Giovanna Cherici

Excitatory Amino Acid Release from Rat Hippocampal Slices as a Consequence of Free-Radical Formation

Abstract: The release of D‐[3H]aspartate, [3H]noradrenaline, and of endogenous glutamate and aspartate from rat hippocampal slices was significantly increased when the slices were incubated with xanthine oxidase plus xanthine to produce superoxide and hydroxyl free radicals locally. Allopurinol, a specific xanthine oxidase inhibitor, the hy‐droxyl‐radical scavenger D‐mannitol, or the superoxide‐radical scavenger system formed by superoxide dismutase plus catalase prevented this release. These results suggest that endogenous excitatory amino acids are released consequent to the formation of free radicals. The excess of glutamate and aspartate released by this mechanism could be one of the factors contributing to the death of neurons after anoxic or ischemic injuries.

Pharmacological characterization of the metabotropic glutamate receptor inhibiting D-[3H]-aspartate output in rat striatum.

1 The effects of several agonists of the metabotropic glutamate receptor (mGluR) were studied in adult rat striatal slices by measuring (i) KCl (30 mm)‐induced output of previously taken up d‐[3H]‐aspartate (Asp), (ii) forskolin (30 μm)‐induced adenosine 3′:5′‐cyclic monophosphate (cyclic AMP) accumulation and (iii) phophoinositide (PI) hydrolysis. 2 K+‐induced efflux of d‐[3H]‐Asp was inhibited by the following mGluR agonists: (1S,3S,4S)‐(carboxycyclopropyl)glycine (l‐CCG‐I), (1S,3R)‐1‐aminocyclopentane‐1,3‐dicarboxylic acid (1S,3R‐ACPD) and quisqualic acid (Quis). 2‐Amino‐4‐phosphonobutyrate (l‐AP4) was inactive up to 300 μm. The maximal inhibition of d‐[3H]‐Asp output was 60 ± 8%. The EC50s of mGluR agonists were: 0.5 μm for l‐CCG‐I, 100 μm for 1S,3R‐ACPD and 100 μm for Quis. 3 Forskolin‐induced cyclic AMP accumulation was also inhibited by mGluR agonists. The maximal inhibition was 50 ± 4% and was obtained at a concentration of 10 μm for l‐CCG‐I and 100 μm for 1S,3R‐ACPD. The EC50s for this inhibition were: 0.9 μm for l‐CCG‐I and 20 μm for 1S,3R‐ACPD. Quis (300 μm) inhibited cyclic AMP accumulation by approximately 20%. l‐AP4 slightly potentiated cyclic AMP accumulation. 4 PI hydrolysis was stimulated by mGluR agonists. The most potent compound was Quis (100 μm), which increased inositol phosphate formation up to 2.2 fold over control values. Its EC50 was 15 μm.l‐CCG‐I and 1S,3R‐ACPD increased inositol phosphate formation by approximately 1.8 fold and their EC50 values were 30 and 25 μm, respectively. l‐AP4 did not affect PI hydrolysis. 5 In conclusion, mGluR agonists that reduce d‐[3H]‐Asp output have a pharmacological profile similar to that of mGluR agonists inhibiting cyclic AMP accumulation. l‐CCG‐I appears to be a relatively selective agonist for the mGluR receptor which inhibits d‐[3H]‐Asp efflux and cyclic AMP accumulation, while Quis appears to act preferentially on the mGluR receptor linked to the metabolism of PIs.

Ischemia does not induce the release of excitotoxic amino acids from the hippocampus of newborn rats

Simulated ischemic conditions or a source of oxygen-derived free radicals, such as xanthine plus xanthine oxidase, released a significant amount of the excitotoxic amino acids Asp and Glu from adult rat hippocampal slices incubated in vitro. The concentrations of Asp and Glu in the incubation medium increased by 20 and 30 times respectively when such slices were exposed to simulated ischemia for a 10-min period. However, preparations obtained from 4- to 9-day-old rats did not release Asp or Glu either when exposed to ischemia or after K+ depolarization. This release appeared 10-15 days after birth and progressively increased up to 13 months of age. No further increase was observed in 25-month-old animals. The exposure of the slices to a source of oxygen-derived free radicals induced a release of excitotoxic amino acids independently from the age of the rats. The massive excitotoxic amino acid release from adult hippocampal slices and the formation of free radicals induced by ischemic insults has been previously associated with degeneration of hippocampal neurons. The lack of ischemia-induced excitotoxic amino acid release from the newborn hippocampus may help to explain why the newborn hippocampus is more resistant than the adult to hypoxic/ischemic insults.

