The in vitro effect of kynurenic acid on the rainbow trout (Oncorhynchus mykiss) leukocyte and splenocyte activity.

Kynurenic acid (KYNA), an endogenous neuroprotectant formed along the kynurenine pathway of tryptophan degradation, is a selective ligand of the GPR35 receptor, which can be found on the surface of various populations of human immune cells. In infections and inflammations, KYNA produces an anti-inflammatory effect through this receptor, by depressing the synthesis of reactive oxygen species and pro-inflammatory cytokines. However, it is still unrecognized whether receptors for kynurenic acid are also localized on immune cells of poikilothermic animals, or whether KYNA is able to affect these cells. The objective of this study has been to determine the effect of different concentrations of kynurenic acid (12.5 microM to 10 mM) on the viability and mitogenic response of lymphocytes and on the activity of phagocytic cells isolated from blood and the spleen of rainbow trout. The results imply low toxicity of kynurenic acid towards fish immune cells, and the proliferative effect observed at the two lowest concentrations of KYNA (12.5 microM and 25 microM) seems indicative of endogenous kynurenic acid being capable of activating fish lymphocytes. Non-toxic, micromole concentrations of KYNA, however, had no influence on the mitogenic response of lymphocytes nor on the activity of phagocytes in rainbow trout under in vitro conditions. There is some likelihood that such an effect could be observed at lower, nanomole concentrations of KYNA.

Tryptophan-kynurenine metabolism and insulin resistance in hepatitis C patients.

Chronic hepatitis C virus (HCV) infection is associated with 50% incidence of insulin resistance (IR) that is fourfold higher than that in non-HCV population. IR impairs the outcome of antiviral treatment. The molecular mechanisms of IR in HCV are not entirely clear. Experimental and clinical data suggested that hepatitis C virus per se is diabetogenic. However, presence of HCV alone does not affect IR. It was proposed that IR is mediated by proinflammatory cytokines, mainly by TNF-alpha. TNF-alpha potentiates interferon-gamma-induced transcriptional activation of indoleamine 2,3-dioxygenase, the rate-limiting enzyme of tryptophan- (TRP-) kynurenine (KYN) metabolism. Upregulation of TRP-KYN metabolism was reported in HCV patients. KYN and some of its derivatives affect insulin signaling pathways. We hypothesized that upregulation of TRP-KYN metabolism might contribute to the development of IR in HCV. To check this suggestion, we evaluated serum concentrations of TRP and KYN and HOMA-IR and HOMA-beta in 60 chronic HCV patients considered for the treatment with IFN-alpha. KYN and TRP concentrations correlated with HOMA-IR and HOMA-beta scores. Our data suggest the involvement of KYN and its metabolites in the development of IR in HCV patients. TRP-KYN metabolism might be a new target for prevention and treatment of IR in HCV patients.

Micromolar concentration of kynurenic acid in rat small intestine.

Kynurenic acid is an antagonist of glutamate and alpha-7 nicotinic acetylcholine receptors and an agonist of the G: -protein-coupled receptor GPR35, which is predominantly expressed in immune and gastrointestinal tissues. In this study, we report that kynurenic acid is present in the lumen of rat small intestine in micromolar concentration sufficient to affect the GPR35 receptor. Moreover, we show that kynurenic acid can be produced by Escherichia coli. We suggest that kynurenic acid may modulate gastrointestinal function and integrity.

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.

Influence of several convulsants on the protective activity of a non-competitive AMPA/kainate antagonist, LY 300164, and lamotrigine against maximal electroshock in mice.

Aminophylline (50-100 mg/kg) and strychnine (0.125-0.5 mg/kg) significantly raised the ED50 values of LY 300164 against maximal electroshock in mice, from 4 to 8 mg/kg (aminophylline 100 mg/kg) and from 3.6 to 11.5 mg/kg (strychnine 0.5 mg/kg). Also, aminophylline (25-50 mg/kg) and strychnine (0.125-0.25 mg/kg) increased the ED50 value of lamotrigine in this test, for instance from 5.5 to 8.0 mg/kg (aminophylline 50 mg/kg) and from 5.2 to 8.9 mg/kg (strychnine 0.25 mg/kg). Moreover, the ED50S values of aminophylline and strychnine for the reduction of the anticonvulsant effect of LY 300164 (7 mg/kg, the dose equal to its ED97 value against maximal electroshock) were 79.9 and 0.2 mg/kg, respectively. The respective ED50 values for the inhibition of the antiseizure action of lamotrigine were 40.9 and 0.2 mg/kg. Neither bicuculline nor picrotoxin affected the protective action of LY 300164 or lamotrigine. Strychnine significantly lowered the plasma concentrations of LY 300164 and this my point to a pharmacokinetic mechanism of the observed interaction. Aminophylline did not affect the plasma concentrations of the studied anticonvulsant drugs and strychnine - that of lamotrigine, so a pharmacokinetic interaction does not seem probable. The present results indicate that the potential of aminophylline and strychnine to attenuate the anticonvulsant activity of conventional antiepileptics is extended to LY 300164 and lamotrigine.

