Tomasz Kocki

Decreased level of kynurenic acid in cerebrospinal fluid of relapsing-onset multiple sclerosis patients

The present study was undertaken to measure cerebrospinal fluid (CSF) levels of kynurenic acid (KYNA) in patients with relapsing-onset multiple sclerosis (MS) during remission or not progressing for at least 2 months. In these patients the levels of CSF KYNA were found to be significantly lower compared with subjects with non-inflammatory neurological diseases, as well as those with inflammatory disease (median (interquartile range): 0.41 (0.3-0.5) pmol/ml, n=26 vs. 0.67 (0.5-1.1), n=23, P<0.01 and 1.7 (1.5-2.6), n=16, P<0.001, respectively). These results provide further evidence of the alterations in the kynurenine pathway during remitting-onset MS.

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, α-ethylglutamic acid or (RS)-α-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.

l-Cysteine sulphinate, endogenous sulphur-containing amino acid, inhibits rat brain kynurenic acid production via selective interference with kynurenine aminotransferase II

In the present study the effect of endogenous sulphur-containing amino acids, L-cysteine sulphinate, L-cysteate, L-homocysteine sulphinate and L-homocysteate, on the production of glutamate receptor antagonist, kynurenic acid (KYNA), was evaluated. The experiments comprised the measurements of (a). KYNA synthesis in rat cortical slices and (b). the activity of KYNA biosynthetic enzymes, kynurenine aminotransferases (KATs). All studied compounds reduced KYNA production and inhibited the activity of KAT I and/or KAT II, thus acting most probably intracellularly. L-Cysteine sulphinate in very low, micromolar concentrations selectively affected the activity of KAT II, the enzyme catalyzing approximately 75% of KYNA synthesis in the brain. L-Cysteine sulphinate potency was higher than other studied sulphur-containing amino acids, than L-aspartate, L-glutamate, or any other known KAT II inhibitor. Thus, L-cysteine sulphinate might act as a modulator of KYNA formation in the brain.

Activation of kynurenine pathway in ex vivo fibroblasts from patients with bipolar disorder or schizophrenia: Cytokine challenge increases production of 3-hydroxykynurenine

Accumulating data suggest a causative link between immune stimulation, disturbed metabolism of tryptophan, and pathogenesis of bipolar disorder and schizophrenia. The goal of this study was to examine the production of kynurenic acid (KYNA), 3-hydroxykynurenine (3-HK) and the expression of kynurenine pathway enzymes involved in their synthesis and metabolism in cultured skin fibroblasts obtained from patients with bipolar disorder, schizophrenia or from healthy control individuals. The assessment was performed under basal conditions or following treatment with interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, or their combinations, in cells exposed to exogenous kynurenine. In both groups of patients, the baseline production of KYNA and 3-HK was increased, as compared to control subjects. Case-treatment analyses revealed significant interactions between bipolar case status and IL-1β, IL-6, IFN-γ + TNF-α, or IFN-γ + IL-1β, as well as between schizophrenia case status and IL-1β, IFN-γ + TNF-α, or IFN-γ + IL-1β, in terms of higher 3-HK. Noteworthy, no case-treatment interactions in terms of KYNA production were found. Observed changes did not appear to correlate with the expression of genes encoding kynurenine aminotransferases (KATs), kynureninase (KYNU) or kynurenine-3-monooxygenase (KMO). The single nucleotide polymorphisms (SNPs), rs1053230 and rs2275163, in KMO influenced KYNA levels yet did not explain the case-treatment discrepancies. In conclusion, our present findings indicate the utility of skin-derived fibroblasts for kynurenines research and support the concept of kynurenine pathway alterations in bipolar disorder and schizophrenia. The increase in ratio between neurotoxic 3-HK and neuroinhibitory/neuroprotective KYNA following exposure to cytokines may account for altered neurogenesis and structural abnormalities characteristic for both diseases.

Effects of pro-inflammatory cytokines on expression of kynurenine pathway enzymes in human dermal fibroblasts

BackgroundThe kynurenine pathway (KP) is the main route of tryptophan degradation in the human body and generates several neuroactive and immunomodulatory metabolites. Altered levels of KP-metabolites have been observed in neuropsychiatric and neurodegenerative disorders as well as in patients with affective disorders. The purpose of the present study was to investigate if skin derived human fibroblasts are useful for studies of expression of enzymes in the KP.MethodsFibroblast cultures were established from cutaneous biopsies taken from the arm of consenting volunteers. Such cultures were subsequently treated with interferon (IFN)-γ 200 U/ml and/or tumor necrosis factor (TNF)-α, 100 U/ml for 48 hours in serum-free medium. Levels of transcripts encoding different enzymes were determined by real-time PCR and levels of kynurenic acid (KYNA) were determined by HPLC.ResultsAt base-line all cultures harbored detectable levels of transcripts encoding KP enzymes, albeit with considerable variation across individuals. Following cytokine treatment, considerable changes in many of the transcripts investigated were observed. For example, increases in the abundance of transcripts encoding indoleamine 2,3-dioxygenase, kynureninase or 3-hydroxyanthranilic acid oxygenase and decreases in the levels of transcripts encoding tryptophan 2,3-dioxygenase, kynurenine aminotransferases or quinolinic acid phosphoribosyltransferase were observed following IFN-γ and TNF-α treatment. Finally, the fibroblast cultures released detectable levels of KYNA in the cell culture medium at base-line conditions, which were increased after IFN-γ, but not TNF-α, treatments.ConclusionsAll of the investigated genes encoding KP enzymes were expressed in human fibroblasts. Expression of many of these appeared to be regulated in response to cytokine treatment as previously reported for other cell types. Fibroblast cultures, thus, appear to be useful for studies of disease-related abnormalities in the kynurenine pathway of tryptophan degradation.