Thiokynurenates: a new group of antagonists of the glycine moduiatory site of the NMBA receptor

Several substituted derivatives of kynurenic acid were tested on the N-methyl-D-aspartate (NMDA) receptor/ion channel complex present in the guinea pig myenteric plexus, on the binding of [3H]glycine and of [3H]N-[1-(2-thienyl)cyclohexyl]piperidine [( 3H]TCP) to rat cortical membranes and on the depolarization of mice cortical wedges induced by NMDA or quisqualic acid (QA). Kynurenic acid derivatives, having a chlorine (CI) or a fluorine atom in position 5 or 7 but not in position 6 or 8 had significantly lower IC50s than the parent compound when tested on the antagonism of glutamate-induced ileal contraction and in the glycine binding assay. A further significant increase in potency was obtained by substituting a thio group for the hydroxy group in position 4 of kynurenic acid: the IC50 was 160 +/- 20 microM of kynurenic acid and 70 +/- 15 microM of thiokynurenic acid in the myenteric plexus whereas these IC50s for glycine binding were 25 +/- 3 and 9 +/- 2 microM respectively. Several thiokynurenic acid derivatives were synthetized and showed an increased affinity for the glycine recognition site over the corresponding kynurenic acid derivatives. Glycine competitively antagonized the actions of the thiokynurenates in the ileum, in cortical wedges and on [3H]TCP binding. In this preparation, 7-Cl-thiokynurenic acid had an IC50 of 8 microM for antagonizing 10 microM NMDA-induced depolarization while 50% of the 10 microM QA depolarization was antagonized at 300 microM. Thus thiokynurenic acid derivatives seem to be a new group of potent and selective antagonists of strychnine-insensitive glycine receptors.

Ammonium acetate inhibits ionotropic receptors and differentially affects metabotropic receptors for glutamate

The effects of ammonium salts in concentration similar to those found in plasma in course of hepatic encephalopathy (2–4 mM) were studied in brain slices in order to clarify how glutamate synapses are affected by this pathological situation. Electrophysiological (mice cortical wedge preparations) and biochemical techniques (inositol phosphates and cyclic AMP measurements) were used so that the function of both the ionotropic and metabotropic glutamate receptors was evaluated. Ammonium acetate (2–4 mM), but not sodium acetate reduced the degree of depolarization of cortical wedges induced by different concentrations of N-methyl-D-aspartic acid (NMDA) or (S)-alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). This reduction was non-competitive in nature and did not reverse during the experimental period (90 min). In a similar manner, ammonium acetate reduced the formation of inositol phosphates induced by (1S,3R)-1-amynocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) (100μM), the prototype agonist of metabotropic glutamate receptors. When the metabotropic glutamate receptors negatively linked to the forskolin-stimulated cyclic AMP formation were evaluated, ammonium acetate significantly hampered forskolin effects and its actions were additive with those of the metabotropic glutamate receptor agonist 1S,3R-ACPD. In conclusion, our results suggest that toxic concentrations of ammonium impair the function of glutamate receptors of NMDA and AMPA type and of the metabotropic glutamate receptors linked to inositol phosphate formation while they functionally potentiate the action of glutamate agonists on the receptors negatively linked to adenylyl cyclase.

Quinoxalines interact with the glycine recognition site of NMDA receptors: studies in guinea‐pig myenteric plexus and in rat cortical membranes