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.

AMPA and GABA(B) receptor antagonists and their interaction in rats with a genetic form of absence epilepsy.

The effects of combined and single administration of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist, 7,8-methylenedioxy-1-(4-aminophenyl)-4-methyl-3-acetyl-4,5-dihydro-2,3-benzodiazepine (LY 300164), and of the GABA(B) receptor antagonist gamma-aminopropyl-n-butyl-phosphinic acid (CGP 36742), on spontaneously occurring spike-wave discharges were investigated in WAG/Rij rats. LY 300164 had minor effects; only the highest dose (16 mg/kg) reduced the number of spike-wave discharges in a short time window. CGP 36742 was more effective as it significantly reduced the number of spike-wave discharges and shortened their duration at the doses of 25 and 100 mg/kg. The ED(50) values for the inhibition of spike-wave discharges by LY 300164 and CGP 36742 in a time window 30-60 min after injection were 15.5 and 16.6 mg/kg, respectively. The ED(50) of CGP 36742 was reduced to 8.0 mg/kg when this antagonist was administered in combination with LY 300164 (6 mg/kg). The interaction between the two antagonists appeared to be additive according to isobolographic analysis. Importantly, CGP 36742 and LY 300164 administered either alone or in combination had no apparent effects on behavior. These results may provide information for a rational approach to polytherapy for the treatment of generalized absence epilepsy.

Amlodipine enhances the activity of antiepileptic drugs against pentylenetetrazole-induced seizures.

Amlodipine (AML), which belongs to the 1,4-dihydropyridine calcium channel antagonists, possesses pharmacological and pharmacokinetic profile that distinguishes it from other agents of this class. Pentylenetetrazole (PTZ)-induced clonic and tonic convulsions in mice were significantly reduced by administration of AML at 10 mg/kg. At this dose AML remained without influence upon the plasma level of PTZ. The ED50 value of AML against clonic seizures induced by PTZ was 5.4 mg/kg. This calcium channel antagonist (at 2.5 mg/kg) combined with ethosuximide (ETX), valproate magnesium (VPA) or phenobarbital (PB) significantly reduced their ED50 values against clonic phase of PTZ-induced seizures. AML administered alone or in combination with antiepileptic drugs (AEDs) worsened the motor performance of mice in the chimney test. However, these treatments remained without significant influence on the retention time in the passive avoidance test. Plasma levels of antiepileptics remained unchanged in the presence of AML. The results indicate that AML does not seem a good candidate for a combination therapy in epileptic patients because of its adverse potential.

Protective effect of adenosine receptor agonists in a new model of epilepsy--seizures evoked by mitochondrial toxin, 3-nitropropionic acid, in mice.

The role of adenosine receptor agonists in the convulsant activity of mitochondrial toxin, 3-nitropropionic acid (3-NPA), was studied in mice. The occurrence of seizures evoked by peripheral application of 3-NPA was inhibited with the use of A1 adenosine receptor agonist, R-N6-phenylisopropyladenosine and A1/A2 agonist, 2-chloroadenosine. Moreover, both drugs prevented 3-NPA-induced mortality. Similarly, A1/A2 agonist, 5'-N-ethylcarboxamidoadenosine, protected against seizures evoked by the intracerebral administration of 3-NPA, and this effect was reversed by the co-application of adenosine receptor antagonist, 8-(p-sulfophenyl)theophylline. Obtained results suggest that A1 adenosine receptor activation may modulate the chain of events leading to the development of 3-NPA-induced seizures.

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.

Kynurenine aminotransferase I activity in human placenta.

