Vincenzo Carlà

Presence of kynurenic acid in the mammalian brain.

Kynurenic acid, a tryptophan metabolite able to antagonize the actions of the excitatory amino acids, has been identified and measured for the first time in the brain of mice, rats, guinea pigs, and humans by using an HPLC method. Its content was 5.8 ± 0.9 in mouse brain, 17.8 ± 2.0 in rat brain, 16.2 ± 1.5 in guinea pig brain, 26.8 ± 2.9 in rabbit brain, and 150 ± 30 in human cortex (pmol/g wet wt, mean ± SE). The regional distribution of this molecule was uneven. In rats, guinea pigs, and rabbits, the brainstem was the area richest in this compound. Tryptophan administration (100–300 mg/kg, i.p.) to rats resulted in a significant increase of the brain content of kynurenic acid. Similarly, 1 h after probenecid administration (200 mg/kg, i.p.), the brain content of kynurenate increased by fourfold, thus suggesting that its turnover rate is relatively fast.

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.

Calcium channel inhibitors suppress the morphine-withdrawal syndrome in rats.

1 The effects of the Ca2+‐channel blockers verapamil and nimodipine, on the behavioural signs of naloxone (1 mg kg−1)‐induced abstinence syndrome in morphine‐dependent rats, were evaluated. The content of noradrenaline (NA) and of its metabolite 3‐methoxy‐4‐hydroxyphenylglycol (MHPG) was measured, using high performance liquid chromatography and electrochemical detection or gas chromatography‐mass spectrometry, in various brain regions of these animals. Possible interactions of nimodipine and verapamil with opioid receptors were evaluated by examining their ability to displace [3H]‐naloxone binding to brain membranes. 2 Verapamil (5, 10 and 50 mg kg−1) and nimodipine (1,5 and 10 mg kg−1) dose‐dependently reduced most of the signs of morphine abstinence. 3 Naloxone‐precipitated abstinence decreased the NA content in the cortex, hippocampus, brainstem and cerebellum. In the same brain regions the content of MHPG increased, suggesting an increased release of the amine during morphine abstinence. 4 Nimodipine (10 mg kg−1 i.v.) did not change the content of NA or MHPG in the cortex, hippocampus and brainstem. However, nimodipine pre‐treatment markedly reduced the changes in NA and MHPG content induced by the abstinence syndrome. 5 Neither verapamil nor nimodipine displaced [3H]‐naloxone from its binding sites. 6 These results suggest that Ca2+‐channel blockers suppress the behavioural and neurochemical expressions of morphine abstinence by a mechanism that differs from those of opioids or α2‐adrenoceptor agonists.

Inhibitors of kynurenine hydroxylase and kynureninase increase cerebral formation of kynurenate and have sedative and anticonvulsant activities

Kynurenate is an endogenous antagonist of the ionotropic glutamate receptors. It is synthesized from kynurenine, a tryptophan metabolite, and a significant increase in its brain concentration could be useful in pathological situations. We attempted to increase its neosynthesis by modifying kynurenine catabolism. Several kynurenine analogues were synthesized and tested as inhibitors of kynurenine hydroxylase (E.C.1.14.13.9) and of kynureninase (E.C.3.7.1.3), the two enzymes which catalyse the conversion of kynurenine to excitotoxin quinolinate. Among these analogues we observed that nicotinylalanine, a compound whose pharmacological properties have previously been reported, had an IC50 of 900 +/- 180 microM as inhibitor of kynurenine hydroxylase and of 800 +/- 120 microM as inhibitor of kynureninase. In the search for more potent molecules we noticed that meta-nitrobenzoylalanine had an IC50 of 0.9 +/- 0.1 microM as inhibitor of kynurenine hydroxylase and of 100 +/- 12 microM as inhibitor of kynureninase. When administered to rats meta-nitrobenzoylalanine (400 mg/kg) significantly increased the concentration of kynurenine (up to 10 times) and kynurenate (up to five times) in the brain. Similar results were obtained in the blood and in the liver. Furthermore meta-nitrobenzoylalanine increased in a dose dependent, long lasting (up to 13 times and up to 4 h) manner the concentration of kynurenate in the hippocampal extracellular fluid, as evaluated with a microdialysis technique. This increase was associated with a decrease in the locomotor activity and with protection from maximal electroshock-induced seizures in rats or from audiogenic seizures in DBA/2 mice. The conclusions drawn from the present study are: (i) meta-nitrobenzoylalanine is a potent inhibitor of kynurenine hydroxylase also affecting kynureninase; (ii) the inhibition of these enzymes causes a significant increase in the brain extracellular concentration of kynurenate; (iii) this increase is associated with sedative and anticonvulsant actions, suggesting a functional antagonism of the excitatory amino acid receptors.

