Grazia Lombardi

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.

GM1 ganglioside stimulates the regeneration of dopaminergic neurons in the central nervous system

The effect of GM1 ganglioside on the recovery of nigro-strital dopaminergic neurons was studied in rats after unilateral hemitransection. We find that repeated administration of GM1 significantly increased the HVA content, the tyrosine hydroxylase activity and the tyrosine hydroxylase-related immunofluorescence in the striatum ipsilateral to the lesion. Futhermore, GM1 reduced the sensitivity of lesioned rats to apomorphine. The data comparable with the view that a functional dopaminergic reinnervation of the striatum is facilitated by GM1 treatments after hemitransection.

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 Release and Neosynthesis of Glutamic Acid Are Increased in Experimental Models of Hepatic Encephalopathy

Abstract: The effects of ammonium ions on the release of glutamic acid from the rat cerebral cortex were measured in vivo using cortical cups and a multiple ion detection technique. The neosynthesis of this amino acid from glucose was also studied in two experimental models of hepatic encephalopathy: (1) rats receiving large amounts of ammonium acetate (i.p.) and (2) rats with a surgically constructed portocaval anastomosis. Intraperitoneal administration of 8 mmol/kg of ammonium acetate increased the cortical release of glutamic acid from 9.1.0.8 to 19.2 (nmol. cm−2. min−1). Moreover, 20 min after ammonium acetate administration the rate of incorporation of 13C2, originating from [13C]glucose, into glutamic acid increased by 65%. In several brain areas of rats bearing a portocaval anastomosis and fed ad libitum for 4 weeks, the content of glutamic acid slightly increased and the rate of formation of [13C2]glutamate from [13C]glucose approximately doubled. These results indicate that ammonium ions increase the release and the formation of glutamic acid in the brain. The resulting increased concentration of this amino acid in the extracellular spaces may be one of the mechanisms of ammonia toxicity in vivo.

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.

Chronic GM1 ganglioside treatment reduces dopamine cell body degeneration in the substantia nigra after unilateral hemitransection in rat

The effect of GM1 ganglioside on the recovery of dopaminergic nigro-striatal neurons was studied in rats after unilateral hemitransection. GM1 treatment partially prevented the decrease of tyrosine hydroxylase (TH) activity caused by hemitransection in the substantia nigra ipsilateral to the lesion. Concomitantly a significant increase of TH-immunoreactivity in the substantia nigra was also detected. In particular, chronic treatment with GM1 prevented the disappearance of TH-positive cell bodies in the substantia nigra and induced the appearance of longer TH-positive dendrites with respect to the saline treatment. These data indicate that GM1 treatment maintains the number of dopaminergic cell bodies in the substantia nigra after hemitransection by protecting against retrograde neuronal degeneration.

Characterization of ionotropic glutamate receptors in human lymphocytes.

The effect of L‐glutamate (Glu) on human lymphocyte function was studied by measuring anti‐CD3 monoclonal antibody (mAb) or phytohaemagglutinin (PHA)‐induced intracellular Ca2+ ([Ca2+]i) rise (Fura‐2 method), and cell proliferation (MTT assay). Glu (0.001 – 100 μM) did not modify basal lymphocyte [Ca2+]i, but significantly potentiated the effects of anti‐CD3 mAb or PHA. Maximal [Ca2+]i rises over resting cells were: 165±8 and 247±10 nM at 3.0×10−2 mg ml−1 anti‐CD3 mAb; 201±4 and 266±9 nM at 5.0×10−2 mg ml−1 PHA, in the absence or presence of 1 μM Glu, respectively. The Glu effect showed a bell‐shape concentration‐dependent relationship, with a maximum (+90±3% for anti‐CD3 mAb and +57±2% for PHA over Glu‐untreated cells) at 1 μM. Non‐NMDA receptor agonists (1 μM) showed a greater efficacy (+76±2% for (S)‐AMPA; +78±4% for KA), if compared to NMDA (+46±2%), or Glu itself. Ionotropic Glu receptor antagonists completely inhibited the effects of the corresponding specific receptor agonists (1 μM). The IC50 values calculated were: 0.9 μM for D‐AP5; 0.6 μM for (+)‐MK801; 0.3 μM for NBQX. Both NBQX and KYNA were able to abolish Glu effect. The IC50s calculated were: 3.4 μM for NBQX; 0.4 μM for KYNA. Glu (0.1 – 1 mM) did not change the resting cell proliferation, whereas Glu (1 mM) significant inhibited (−27±4%) PHA (1.0×10−2 mg ml−1)‐induced lymphocyte proliferation at 72 h. In conclusion, human lymphocytes express ionotropic Glu receptors functionally operating as modulators of cell activation.

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.

The excitotoxin quinolinic acid is present in the brain of several mammals and its cortical content increases during the aging process.

The distribution of the excitotoxin quinolinic acid (QUIN) has been evaluated in the brains of rabbit, guinea pig and rat, using a mass spectrometric method. Furthermore, the cortical content of this molecule has been measured during the development and the aging of the rat. The cortex contained the highest concentration of QUIN in the three species studied. During the rat development the concentration of this molecule increased and unusually high amounts of it were found in approximately 50% of 30-month-old rats.

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.