The aim of the study was to detect and characterize placental kynurenine aminotransferase I (KAT I) activity in physiological pregnancy at term. Placental KAT I was inhibited by l -glutamine, l -tryptophan, and l -phenylalanine and reached optimum activity at pH 9.8. When pyruvate was used as a co-factor, the KAT I activity was significantly higher than the activity of this enzyme in the presence of 2-oxoglutarate. In the light of our findings placental KAT I seems to have biochemical characteristics of KAT I detected in human brain.

On the interactions between antimuscarinic atropine and NMDA receptor antagonists in anticholinesterase-treated mice.

Both organophosphate (OP) and carbamate pesticides may produce seizures and death commonly attributed to the inhibition of acetylcholinesterase (AChE) and subsequent excess of acetylcholine (ACh). The anticonvulsant and neuroprotective properties of N-methyl-D-aspartate (NMDA) receptor antagonists in animals encouraged us to investigate their effects on the toxic and convulsant properties of OP and carbamate pesticides. Adult Swiss mice were systemically injected with the OP pesticide, chlorfenvinphos (CVP), or the carbamate pesticide, methomyl (MET). Both CVP and MET induced dose-dependent seizure activity and death in mice. Pretreatment with the muscarinic antagonist, atropine (ATR), at a dose of 1.8 mg/kg did not prevent seizures but decreased the lethal effects of CVP and MET. Pretreatment with the NMDA antagonists, dizocilpine (MK-801) at a dose of 1 mg/kg or 3-((R,S)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) at a dose of 10 mg/kg, influenced neither MET-induced seizures nor CVP- or MET-induced death. However, both MK-801 and CPP blocked CVP-induced seizures. Concurrent administration of ATR and the NMDA antagonists prevented seizures produced by CVP, but not those produced by MET. Nevertheless, both MK801 and CPP coadministered with ATR markedly enhanced its antilethal effects in CVP- and MET-intoxicated mice. The antidotes had no influence upon brain AChE activities in mice treated with saline or CVP or MET. It seems that combined treatment with ATR and NMDA receptor antagonists might be of clinical relevance.

Evaluation of interaction between valproate and baclofen in the formalin test in mice.

Valproate and baclofen dose-dependently inhibited both phases of the formalin test. Combination of valproate and baclofen exerted the additive antinociceptive effect on both phases of the formalin test.

Isobolographic analysis of interaction between vigabatrin and baclofen in the formalin test in mice.

Vigabatrin and baclofen given together, at doses not affecting motor performance, produced dose-dependent inhibition of both phases in the formalin test in mice. Isobolographic analysis revealed a significant synergy between both drugs in both phases of the forrmalin test.

Proconvulsive effects of the mitochondrial respiratory chain inhibitor--3-nitropropionic acid.

The role of impaired mitochondrial function in processes leading to the generation of seizures was studied in mice. An inhibitor of mitochondrial complex III, 3-nitropropionic acid, which is known to evoke convulsions per se, and was used here in subthreshold dose, enhanced seizures generated by electric current and application of 4-aminopyridine. In contrast, 3-nitropropionic acid did not affect convulsions induced by gamma-aminobutyric acid (GABA) receptor antagonists - bicuculline, pentylenetetrazol and picrotoxin, glycine antagonist - strychnine, cholinomimetic drug-pilocarpine, and kynurenine aminotransferase inhibitor - aminooxyacetic acid. It is hypothesised that deranged mitochondrial metabolism renders the central nervous system more susceptible to factors inducing seizures via direct depolarization.

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.

Pilocarpine-induced seizures in rodents--17 years on.

In 1983, we reported that intracerebral or systemic administration of cholinergic agents produced seizures and seizure-related brain damage in rodents. During the following 17 years, seizures induced by cholinomimetic drugs became a popular model of epilepsy. Seizures can by produced by cholinergic agonists acting directly at muscarinic receptors or by drugs enhancing cholinergic transmission due to the inhibition of acetylcholinesterase activity. Status epilepticus evoked by pilocarpine in rodents triggers long-lasting changes which can be described as: (I) acute-onset seizures lasting for several hours, (II) a silent period characterized by normalization of electroencephalographic patterns lasting for days, and (III) spontaneous recurrent seizures lasting for life. Therefore, seizures induced by cholinomimetics in rodents are of value for studies of basic mechanisms of epileptogenesis and action of antiepileptic drugs.