Astrocytic activation in relation to inflammatory markers during clinical exacerbation of relapsing-remitting multiple sclerosis.

The study aimed to assay the cerebrospinal fluid (CSF) levels of protein S100B, a biomarker of astrocyte activation in relation to kynurenic acid (KYNA) and nitric oxide (NO) metabolites, nitrate/nitrite (NOx) concentrations in acute relapse multiple sclerosis (MS) patients. Twenty relapsing-remitting MS (RR-MS) patients and 10 controls were enrolled. RR-MS patients were assessed on the expanded disability status scale (EDSS) and underwent lumbar puncture. The CSF KYNA, NOx and S100B levels were significantly higher in RR-MS group compared to controls (p = 0.01, 0.001, 0.04, respectively). There was a significant correlation between CSF S100B and KYNA (p = 0.01) but not NOx (p > 0.05) in RR-MS. CSF KYNA, NOx or S100B concentrations did not correlate with disease characteristics of MS patients. Our study suggests the activation of the kynurenine pathway leading to the increase of neuroprotective KYNA in the CSF of MS patients during acute relapse what contrasts with chronic phases of the disease.

Kynurenic acid synthesis in bovine retinal slices--effect of glutamate agonists.

The purpose of the present study was to investigate the effect of glutamate agonists upon kynurenic acid (KYNA) production in bovine retinal slices. Quantitative analysis of newly synthesized KYNA was carried out using an HPLC system and detected fluorimetrically. Glutamate at the concentration of 0.01, 0.1 and 1 mM reduced KYNA synthesis in the retinal slices to 70% (p < 0.05), 35% (p < 0.01) and 23% (p < 0.001), respectively. The concentration of glutamate reducing production of KYNA by 50% (IC(50)) was 0.035 mM (0.02-0.06). Aspartate at the concentration of 0.01, 0.1 and 1 mM lowered KYNA synthesis in the retinal slices to 80% (p < 0.01), 57% (p < 0.001) and 43% (p < 0.001), respectively. In contrast, kainic acid (up to 5 mM), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) (up to 1 mM) and 1-aminocyclopentane-trans-1,3-dicarboxylic acid (t-ACPD) (up to 3 mM) turned out to be ineffective in diminishing KYNA synthesis. These data demonstrate that glutamate, aspartate and N-methyl-D-aspartate (NMDA) inhibit KYNA synthesis in bovine retinal slices with different potency.

Dual effect of DL-homocysteine and S-adenosylhomocysteine on brain synthesis of the glutamate receptor antagonist, kynurenic acid.

Increased serum level of homocysteine, a sulfur-containing amino acid, is considered a risk factor in vascular disorders and in dementias. The effect of homocysteine and metabolically related compounds on brain production of kynurenic acid (KYNA), an endogenous antagonist of glutamate ionotropic receptors, was studied. In rat cortical slices, DL-homocysteine enhanced (0.1-0.5 mM) or inhibited (concentration inducing 50% inhibition [IC50]=6.4 [5.5-7.5] mM) KYNA production. In vivo peripheral application of DL-homocysteine (1.3 mmol/kg intraperitoneally) increased KYNA content (pmol/g tissue) from 8.47 +/- 1.57 to 13.04 +/- 2.86 (P <0.01; 15 min) and 11.4 +/- 1.72 (P <0.01; 60 min) in cortex, and from 4.11 +/- 1.54 to 10.02 +/- 3.08 (P <0.01; 15 min) in rat hippocampus. High concentrations of DL-homocysteine (20 mM) applied via microdialysis probe decreased KYNA levels in rabbit hippocampus; this effect was antagonized partially by an antagonist of group I metabotropic glutamate receptors, LY367385. In vitro, S-adenosylhomocysteine acted similar to but more potently than DL-homocysteine, augmenting KYNA production at 0.03-0.08 mM and reducing it at > or =0.5 mM. The stimulatory effect of S-adenosylhomocysteine was abolished in the presence of the L-kynurenine uptake inhibitors L-leucine and L-phenyloalanine. Neither the N-methyl-D-aspartate (NMDA) antagonist CGS 19755 nor L-glycine influenced DL-homocysteine- and S-adenosylhomocysteine-induced changes of KYNA synthesis in vitro. DL-Homocysteine inhibited the activity of both KYNA biosynthetic enzymes, kynurenine aminotransferases (KATs) I and II, whereas S-adenosylhomocysteine reduced only the activity of KAT II. L-Methionine and L-cysteine, thiol-containing compounds metabolically related to homocysteine, acted only as weak inhibitors, reducing KYNA production in vitro and inhibiting the activity of KAT II (L-cysteine) or KAT I (L-methionine). The present data suggest that DL-homocysteine biphasically modulates KYNA synthesis. This seems to result from conversion of compound to S-adenosylhomocysteine, also acting dually on KYNA formation, and in part from the direct interaction of homocysteine with metabotropic glutamate receptors and KYNA biosynthetic enzymes. It seems probable that hyperhomocystemia-associated brain dysfunction is mediated partially by changes in brain KYNA level.