The excitotoxin quinolinic acid is present and unevenly distributed in the rat brain

The presence of quinolinic acid (2,3-pyridinedicarboxylic acid, QA) in the rat brain has been demonstrated using a mass-spectrometric method. Distribution studies indicate that this molecule is more concentrated in the cortex (2.1 nmol/g wet weight) than in other brain areas. Tryptophan, a possible QA precursor, administered in large doses, increases the cortical content of QA. The contrary occurs when rats are pretreated with p-chlorophenylalanine, a drug capable of decreasing brain tryptophan concentration. The neurotoxin 5,7-dihydroxytryptamine is inactive. Our findings support the idea that QA merits special attention as a potential transmitter and as an endogenous excitotoxin in brain.

Activation of type 5 metabotropic glutamate receptors enhances NMDA responses in mice cortical wedges

We measured the effects of agonists and antagonists of metabotropic glutamate (mGlu) receptors (types 1 and 5) on NMDA‐induced depolarization of mouse cortical wedges in order to characterize the mGlu receptor type responsible for modulating NMDA responses. We also characterized a number of mGlu receptor agents by measuring [3H]‐inositol phosphate (IP) formation in cortical slices and in BHK cells expressing either mGlu 1 or mGlu 5 receptors. (S)‐3,5‐dihydroxyphenylglycine (DHPG), an agonist of both mGlu 1 and mGlu 5 receptors, at concentrations ranging from 1 – 10 μM, enhanced up to 105±15% the NMDA‐induced depolarization. Larger concentrations (100 – 300 μM) of the compound were inactive in this test. When evaluated on [3H]‐IP synthesis in cortical slices or in cells expressing either mGlu 1 or mGlu 5 receptors, DHPG responses (1 – 300 μM) increased in a concentration‐dependent manner. (RS)‐2‐chloro‐5‐hydroxyphenylglycine (CHPG) and (S)‐(+)‐2‐(3′‐carboxybicyclo[1.1.1]pentyl)‐glycine (CBPG), had partial agonist activity on mGlu 5 receptors, with maximal effects reaching approximately 50% that of the full agonists. These compounds, however, enhanced NMDA‐evoked currents with maximal effects not different from those induced by DHPG. Thus the enhancement of [3H]‐IP synthesis and the potentiation of NMDA currents were not directly related. 2‐methyl‐6‐(phenylethynyl)‐pyridine (MPEP, 1 – 10 μM), a selective mGlu 5 receptor antagonist, reduced DHPG effects on NMDA currents. 7‐(hydroxyimino)cyclopropan[b]‐chromen‐1a‐carboxylic acid ethylester (CPCCOEt, 30 μM), a preferential mGlu 1 receptor antagonist, did not reduce NMDA currents. These results show that mGlu 5 receptor agonists enhance while mGlu 5 receptor antagonists reduce NMDA currents. Thus the use of mGlu 5 receptor agents may be suggested in a number of pathologies related to altered NMDA receptor function.

Kynurenic acid is present in the rat brain and its content increases during development and aging processes.

