Haruhiko Shinozaki

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.

Metabotropic glutamate receptor agonist DCG-IV as NMDA receptor agonist in immature rat hippocampal neurons

The electrophysiological action of (2S,1R;2R,3R)-2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV) on the NMDA receptor was investigated with the help of concentration-jump experiments on freshly dissociated hippocampal CA1 and CA3 neurons of rats. Inward currents elicited by a pulse of DCG-IV were insensitive to 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and could be blocked by D-(-)-2-amino-5-phosphonopentanoic acid (D-AP5) and magnesium and enhanced by glycine. The substance displayed cross-desensitization with NMDA but not with kainate or (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). Dose-response curves showed that DCG-IV was weaker than NMDA but more potent than glutamate in eliciting agonist-gated currents. From these data we conclude that DCG-IV has to be used with caution as a tool for the investigation of metabotropic glutamate receptors.

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.

Characterization of metabotropic glutamate receptors negatively linked to adenylyl cyclase in brain slices

We have characterized the pharmacological profile of activation of metabotropic glutamate receptors negatively linked to adenylyl cyclase (mGluR decreases cAMP) in brain slices. Among the putative mGluR agonists, (2S,1R,2R,3R)-2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV) and (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD), were the most potent inhibitors of forskolin-stimulated cAMP formation in hippocampal slices, followed by ibotenate, L-2-amino-3-phosphonopropionate (AP3), quisqualate, L-glutamate and beta-N-methylamino-L-alanine (BMAA). Inhibition of forskolin-stimulated cAMP formation by DL-2-amino-4-phosphonobutanoate (AP4) was biphasic, suggesting that the drug interacts with more than one mGluR decreases cAMP subtype. Both L-AP4 and L-serine-O-phosphate (a restricted analogue of AP4) were much more effective in inhibiting forskolin-stimulated cAMP formation than their D-isomers, indicating that interaction of these drugs with the mGluR decreases cAMP is stereoselective. Despite the fact that DCG-IV and ibotenate behave as NMDA receptor agonists, their effect was insensitive to MK-801. The regional pattern of expression of mGluR decreases cAMPS, as estimated by using 1S,3R-ACPD as an agonist, did not correlate with the steady-state levels of mGluR2 mRNA. Thus, 1S,3R-ACPD inhibited forskolin-stimulated cAMP in slices from hippocampus, cerebral cortex, corpus striatum, olfactory tubercle or hypothalamus, but not in slices from olfactory bulb or cerebellum; in contrast, mGluR2 mRNA levels were high in the olfactory bulb and very low in the corpus striatum. 1S,3R-ACPD also inhibited forskolin-stimulated cAMP formation in cortical membranes, excluding the involvement of trans-synaptic mechanisms in the activity of mGluR decreases cAMPS.(ABSTRACT TRUNCATED AT 250 WORDS)

Novel kainate derivatives: potent depolarizing actions on spinal motoneurones and dorsal root fibres in newborn rats