Endogenous protectant kynurenic acid in amyotrophic lateral sclerosis.

OBJECTIVES: Excitotoxicity may play a role in neurodegeneration in amyotrophic lateral sclerosis (ALS). Kynurenic acid (KYNA), an endogenous antagonist of excitatory amino acid receptors, may inhibit excitotoxic lesions. The aim of this study was to determine the concentration of KYNA in ALS patients. MATERIAL AND METHODS: KYNA was measured by high-performance liquid chromatography in the serum and cerebrospinal fluid (CSF) from ALS and control patients. RESULTS: Our study revealed that CSF KYNA concentration was significantly higher in patients with bulbar onset of ALS compared to controls, and compared to patients with limb onset of the disease. CSF KYNA was also higher in patients with severe clinical status compared to controls. Serum KYNA was significantly lower in ALS patients with severe clinical status compared to controls, and compared to patients with mild clinical status. There were no significant differences in CSF and serum KYNA concentration between the whole ALS group of patients and controls. There was no difference in CSF KYNA concentration between males and females, and there was no correlation between KYNA concentration and age of patients, and duration of ALS. CONCLUSIONS: An increased CSF KYNA concentration in patients with bulbar onset of ALS and in patients with severe clinical status may indicate neuroprotective role of KYNA against excitotoxicity. The difference of KYNA concentration in CSF of patients with bulbar and limb onset of ALS suggests that these two variants of motor neuron disease may have different etiopathogenetic mechanisms.

Presence of kynurenic acid and kynurenine aminotransferases in the inner retina.

Kynurenine aminotransferases (KATs I and II) are pivotal to the synthesis of kynurenic acid (KYNA), the only known endogenous glutamate receptor antagonist and neuroprotectant. This study is the first to identify KYNA in the rat retina and to examine immunohistochemically the distribution of KAT isoforms. As determined by HPLC, KYNA concentration in the retina was 99.9 +/- 24.6 pmol/g wet wt. Immunohisto- chemical experiments showed that both KATs were present in the retina. KAT I was preferentially localised on Müller cell endfeet while KAT II was expressed in cells within the ganglion cell layer. In conclusion, KYNA is present and synthesised in the inner retina. This may suggest a modulatory role in glutamate-mediated retinal neurotransmission.

Evidence for intraocular synthesis of kynurenic acid, a putative endogenous neuroprotectant.

Kynurenic acid (KYNA), an excitatory amino acid antagonist preferentially active at glycine binding site of the NMDA receptor, has been previously identified in the brain. This study was designed to examine its presence in the rabbit vitreous humor. Mean (+/- SD) level of KYNA in the vitreous was 22.3 +/- 3.9 pmol/ml as determined by HPLC. Intravitreal administration of 10 mmol aminooxyacetic acid (AOAA), a KYNA synthesis inhibitor, diminished its production by 9.6% after 2 h, 47.8% after 24 h and 21.5% after 48 h. It can be concluded that AOAA decreases the intravitreal concentration of KYNA, providing evidence of its intraocular origin.

N(G)-nitro-L-arginine and its methyl ester inhibit brain synthesis of kynurenic acid possibly via nitric oxide-independent mechanism.