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

SummaryThe 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 and kynurenine aminotransferases expression in colon derived normal and cancer cells

Abstract Background. Kynurenic acid (KYNA), a tryptophan metabolite, was found in human saliva, gastric juice, bile, pancreatic juice and mucus of rat small intestine. Methods. KYNA content in mucus aspirated from human caecum or colon ascendens and KYNA production in colon epithelial and cancer cells were determined using HPLC. Moreover, biological properties of KYNA and kynurenine aminotransferases (KATs) expression in colon epithelial and colon cancer cells were studied. Results. Considerably higher KYNA concentration was detected in samples from patients diagnosed with colon carcinoma (269.40 ± 107.00 pmol/ml, N = 4), Adenoma tubulovillosum (200.50 ± 36.72, N = 10) or Adenoma tubulare (243.50 ± 38.09, N = 9) than in control group (82.22 ± 7.61 pmol/ml, N = 30). Moreover, colon epithelium CCD 841 CoTr cells actively synthesized KYNA in a concentration- and time-dependent manner. This process was decreased by aminooxyacetic acid and L-glutamate in opposite to 4-aminopyridine treatment. Interestingly, KYNA production in colon cancer cells (HT-29 1.39 ± 0.27, LS-180 1.18 ± 0.15 and Caco-2 4.21 ± 0.30 pmol/1 × 105 cells/2 h) was considerably higher in comparison to normal colon epithelial cells (0.70 ± 0.07 pmol/1 × 105 cells/2 h). However, KATs I and II were expressed at similar level in both colon epithelium and cancer cells. Furthermore, KYNA exerted an antiproliferative effect at higher micro- and milimolar concentrations against colon cancer cells with the IC50 of 0.9, 0.2 and 1.2 mM for HT-29, LS-180 and Caco-2 cells, respectively. Conclusion. Summarizing, this is the first report presenting KYNA synthesis and KAT expression in colon derived normal and cancer cells.

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 ≥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.

Demonstration of kynurenine aminotransferases I and II and characterization of kynurenic acid synthesis in cultured cerebral cortical neurons

The present study characterizes the synthesis of kynurenic acid (KYNA) from exogenously added kynurenine and its regulation by extrinsic factors, in cultured cerebral cortical neurons and, for comparison, in astrocytes incubated under identical conditions. The neuronal culture showed positive immunostaining for both kynurenic acid aminotransferase (KAT) isoforms I and II. Neurons synthesized KYNA at a rate about 2.3 times higher than astrocytes. Neuronal, but not astrocytic, KYNA synthesis was lowered ∼30% by ionotropic glutamate receptor agonists [(R,S)‐3‐hydroxy‐5‐methoxyloxasole‐4‐propionic acid (AMPA; 100 μM) and N‐methyl‐D‐aspartic acid (NMDA; 100 μM)] and depolarizing agents [KCl (50 mM) and 4‐aminopyridine (4‐AP; 10 μM)]. Neuronal and astrocytic synthesis alike were vulnerable to inhibition exerted by the aminotransferase inhibitor aminooxyacetic acid (AOAA), glutamate (IC50: 31 and 85 μM, respectively), substrates of the L‐amino transport system [leucine (Leu); IC50: 19 and 42 μM, respectively] and 2‐aminobicyclo[2,2,1]heptane‐2‐carboxylic acid (BCH; IC50: 19 and 28 μM, respectively). Glutamine (Gln), which is a metabolic precursor of glutamate in astrocytes and L‐system substrate in both cell types, inhibited KYNA synthesis both in neurons and in astrocytes (IC50: 268 and 318 μM, respectively). α‐Ketoisocaproic acid (KIC), a Leu transamination product that is produced mainly in astrocytes and shuttled to neurons to modulate intraneuronal concentration of glutamate, stimulated KYNA synthesis in neurons but did not affect the synthesis in astrocytes. In conclusion, this study is the first to demonstrate active, regulation‐prone KYNA synthesis in neurons.

Alterations of kynurenic acid content in the retina in response to retinal ganglion cell damage.

The present study is the first to examine the modulation of retinal kynurenic acid (KYNA) content in response to N-methyl-D-aspartate (NMDA)-induced cell death in adult rat retinal ganglion cells (RGC). Adult Brown Norway rats were intravitreally injected with NMDA or PBS. Surviving RGC were retrogradely labeled with fluorogold and counted in wholemounts of retinas 2, 7 and 14 days after injection. Retinal KYNA content was measured by HPLC at the same time points. RGC numbers decreased significantly 2, 7 and 14 days after NMDA injection if compared to control retinas. KYNA concentration increased significantly two days after NMDA-injection. However, 7 and 14 days after injection retinal KYNA content was found markedly decreased in NMDA-treated eyes as compared to controls. It is conceivable that KYNA deficiency is causally related to the pathology of excitotoxic retinal diseases.