutamine were used as labeled precursors for monitoring metabolic pathways by nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography-mass spectrometry (LC-MS) technologies. To characterize release mechanisms and influence of glutamate transporters on maintenance of homeostasis in the glutamatergic synapse, a quantification was performed by HPLC analysis of the amounts of glutamate and aspartate released in response to depolarization by potassium (55 mM) in the absence and presence of DL-threo-beta-benzyloxyaspartate (TBOA) and in response to L-trans-pyrrolidine-2,4-dicarboxylate (t-2,4-PDC), a substrate for the glutamate transporter. Based on labeling patterns of glutamate the biosynthesis of the intracellular pool of glutamate from glutamine was found to involve the TCA cycle to a considerable extent (approximately 50%). Due to the mitochondrial localization of PAG this is unlikely only to reflect amino acid exchange via the cytosolic aspartate aminotransferase reaction. The involvement of the TCA cycle was significantly lower in the synthesis of the released vesicular pool of glutamate. However, in the presence of TBOA, inhibiting glutamate uptake, the difference between the intracellular and the vesicular pool with regard to the extent of involvement of the TCA cycle in glutamate synthesis from glutamine was eliminated. Surprisingly, the intracellular pool of glutamate was decreased after repetitive release from the vesicular pool in the presence of TBOA indicating that neuronal reuptake of released glutamate is involved in the maintenance of the neurotransmitter pool and that 0.5 mM glutamine exogenously supplied is inadequate to sustain this pool.

A potent metabotropic glutamate receptor agonist: electrophysiological actions of a conformationally restricted glutamate analogue in the rat spinal cord and Xenopus oocytes

The (2S,3S,4S) isomer of alpha-(carboxycyclopropyl)glycine (L-CCG-I), a conformationally restricted glutamate analogue, caused a marked depolarization of motoneurons in the isolated rat spinal cord, which was almost insensitive to CPP and CNQX. Depolarizing responses to L-CCG-I were markedly decreased by reducing the temperature of the bathing fluid. Similar results were obtained in the case of trans-ACPD, which is a metabotropic glutamate receptor agonist, but the depolarizing action of L-CCG-I was more potent than that of trans-ACPD. In Xenopus oocytes injected with poly(A)+ mRNA extracted from the rat brain, L-CCG-I induced significant oscillatory chloride currents, suggesting that L-CCG-I is a potent agonist for metabotropic-type glutamate receptors.

Differing effects of substrate and non-substrate transport inhibitors on glutamate uptake reversal

Na+‐dependent excitatory amino acid transporters (EAATs) normally function to remove extracellular glutamate from brain extracellular space, but EAATs can also increase extracellular glutamate by reversal of uptake. Effects of inhibitors on EAATs can be complex, depending on cell type, whether conditions favor glutamate uptake or uptake reversal and whether the inhibitor itself is a substrate for the transporters. The present study assessed EAAT inhibitors for their ability to inhibit glutamate uptake, act as transporter substrates and block uptake reversal in astrocyte and neuron cultures. lthreo‐β‐hydroxyaspartate (l‐TBHA), dlthreo‐β‐benzyloxyaspartate (dl‐TBOA), ltrans‐pyrrolidine‐2,4‐dicarboxylic acid (ltrans‐2,4‐PDC) (+/–)‐cis‐4‐methy‐trans‐pyrrolidine‐2,4‐dicarboxylic acid (cis‐4‐methy‐trans‐2,4‐PDC) and lantiendo‐3,4‐methanopyrrolidine‐2,4‐dicarboxylic acid (lantiendo‐3,4‐MPDC) inhibited l‐[14C]glutamate uptake in astrocytes with equilibrium binding constants ranging from 17 µm (dl‐TBOA and l‐TBHA) – 43 µm (cis‐4‐methy‐trans‐2,4‐PDC). Transportability of inhibitors was assessed in astrocytes and neurons. While l‐TBHA, ltrans‐2,4‐PDC, cis‐4‐methy‐trans‐2,4‐PDC and lantiendo‐3,4‐MPDC displayed significant transporter substrate activities in neurons and astrocytes, dl‐TBOA was a substrate only in astrocytes. This effect of dl‐TBOA was concentration‐dependent, leading to complex effects on glutamate uptake reversal. At concentrations low enough to produce minimal dl‐TBOA uptake velocity (≤ 10 µm), dl‐TBOA blocked uptake reversal in ATP‐depleted astrocytes; this blockade was negated at concentrations that drove substantial dl‐TBOA uptake (> 10 µm). These findings indicate that the net effects of EAAT inhibitors can vary with cell type and exposure conditions.