1 The effects of several quinoxalines, including 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) and 6,7,dinitroquinoxaline‐2,3‐dione (DNQX), and of two kynurenates, kynurenate (KYNA) and 7‐Cl‐kynurenate (7‐Cl‐KYNA), have been evaluated on the N‐methyl‐D‐aspartate (NMDA) receptors present in the guinea‐pig ileum myenteric plexus preparation and on the strychnine‐insensitive [3H]‐glycine binding sites of cortical membranes. 2 Quinoxalines and kynurenates antagonized in a non‐competitive manner L‐glutamate‐induced contraction. Their IC50s were (in μm): 5 for 7‐Cl‐KYNA, 7.5 for 6,7‐Cl‐3‐hydroxy‐2‐quinoxaline carboxylate (6,7‐Cl‐HQCA), 20 for DNQX, 50 for CNQX, 76 for KYNA and 125 for 3‐hydroxy‐2‐quinoxaline carboxylate (HQCA). 3 Glycine (5–50 μm) completely reversed the antagonism displayed by both quinoxalines and kynurenates. The interaction between glycine and the tested compounds appeared to be competitive in nature. 4 Quinoxalines and kynurenates displaced, in a concentration‐dependent manner, [3H]‐glycine from its strychnine‐insensitive binding sites present in rat cortical membranes. Their IC50s for this action were (in μm): 0.45 for 7‐Cl‐KYNA, 0.6 for 6,7‐Cl‐HQCA, 2.4 for DNQX, 3.5 for CNQX, 20 for KYNA and 40 for HQCA. 5 When the IC50s for the displacement effect of [3H]‐glycine binding were plotted against the IC50s obtained in the myenteric plexus, a significant correlation was found. 6 These data show that quinoxalines and kynurenates may antagonize the responses to L‐glutamate by interacting with the glycine recognition sites of the NMDA receptor ion channel complex.

The depolarization-induced outflow of d-[3H]aspartate from rat brain slices is modulated by metabotropic glutamate receptors

Rat brain slices were used to study the effects of different metabotropic glutamate receptor ligands on (i) the depolarization (30 mM KCl)-induced outflow of previously taken up D-[3H]aspartate; (ii) the inhibition of forskolin (30 microM)-induced cyclic AMP accumulation; and (iii) the hydrolysis of phosphoinositides. In addition, the localization of mRNAs coding for different metabotropic glutamate receptor subtypes was detected using in situ hybridization. (1S-3R)-1-Aminocyclopentane-1,3-dicarboxylic acid (30-300 microM), a non selective metabotropic glutamate receptor agonist, significantly increased the KCl-induced output of radioactivity from cortical slices, whereas it inhibited the output from striatal slices. Conversely, (1S,3S,4S)-carboxycyclopropylglycine (0.1-1 microM), a relatively selective agonist of the mGluR2 metabotropic glutamate receptor subtype, had an inhibitory effect on the output of D-[3H]aspartate from both cortical and striatal slices and proved to be the most potent metabotropic glutamate receptor agonist in inhibiting cyclic AMP accumulation, but not in stimulating phosphoinositide hydrolysis. Since 2-amino-4-phosphonobutyrate (a mGluR4, mGluR6 and mGluR7 agonist) was not active in any of the assays tested, we hypothesized that the mGluR2 subtype could be involved in these events. Accordingly, mGluR2 mRNA expression was abundant in cortical neurons projecting to the striatum. Our experiments suggest that the stimulation of metabotropic glutamate receptors may either decrease or increase transmitter release depending on the subtype that prevails in the region under study.

Glycine and kynurenate modulate the glutamate receptors in the myenteric plexus and in cortical membranes.

The responses evoked by stimulation of the N-methyl-D-aspartate receptors in the guinea-pig myenteric plexus were potentiated by micromolar concentrations of glycine and were non-competitively antagonized by kynurenate (IC50: 60 microM). The effects of kynurenate were competitively prevented by glycine. Furthermore, kynurenate displaced [3H]glycine from its binding sites on rat cortical membranes (IC50: 20 microM). Kynurenate and glycine, therefore, probably act at the same site, evoking opposite effects on the function of the ion channel complex of the N-methyl-D-aspartate receptor.

Release of Glutamate from Striatum of Freely Moving Rats by pros‐Methylimidazoleacetic Acid

Abstract: The effect of pros‐methylimidazoleacetic acid (p‐MIAA) was measured on the release of glutamate and aspartate from cerebral cortex, hippocampus, and striatum of freely moving rats, and on the uptake of 14C by striatal slices incubated in the presence of l‐[14C]‐glutamate. Twenty‐four hours after implantation of a dialysis fiber, striatum, hippocampus, or cerebral cortex spontaneously released both glutamate and aspartate in the micromolar range. p‐MIAA (1 µM to 1 mM), added to the dialysis perfusate, elicited a concentration‐dependent increase of glutamate release from striatum with a maximal increase of about threefold. This effect did not occur in hippocampus or cortex. In none of these regions did p‐MIAA increase aspartate release significantly. The p‐MIAA effect was not mimicked by its isomer tele‐methylimidazoleacetic acid. p‐MIAA did not influence the uptake of glutamate by striatal slices. The glutamate‐releasing action of p‐MIAA may affect striatal function and explain the positive correlation between levels of p‐MIAA in CSF and the severity of Parkinsons disease.