The content of kynurenic acid, a tryptophan (TRP) metabolite which acts as an antagonist of the excitatory amino acid receptors, was measured in the brain, blood, liver and kidney of rats of different ages, using a sensitive and specific method based on ion exchange chromatography and high-performance liquid chromatography (HPLC). In a portion of the cortex of these animals the content of serotonin (5-HT), 5-hydroxyindole-3-acetic acid (5-HIAA) and of TRP was also measured using HPLC and electrochemical detection. The brain content of kynurenic acid was extremely low during the first week of life measuring 15 +/- 3 pmol/g protein (mean +/- S.E.M.), was found to be 320 +/- 31 at 3 months and continued to increase reaching levels of 747 +/- 116 at 18 months of age. Aging failed to change kynurenate content in the liver and kidney while blood kynurenate concentration increased from 28 +/- 5 (pmol/ml) in 3 months old rats to 65 +/- 10 in those 18 months old. The accumulation of kynurenate in the brain was not due to an increased availability of TRP to the central nervous system of aged rats. In fact, the cortical content of this amino acid was slightly lower in animals 18 months old than in those 3 months old. These large changes of the brain content of an electrophysiologically active TRP metabolite such as kynurenic acid could help explain the functional differences present in the brain of newborn and aged animals.

Increase in the content of quinolinic acid in cerebrospinal fluid and frontal cortex of patients with hepatic failure.

Abstract: Quinolinic acid (QUIN), an excitotoxic tryptophan metabolite, has been identified and measured in human cerebrospinal fluid (CSF) using a mass‐fragmentographic method. Furthermore, its content has been evaluated in frontal cortex obtained at autopsy from the cadavers of patients who died after hepatic coma. During the coma, the concentration of QUIN in the CSF was 152 ± 38 pmol ml‐1. In contrast, the concentration in control patients affected by different pathologies was 22 ± 7 pmol ml‐1. In the frontal cortex of patients who died after episodes of hepatic encephalopathy, the content of QUIN was three times higher than in controls (2.6 ± 0.6 versus 0.80 ± 0.08 nmol/g wet weight). As a result of these investigations we are now able to extend our previous observations on the increase of QUIN in the brains of rats used as experimental models of hepatic encephalopathy to man. QUIN should therefore be added to the list of compounds possibly involved in the pathogenesis and symptomatology of brain disorders associated with liver failure.

General anaesthetics inhibit the responses induced by glutamate receptor agonists in the mouse cortex

The effects of several general anaesthetics on the responses evoked by the excitatory amino acid agonists N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) were investigated in mouse cortical wedges placed in a two compartment bath and superfused with a nominally Mg(2+)-free Krebs solution. Chloroform (3-6 mM) and halothane (1-3 mM) preferentially antagonized AMPA responses; thiopental (0.1-0.4 mM), diethyl ether (10-30 mM) and isoflurane (1-3 mM) antagonized both AMPA or NMDA responses while ketamine antagonized NMDA responses in a highly selective way. The antagonism of the excitatory amino acid responses exerted by the anaesthetics was non competitive in nature. The inhibition of excitatory amino acid receptor function may be one of the mechanisms whereby general anaesthesia is produced.

Content of quinolinic acid and of other tryptophan metabolites increases in brain regions of rats used as experimental models of hepatic encephalopathy

Abstract: The content of the tryptophan metabolites quinolinic acid (QUIN), 5‐hydroxytryptamine (5‐HT), and 5‐hydroxyindoleacetic acid (5‐HIAA) was measured in various brain areas of rats bearing a portocaval anastomosis (PCA) for 4 weeks, using mass fragmentography or HPLC. In these animals, the content of the excitotoxic compound QUIN increased by 75% in the cortex and 125% in the cerebellum. The content of 5‐HT increased by 27% in the brainstem. No changes occurred in other brain areas. On the other hand, the content of 5‐HIAA increased by 66% in the cortex, 65% in the caudate, 64% in the hippocampus, 120% in the diencephalon, and 185% in the brainstem. Probenecid administration caused a larger increase of 5‐HIAA accumulation in various brain areas of PCA‐bearing rats than in those of sham‐operated controls. The cortical content of QUIN and 5‐HIAA increased after administration of ammonium acetate (7 mmol/kg), whereas an equimolar amount of sodium acetate was inactive. These results confirm that profound changes in the disposition of tryptophan occur in the brains of experimental animals used as models of hepatic encephalopathy. Furthermore, this study adds the excitotoxic compound QUIN to the list of molecules possibly involved in the pathogenesis of this brain disorder.