1 Neuropharmacological actions of several kainate derivatives (kainoids) were examined for electrophysiological effects in the isolated spinal cord and the dorsal root fibre of the newborn rat. 2 Some kainoids caused depolarization of the motoneurone much more effectively than kainic acid or domoic acid and others were weaker. The rank order of the depolarizing activities of the kainoids tested here is as follows: 4‐(2‐methoxyphenyl)‐2‐carboxy‐3‐pyrrolidineacetic acid (MFPA) > acromelic acid A > domoic acid ≧ 4‐(2‐hydroxyphenyl)‐2‐carboxy‐3‐pyrrolidineacetic acid (HFPA) ≧ acromelic acid B > kainic acid. 3 In the isolated dorsal root fibre, domoic acid caused the most significant depolarization. There were distinct differences with regard to the rank order of the depolarizing activity between the motoneurone and the dorsal root fibre. The rank order in the dorsal root fibre is domoic acid > acromelic acid B > 5‐bromowillardiine ≧ MFPA > acromelic acid A > HFPA > kainic acid. 4 Significant desensitization of kainate receptors was observed in the isolated dorsal root fibre during prolonged application of l‐glutamate, kainate and its derivatives. Cross desensitization was also observed among these excitatory amino acids. Receptors desensitized by kainate did not respond to MFPA, HFPA and acromelic acids, suggesting that these kainate derivatives activated common kainate receptors in the dorsal root fibre. 5 In both motoneurones and dorsal root fibres, 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) effectively depressed the depolarization induced by kainoids, and neither 3‐[(±)‐2‐carboxypiperazin‐4‐yl]propyl‐1‐phosphonic acid (CPP) nor picrotoxin blocked or affected the depolarization, but there were some differences in pharmacological potencies of glutamate antagonists between both preparations. 6 MFPA, HFPA and acromelic acids should provide valuable pharmacological tools for analysis of physiological functions of excitatory amino acids, in particular, as specific agonists for some subtypes of kainate receptors.

(2S,3S,4R)-2-(Carboxycyclopropyl)glycine, a potent and competitive inhibitor of both glial and neuronal uptake of glutamate

The effects of several diastereoisomers of L-2-(carboxycyclopropyl)glycine (CCG) on L-glutamate uptake were compared among three different preparations, glial plasmalemmal vesicles (GPV), synaptosomes and cultured astrocytes from rat hippocampus. The (2S,3S,4R)-isomer (L-CCG-III) inhibited a Na(+)-dependent high-affinity L-glutamate uptake in GPV and synaptosomes in a dose dependent manner at a micromolar range. The potency was quite similar to that of L-threo-beta-hydroxyaspartate in both subcellular fractions and much higher than L-aspartate-beta-hydroxamate, which were known as potent inhibitors of glutamate uptake. The (2S,3R,4S)-isomer (L-CCG-IV) also inhibited the glutamate uptake in GPV and synaptosomes, but it was about 100 times less active than L-CCG-III. The (2S,3S,4S)- and (2S,3R,4R)-isomers (L-CCG-I and L-CCG-II, respectively) hardly showed any inhibitory action on the glutamate uptake. Dixon plot analysis of the initial uptake rate revealed that the inhibition was in a competitive manner and the value of the inhibition constant (Ki) was about 1 microM in both GPV and synaptosomes. L-CCG-III effectively inhibited the glutamate uptake by cultured hippocampal astrocytes as well. These results suggested that L-CCG-III inhibited the glutamate uptake in both neurones and glial cells of the mammalian central nervous system in a similar manner.

(2S,1′R,2′R,3′R)-2-(2,3-Dicarboxycyclopropyl) glycine enhances quisqualate-stimulated inositol phospholipid hydrolysis in hippocampal slices

In adult rat hippocampal slices, (2S,1R,2R,3R)-2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV) did not affect the basal hydrolysis of polyphosphoinositides but dramatically enhanced (EC50 value = 30 nM) the stimulation of [3H]inositol monophosphate (InsP) formation by quisqualate, without substantially affecting the stimulation produced by maximal concentrations of 1S,3R-amino-cyclopentane-1,3-dicarboxylic acid (ACPD) or carbamylcholine. alpha-Amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) was virtually devoid of activity on [3H]InsP formation, either in the absence or presence of DCG-IV. These results suggest that DCG-IV acts, directly or indirectly, as a positive modulator of metabotropic glutamate receptors in the rat hippocampus.

(2S,1'R,2'R,3'R)-2-(2,3-dicarboxycyclopropyl) glycine positively modulates metabotropic glutamate receptors coupled to polyphosphoinositide hydrolysis in rat hippocampal slices.

