The preferred route of kynurenine metabolism in the rat

It has been suggested (Ueda, T., Otsuka, H. and Goda, K. (1978) J. Biochem. 84, 687–696) that direct cleavage of kynurenine, catalysed by kynureninase, followed by microsomal hydroxylation of the resultant anthranilic acid, may provide an alternative to the established pathway of kynurenine metabolism that involves direct hydroxylation followed by cleavage to 3-hydroxyanthranilic acid. To test this suggestion, anthranilic acid was administered to rats; there was no increase in either the concentration of nicotinamide nucleotides in the liver or the urinary excretion of N¹-methyl nicotinamide. However, injection of either kynurenine or 3-hydroxyanthranilic acid did increase the concentration of nicotinamide nucleotides in the liver. The kinetics of kynurenine hydroxylase (K_m = 1.8±0.6·10^?5 mol/l) and kynureninase (K_m = 2.5±0.8·10^?4 mol/l, liver steady-state kynurenine = 4.9±0.9 μmol/kg) are such that the preferred route of kynurenine metabolism is probably by way of hydroxylation rather than cleavage.     

The kynurenine pathway modulates neurodegeneration in a Drosophila model of Huntington's disease

Neuroactive metabolites of the kynurenine pathway (KP) of tryptophan degradation have been implicated in the pathophysiology of neurodegenerative disorders, including Huntington's disease (HD) [1]. A central hallmark of HD is neurodegeneration caused by a polyglutamine expansion in the huntingtin (htt) protein [2]. Here we exploit a transgenic Drosophila melanogaster model of HD to interrogate the therapeutic potential of KP manipulation. We observe that genetic and pharmacological inhibition of kynurenine 3-monooxygenase (KMO) increases levels of the neuroprotective metabolite kynurenic acid (KYNA) relative to the neurotoxic metabolite 3-hydroxykynurenine (3-HK) and ameliorates neurodegeneration. We also find that genetic inhibition of tryptophan 2,3-dioxygenase (TDO), the first and rate-limiting step in the pathway, leads to a similar neuroprotective shift toward KYNA synthesis. Importantly, we demonstrate that the feeding of KYNA and 3-HK to HD model flies directly modulates neurodegeneration, underscoring the causative nature of these metabolites. This study provides the first genetic evidence that inhibition of KMO and TDO activity protects against neurodegenerative disease in an animal model, indicating that strategies targeted?at?two key points within the KP may have therapeutic relevance in HD, and possibly other neurodegenerative disorders.     

Alteration of kynurenic acid concentration in rat plasma following optically pure kynurenine administration: a comparative study between enantiomers

L-Kynurenine (KYN), a tryptophan metabolite, is metabolized to kynurenic acid (KYNA), which is an antagonist of N-methyl-D-aspartate and alpha7 nicotinic acetylcholine receptors, by kynurenine aminotransferase (KAT) I and KAT II. In this study, optically pure KYN, namely L-KYN or D-KYN, was administered intraperitoneally to male Sprague-Dawley rats (16.3 micromol kg(-1)), and the change in plasma KYNA was investigated by using column-switching high-performance liquid chromatography (HPLC) with fluorescence detection. Unexpectedly, no remarkable alteration in the plasma KYNA was observed when a natural isomer, L-KYN, was administered, whereas plasma KYNA concentration was unequivocally increased when an unnatural isomer, D-KYN, was administered. Serum protein bindings of L-KYN and D-KYN were also studied, and the protein binding of L-KYN (approximately 65%) in rat serum was larger than that of D-KYN (approximately 12%), suggesting that D-KYN may be easily incorporated and metabolized in tissues during blood circulation to generate KYNA in mammals. In addition, the increase in plasma KYNA by the administration of D-KYN was suppressed in rats pretreated with a selective inhibitor of D-amino acid oxidase (DAAO), 5-methylpyrazole-3-carboxylic acid (80 mg/kg). These results suggest that DAAO might be responsible for the production of KYNA from D-KYN in vivo.     