Characterization of the glutamatergic system for induction and maintenance of allodynia

Glutamate is the main excitatory neurotransmitter in the central nervous system and has been shown to be involved in spinal nociceptive processing. We previously demonstrated that intrathecal (i.t.) administration of prostaglandin (PG) E(2) and PGF(2 alpha) induced touch-evoked pain (allodynia) through the glutamatergic system by different mechanisms. In the present study, we characterized glutamate receptor subtypes and glutamate transporters involved in induction and maintenance of PGE(2)- and PGF(2 alpha)-evoked allodynia. In addition to PGE(2) and PGF(2 alpha), N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), but not kainate, induced allodynia. PGE(2)- and NMDA-induced allodynia were observed in NMDA receptor epsilon 4 (NR2D) subunit knockout (GluR epsilon 4(-/-)) mice, but not in epsilon 1 (NR2A) subunit knockout (GluR epsilon 1(-/-)) mice. Conversely, PGF(2 alpha)- and AMPA-induced allodynia were observed in GluR epsilon 1(-/-) mice, but not in GluR epsilon 4(-/-) mice. The induction of allodynia by PGE(2) and NMDA was abolished by the NMDA receptor epsilon 2 (NR2B) antagonist CP-101,606 and neonatal capsaicin treatment. PGF(2 alpha)- and AMPA-induced allodynia were not affected by CP-101,606 and by neonatal capsaicin treatment. On the other hand, the glutamate transporter blocker DL-threo-beta-benzyloxyaspartate (DL-TBOA) blocked all the allodynia induced by PGE(2), PGF(2 alpha), NMDA, and AMPA. These results demonstrate that there are two pathways for induction of allodynia mediated by the glutamatergic system and suggest that the glutamate transporter is essential for the induction and maintenance of allodynia.

Comparison of Effects of DL-Threo-β-Benzyloxyaspartate (DL-TBOA) and L-Trans-Pyrrolidine-2,4-Dicarboxylate (t-2,4-PDC) on Uptake and Release of [3H]D-Aspartate in Astrocytes and Glutamatergic Neurons

Uptake and release processes in cerebellar astrocytes and granule neurons (glutamatergic) for glutamate were investigated by the use of [3H]D-aspartate, a non-metabolizable glutamate analog. The effects of DL-threo-β-benzyloxyaspartate (DL-TBOA) and L-trans-pyrrolidine-2,4-dicarboxylate (t-2,4-PDC) on uptake and release of [3H]D-aspartate were studied. Both compounds inhibited potently uptake of [3H]D-aspartate in neurons and astrocytes (IC50 values 10-100 μM), DL-TBOA being slightly more potent than t-2,4-PDC. Release of preloaded [3H]D-aspartate from neurons or astrocytes could be stimulated by addition of excess t-2,4-PDC whereas addition of DL-TBOA had no effect on [3H]D-aspartate efflux. Moreover, DL-TBOA inhibited significantly the depolarization-induced (55 mM KCl) release of preloaded [3H]D-aspartate in the neurons. The results reflect the fact that DL-TBOA is not transported by the glutamate carriers while t-2,4-PDC is a substrate which may heteroexchange with [3H]D-aspartate. It is suggested that DL-TBOA may be used to selectively inhibit depolarization coupled glutamate release mediated by reversal of the carriers.

Potent NMDA-like actions and potentiation of glutamate responses by conformational variants of a glutamate analogue in the rat spinal cord.

1 Neuropharmacological actions of all possible‐state isomers of α‐(carboxycyclopropyl)glycine (CCG), conformationally restricted analogues of glutamate, were examined for electrophysiological effects in the isolated spinal cord of the newborn rat. 2 Eight CCG stereoisomers demonstrated a large variety of depolarizing activities. Among them, the (2R, 3S, 4S) isomers of CCG (D‐CCG‐II) showed the most potent depolarizing activity, followed by the (2S, 3R, 4S) isomer (L‐CCG‐IV). 3 The depolarization evoked by L‐CCG‐IV, D‐CCG‐II and other D‐CCG isomers was effectively depressed by N‐methyl‐D‐aspartate (NMDA) antagonists. D‐CCG‐II was about 5 times more potent than NMDA in causing a depolarization. 4 The (2S, 3S, 4S) isomer of CCG (L‐CCG‐I) was more potent than L‐glutamate in causing a depolarization of spinal motoneurones. The depolarization was slightly depressed by NMDA antagonists, but residual amplitudes of responses to L‐CCG‐I in the presence of NMDA antagonists were almost insensitive to 6,7‐dinitro‐quinoxaline‐2,3‐dione (DNQX) or 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX), suggesting that L‐CCG‐I might be a novel potent agonist. 5 After application of the (2S, 3S, 4R) isomer of CCG (L‐CCG‐III), responses to L‐glutamate, D‐and L‐aspartate were markedly enhanced. The enhancement lasted for a period of several hours without a further application of L‐CCG‐III. 6 L‐CCG‐III also caused a depolarization, but it seemed unlikely that the potentiation of the glutamate response was directly related to the depolarization evoked by L‐CCG‐III. 7 The potentiation might be due to inhibition of uptake processes, but L‐CCG‐III was superior to L‐(−)‐threo‐3‐hydroxyaspartate, a potent uptake inhibitor of L‐glutamate and L‐aspartate, in enhancing the response to L‐glutamate in terms of amplitude and duration of responses. 8 CCG isomers should provide useful pharmacological tools for analysis of glutamate neurotransmitter systems.