In rat hippocampal slices, the novel metabotropic glutamate receptor (mGluR) ligand, (1S,1R,2R,3R)-2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV) enhanced the stimulation of polyphosphoinositide (PPI) hydrolysis elicited by quisqualate or by submaximal concentrations of ibotenate or (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD). The enhancing effect of DCG-IV was (i) specific for mGluR agonists, (ii) restricted to hippocampal slice preparation, (iii) reversible, and (iv) not subject to homologous desensitization, in addition, DCG-IV did not interact with L-2-amino-4-phosphonobutanoate (AP4), a noncompetitive antagonist of mGluRs coupled to PPI hydrolysis in brain slices [32]. The action of DCG-IV on quisqualate-stimulated PPI hydrolysis was insensitive to antagonists of ionotropic glutamate receptors and did not appear to be a consequence of a reduction in the intracellular levels of cAMP [14]. When the stimulation of PPI hydrolysis was measured as a function of the incubation time, DCG-IV potentiated quisqualate-stimulated PPI hydrolysis after 60 min of incubation, when quisqualate had already reached its maximal effect. Knowing that activation of protein kinase C (PKC) limits the extent of mGluR agonist-stimulated PPI hydrolysis over time, we have studied the enhancing effect of DCG-IV in the presence of the PKC activator, 12-O-tetradecanoylphorbol-13-acetate (TPA). As expected [9], TPA reduced quisqualate-stimulated PPI hydrolysis in control slices, but was inactive in slices incubated in the presence of DCG-IV. Taken collectively, these results suggest that DCG-IV positively modulates the activity of mGluRs coupled to PPI hydrolysis through a mechanism, which involves PKC-mediated phosphorylation processes.

A novel antagonist, phenylbenzene ω‐phosphono‐α‐amino acid, for strychnine‐sensitive glycine receptors in the rat spinal cord

1 3‐[2′‐Phosphonomethyl[1,1′‐biphenyl]‐3‐yl]alanine (PMBA) is a novel glycine antagonist at strychnine‐sensitive receptors. The chemical structure of PMBA, possessing both a glycine moiety and a phosphono group, is quite different from that of strychnine. 2 In the spinal motoneurone of newborn rats, glycine (100 μm‐1 mM) induced depolarizing responses in a concentration‐dependent manner. PMBA effectively inhibited depolarizing responses to glycine and other agonists, such as taurine and β‐alanine. The dose‐response curves for glycine were shifted to the right in an almost parallel manner (pA2 value: 5.30 ± 0.23, n = 5) by PMBA which was about 60 times less potent than strychnine (pA2 value: 7.08 ± 0.21, n = 5) as a glycine antagonist. 3 PMBA (1 − 100 μm) did not interact with modulatory glycine sites on N‐methyl‐D‐aspartate (NMDA) receptors, which suggests a high selectivity of PMBA for strychnine‐sensitive glycine receptors. At considerably high concentrations (0.1 mM‐1 mM), PMBA depressed responses to GABA (pA2 value: 3.57 ± 0.24, n = 3). 4 PMBA inhibited the binding of [3H]‐strychnine to synaptosomes from adult rat spinal cords; the IC50 values of PMBA, glycine and strychnine were 8 ± 2, 9 ± 3 and 0.08 ± 0.04 μm, respectively (n = 5) for [3H]‐strychnine (4.8 nM). 5 PMBA is a central excitant drug with relatively high potency and selectivity and should be useful as a pharmacological probe for analysing the mechanisms underlying physiological functions of glycine receptors.

A new metabotropic glutamate receptor agonist: Developmental change of its sensitivity to receptors in the newborn rat spinal cord

A novel metabotropic glutamate receptor agonist, (2S,1R,2R,3R)-2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV), reduced the monosynaptic excitation in newborn rat spinal cord rather than polysynaptic discharges at the nanomolar range without causing postsynaptic depolarization of motoneurones. Its inhibitory action on the monosynaptic excitation reduced in due course of time after birth. On the contrary, the inhibitory action of a metabotropic GABAB receptor agonist, baclofen, did not show marked developmental change. DCG-IV should be expected to have the potential to provide further useful information on the physiological function of metabotropic glutamate receptors.