Perinatal kynurenine pathway metabolism in the normal and asphyctic rat brain

Summary. The kynurenine pathway of tryptophan degradation contains several metabolites which may influence brain physiology and pathophysiology. The brain content of one of these compounds, kynurenic acid (KYNA), decreases precipitously around the time of birth, possibly to avoid deleterious N-methyl-D-aspartate (NMDA) receptor blockade during the perinatal period. The present study was designed to determine the levels of KYNA, the free radical generator 3-hydroxykynurenine (3-HK), and their common precursor L-kynurenine (L-KYN) between gestational day 16 and adulthood in rat brain and liver. The cerebral activities of the biosynthetic enzymes of KYNA and 3-HK, kynurenine aminotransferases (KATs) I and II and kynurenine 3-hydroxylase, respectively, were measured at the same ages. Additional studies were performed to assess whether and to what extent kynurenines in the immature brain derive from the mother, and to examine the short-term effects of birth asphyxia on brain KYNA and 3-HK levels. The results revealed that 1) the brain and liver content of L-KYN, KYNA and 3-HK is far higher pre-term than postnatally; 2) KAT I and kynurenine 3-hydroxylase activities are quite uniform between E-16 and adulthood, whereas KAT II activity rises sharply after postnatal day 14; 3) during the perinatal period, KYNA, but not L-KYN, may originate in part from the maternal circulation; and 4) oxygen deprivation at birth affects the brain content of both KYNA and 3-HK 1 h but not 24 h later. Received August 31, 1999 Accepted September 20, 1999     

Subcellular distribution and properties of kynurenine pyruvate transaminase in rat kidney.

Kynurenine transaminase activity in rat kidney was found in both the mitochondrial and supernatant fractions. These fractions contained (a) kynurenine pyruvate transaminase, which showed a preference for pyruvate as amino acceptor, and had a pH optimum between 8.0 and 8.5, and (b) kynurenine 2-oxoglutarate transaminase, with a preference for 2-oxoglutarate and a pH optimum between 6.0 and 6.5. The apparent Km value of the former enzyme for L-kynurenine was much lower than that of the latter enzyme.     

Tryptophan metabolism through the kynurenine pathway in rat brain and liver slices

We hypothesized that hyperbaric oxygen (HBO) enhances tryptophan (TRP) flux through the kynurenine (KYN) pathway because oxygen is a substrate for four pathway enzymes. Our objective was to compare the biosynthesis of KYN pathway intermediates by rat brain and liver slices with air or HBO as the gas phase. One-millimeter thick liver and brain slices were obtained from male Sprague-Dawley rats and incubated individually in chambers containing Hanks'-HEPES- buffer with (3)H-TRP (30 Ci/mmol) for 2 h (37 degrees C) in either room air or oxygen (1.2 or 5.2 atmospheres absolute [ATA] oxygen). After incubation, tissue was snap-frozen and analyzed for protein content while medium was extracted for high-performance liquid chromatography analysis. Radiolabeled nicotinamide adenine dinucleotide (NAD) was produced by brain and liver; liver (with air as the gas phase) also produced quinolinic acid (QA). HBO at 1.2 and 5.2 ATA caused increased QA and NAD from liver slices. HBO did not affect KYN metabolism in brain slices, although there was decreased production of NAD during high oxygen. We conclude that rat brain and liver contain the complete KYN pathway and that HBO enhances KYN flux in liver tissue.     

Studies on the kynurenine adminotransferase activity in rat liver and kidney.