The effect of nitric oxide synthase (NOS) inhibitors on the brain production of endogenous glutamate receptor antagonist, kynurenic acid, was estimated in vitro. Under standard incubation conditions N(G)-nitro-L-arginine, but not N(G)-nitro-L-arginine methyl ester, up to 5 mM, or 7-nitroindazole, up to 100 microM, inhibited de novo synthesis of kynurenic acid in cortical slices. However, during prolonged incubation, N(G)-nitro-L-arginine methyl ester also reduced the production of kynurenic acid. The substrate for NOS, L-arginine (up to 5 mM), did not influence kynurenic acid synthesis and did not reverse the N(G)-nitro-L-arginine-evoked changes, suggesting that the observed effects are not related to disturbed generation of NO. Enzymatic studies revealed that N(G)-nitro-L-arginine and its methyl ester blocked the activity of brain kynurenine aminotransferase (KAT) I. The activity of KAT II was diminished only by N(G)-nitro-L-arginine. Kinetic analyses have shown that N(G)-nitro-L-arginine and its methyl ester reduce Vmax and increase Km of KAT I, whereas N(G)-nitro-L-arginine diminishes Vmax of KAT II. In conclusion, we report that N(G)-nitro-L-arginine and its methyl ester impair brain synthesis of kynurenic acid, probably via NO-independent mechanism, what could contribute, at least partially, to the enhancement of neurotoxicity or seizures observed in some experimental designs based on their use.

Formation of endogenous glutamatergic receptors antagonist kynurenic acid--differences between cortical and spinal cord slices.

Rat spinal cord slices produced kynurenic acid (KYNA) upon exposure to L-kynurenine. Aminooxyacetic acid, non-selective aminotransferase inhibitor, and L-glutamate, but neither N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-metyloisoxazolo-4-propionate (AMPA), nor kainate, diminished synthesis of KYNA. L-Glutamate action was less potent in spinal than in cortical slices. Metabotropic agonists, L-(+)-2-amino-4-phosphonobutyrate (L-AP4) and (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (t-ACPD), used in concentrations inhibiting cortical KYNA synthesis, were ineffective in spinal cord. Spinal KYNA production seems less susceptible to inhibitory modulation.

Investigations on the synthesis and properties of N-phenyl derivatives of 1,4-dioxo(1,4,5-trioxo)-1,2,3,4-tetra(1,2,3,4,5,6-hexa) hydropyrido-[3,4-d]pyridazines and allied compounds.

2-(1-Piperidino)- and 2-(4-methyl-1-piperazinyl)-6-methyl-3,4-pyridinedicarboximides (1, 2) react with N-phenylhydrazine yielding N-phenylamino-3,4-pyridinedicarboximides (7, 8). The same reaction with 1,6-dimethyl-2-oxo-1,2-dihydro- and 2-chloro-6-methyl-3,4-pyridinedicarboximides (3, 17) gives the salts of the corresponding N-phenylpyridopyridazines with phenylhydrazine (13, 18), which transform into N-phenylaminoimides (14, 19) during boiling in 80% acetic acid. Compounds 7, 8 and 14 isomerize to the corresponding 2-phenyl-1,4-dioxo(1,4,5-trioxo)-1,2,3,4-tetra(1,2,3,4,5,6-hexa) hydropyrido[3,4-d]pyridazines (9, 10, 15) under the influence of heating in alcoholic solution of C2H5ONa or CH3ONa. Only in the case of imide 19 are 2- and 3-phenyl isomers (20 and 21) formed under these conditions. Some of the obtained compounds were pharmacologically active.

Acute ammonia treatment in vitro and in vivo inhibits the synthesis of a neuroprotectant kynurenic acid in rat cerebral cortical slices.

The synthesis of kynurenic acid (KYNA) from kynurenine was measured in the cerebral cortical slices. In vitro, ammonium acetate at the subtoxic to toxic concentration range from 1 mM to 10 mM dose-dependently inhibited KYNA synthesis (IC50=2.99 mM). Ammonia treatment in vivo decreased KYNA synthesis by 30%. These results suggest that impaired neuroprotection exerted by KYNA might be a potential contributor to the glutamate receptor-mediated aspect of acute ammonia neurotoxicity.

Impairment of brain kynurenic acid production by glutamate metabotropic receptor agonists.

The role of glutamatergic mechanisms in kynurenic acid (KYNA) production was evaluated in vitro. The selective ionotropic agonists NMDA, kainate and AMPA did not affect KYNA synthesis. Agonists of metabotropic (mGLU) and ionotropic receptors: quisqualate, L-glutamate and L-aspartate as well as agonists of mGLU receptors: (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (t-ACPD) and L-(+)-2-amino-4-phosphonobutyric acid (L-AP4) diminished KYNA production with different potency. None of the studied mGLU antagonists such as (S)-4-carboxyphenylglycine, alpha-ethylglutamic acid or (RS)-alpha-methylserine-O-phosphate affected the basic or L-glutamate-inhibited synthesis of KYNA. It might be hypothesized that the impairment of KYNA production following the application of mGLU receptor agonists is related to their effects exerted upon the novel subtype of mGLU receptor.