Marina Alesiani

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.

Nicotinylalanine Increases the Formation of Kynurenic Acid in the Brain and Antagonizes Convulsions

Abstract: Kynurenic acid (KYNA) was quantified in the extracellular spaces of the rat hippocampus using microdialysis and HPLC (fluorimetric detection) to study the possible role of this tryptophan metabolite in the modulation of the function of the N‐methyl‐D‐aspartate (NMDA) receptor. Addition of probenecid (1 mM), which is an inhibitor of the organic acid transport system, to the Ringers solution perfusing the dialysis probe increased the KYNA concentration in the dialysate from 10.4 ± 0.9 to 48 ± 6 nM. Addition of 2 mM aminooxyacetic acid, a nonspecific inhibitor of KYNA synthesis, reduced this concentration by 50%. These data suggest that KYNA is continuously synthesized in the rat hippocampus. Nicotinylalanine (NAL), 200–400 mg/kg i.p., an analogue of kynurenine that is able to direct the flow of tryptophan metabolites toward the synthesis of KYNA, significantly increased the KYNA concentration in the hippocampal dialysate and significantly potentiated the effect of tryptophan on the accumulation of KYNA in the brain and other organs. This increase resulted in pharmacological actions compatible with an antagonism of the NMDA receptors. In fact, NAL antagonized sound‐induced seizures and prevented death in DBA/2 mice. Pretreatment of the mice with D‐serine (100 μg intracerebroventricularly), a glycine agonist and a competitive antagonist of KYNA, completely prevented the anticonvulsive action of NAL. These data suggest that changes in the extracellular concentration of KYNA in the brain are associated with a modulation of NMDA receptor function.

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.

Thiokynurenates prevent excitotoxic neuronal death in vitro and in vivo by acting as glycine antagonists and as inhibitors of lipid peroxidation

Several derivatives of kynurenic and thiokynurenic acids were synthesized and tested for their ability to protect primary cultures of cerebellar granule cells against excitotoxic damage, and to affect the binding of [3H]glycine ([3H]Gly), [3H]alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ([3H]AMPA), [3H]3-(2-carboxypiperazine-4-yl-)propyl-1-phosphonic acid ([3H]CPP), [3H]kainic acid and [3H]N-[1-(2-thienyl)cyclohexyl]-3,4-piperidine ([3H]TCP) to rat cortical membranes. Kynurenic and thiokynurenic acid derivatives with one or two halogens in position 5 or 7 were selective glycine antagonists, failing to affect N-methyl-D-aspartate (NMDA), kainate or AMPA sites at micromolar concentrations. 7-Cl-kynurenic, 7-Cl-thiokynurenic, 5,7-diCl-kynurenic and 5,7-diCl-thiokynurenic acids had similar IC50s for displacing [3H]Gly from its strychnine-insensitive site and for reducing the stimulated (0.5 microM NMDA and 1 microM glycine) [3H]TCP binding to cortical membranes. However, 7-Cl-thiokynurenic acid was particularly potent to prevent excitotoxic neuronal death in cultured cerebellar granule cells. This action may be ascribed to inhibition of lipid peroxidation, a property which was demonstrated for the 5- or 7-Cl derivatives of thiokynurenic acid. Furthermore, 7-Cl-thiokynurenic acid reduced excitotoxic damage caused by the injection of quinolinic acid in the rat striatum. Thus, 7-Cl-thiokynurenic acid appears to be a new compound with interesting antiexcitotoxic properties both in vitro and in vivo.

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.

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.

Sulfate esters of hydroxy amino acids as stereospecific glutamate receptor agonists

Enantiomerically pure sulfate esters of the hydroxy amino acids homoserine, hydroxyproline and 4-hydroxypipecolic acid were synthesized and tested on alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptors present in the mice cortical wedge preparation and on NMDA receptors present in the myenteric plexus of the guinea pig with the aim of finding new possible endogenous ligands (either agonists or antagonists) for excitatory amino acid receptors. The linear and flexible compound S-homoserine sulfate caused a depolarization of both AMPA and NMDA receptors. In the cortex its agonist action had an EC50 of 150 microM for NMDA and 300 microM for AMPA receptors and in the myenteric plexus its EC50 was 600 microM. The stereoisomer R-homoserine sulfate did not depolarize the cortical wedges and failed to cause ileal contraction up to a concentration of 500 microM. Among the four possible stereoisomers of 4-hydroxyproline sulfate, which are rigid structures and may be regarded as cyclization forms of homoserine sulfate, t-S-hydroxyproline sulfate was a selective AMPA receptor agonist with an EC50 of 70 microM in the cortex. The other three isomers were not active as agonists up to 500 microM and none of them had antagonist activity. Finally, t-4-hydroxy-S-pipecolic acid-4-sulfate, a superior homologue of t-S-hydroxyproline sulfate, was found to be one of the most potent and selective NMDA receptor agonists so far described with an EC50 of 0.7 microM in the cortex and 250 microM in the myenteric plexus.(ABSTRACT TRUNCATED AT 250 WORDS)