The kynurenine aminotransferase activity of supernatant and mitochondrial fractions obtained from rat liver and kidney was studied with L-kynurenine and L-3-hydroxykynurenine as substrates. A substrate inhibition with L-kynurenine at concentrations higher than 6-7mM was observed with all four enzyme preparations. This did not happen with L-3-hydroxykynurenine as a substrate. Moreover, the liver mitochondrial enzyme shows a Km for pyridoxal phosphate 2-4 times smaller than the other preparations when assayed with L-3-hydroxykynurenine as a substrate. Therefore, the accumulation of xanthurenic acid and not of kynurenic acid in B6 deficiency could be related both to this high activity of liver mitochondrial kynurenine aminotransferase with L-3-hydroxykynurenine, even at small concentrations of B6, and to substrate inhibition observed with L-kynurenine and not with L-3-hydroxykynurenine.     

Endogenous hydrogen sulfide reduces airway inflammation and remodeling in a rat model of asthma

Endogenous hydrogen sulfide (H₂S) is hypothesized to have an important role in systemic inflammation. We investigated if endogenous H₂S may be a crucial mediator in airway inflammation and airway remodeling in a rat model of asthma and if endogenous H₂S may exert its anti-inflammatory effect by inhibiting inducible nitric oxide synthase (iNOS)/NO pathway. Cystathionine-γ-lyase (CSE; a H₂S-synthesizing enzyme) was mainly expressed in airway and vascular smooth muscle cells in rat lung tissue. Levels of endogenous H₂S was decreased in pulmonary tissue in ovalbumin (OVA)-treated rats. Exogenous administration of NaHS alleviated airway inflammation and airway remodeling: peak expiratory flow (PEF) increased, goblet cell hyperplasia and collagen deposition score decreased, with decreased total cells recovered from bronchoalveolar fluid (BALF) and influx of eosinophils and neutrophils. The H₂S levels of serum and lung tissue were positively correlated with PEF and negatively correlated with the level of eosinophils and neutrophils in BALF, score of lung pathology. NaHS treatment significantly attenuated pulmonary iNOS activation in OVA-treated rats. These results suggest that the CSE/H₂S pathway plays an anti-inflammatory and anti-remodeling part in asthma pathogenesis and could be a novel target in prevention and treatment of asthma.     

Effects of calcium ions and quinolinic acid on rat kidney mitochondrial kynurenine aminotransferase

At pH 6.4, rat kidney mitochondrial kynurenine aminotransferase activity is enhanced several-fold by the addition of CaCl₂, apparently because Ca⁺⁺ facilitates the translocation of α-ketoglutarate, one of the substrates, across the mitochondrial inner membrane. Chloride salts or Mg⁺⁺, Mn⁺⁺, Na⁺, K⁺, and NH₄⁺ did not have this effect. At pH 6.8, the enzyme activity was near maximal even without added Ca⁺⁺ but was strongly depressed by either of two calcium chelating agents, quinolinic acid (Q.A.) and ethyleneglycol-bis(β-aminoethyl ether)N,N′-tetraacetic acid (EGTA). These observations support the view that Ca⁺⁺ is involved in regulating kidney mitochondrial translocation of α-ketoglutarate and that the reported interference of polycarboxylate anion translocation by Q.A. $\text{in vivo}$ depends on the ability of that agent to chelate Ca⁺⁺.     

Effects of S100A9 in a rat model of asthma and in isolated tracheal spirals

S100A9 is a member of the S100 family of proteins that contain two EF-hand calcium-binding motifs. We previously reported that S100A9 was differentially expressed during the early airway response phase of asthma and can be regulated by acupuncture. To understand the possible role of S100A9 in asthma, the effects of the S100A9 were investigated in a rat model of asthma and in isolated tracheal spirals. The pulmonary function and isometric tension were measured after the administration of purified recombinant S100A9. The results of in vivo experiments showed that S100A9 (0.1 μg/kg) significantly decreased the pulmonary resistance and increased the dynamic compliance. The in vitro experimental results showed that S100A9 (100, 200, 400, or 800 ng/ml, final concentrations) significantly reduced the isometric tension of isolated tracheal spirals. These results suggest that S100A9 elicits dose-dependent anti-asthmatic effects and may provide further insight into the treatment of asthma.