Kynurenine 3-hydroxylase inhibition in rats: effects on extracellular kynurenic acid concentration and N-methyl-D-aspartate-induced depolarisation in the striatum

Inhibition of kynurenine 3-hydroxylase suppresses quinolinic acid synthesis and, therefore, shunts all kynurenine metabolism toward kynurenic acid (KYNA) formation. This may be a pertinent antiexcitotoxic strategy because quinolinic acid is an agonist of NMDA receptors, whereas kynurenic acid antagonises all ionotropic glutamate receptors with preferential affinity for the NMDA receptor glycine site. We have examined whether the kynurenine 3-hydroxylase inhibitor Ro 61-8048 increases extracellular (KYNA) sufficiently to control excessive NMDA receptor function. Microdialysis probes incorporating an electrode were implanted into the striatum of anaesthetised rats, repeated NMDA stimuli were applied through the probe, and the resulting depolarisation was recorded. Changes in extracellular KYNA were assessed by HPLC analysis of consecutive dialysate samples. Ro 61-8048 (42 or 100 mg/kg) markedly increased the dialysate levels of KYNA. The maximum increase (from 3.0 +/- 1.0 to 31.0 +/- 6.0 nM; means +/- SEM, n = 6) was observed 4 h after administration of 100 mg/kg Ro 61-8048, but the magnitude of the NMDA-induced depolarisations was not reduced. A separate study suggested that extracellular KYNA would need to be increased further by two orders of magnitude to become effective in this preparation. These results challenge the notion that kynurenine 3-hydroxylase inhibition may be neuroprotective, primarily through accumulation of KYNA and subsequent attenuation of NMDA receptor function.     

Metabolism of [5-3H]Kynurenine in the Rat Brain In Vivo: Evidence for the Existence of a Functional Kynurenine Pathway

Abstract: The incorporation of tritium label into quinolinic acid (QUIN), kynurenic acid (KYNA), and other kynurenine (KYN) pathway metabolites was studied in normal and QUIN-lesioned rat striata after a focal injection of [5-³H]KYN in vivo. The time course of metabolite accumulation was examined 15 min to 4 h after injection of [5-³H]KYN, and the concentration dependence of KYN metabolism was studied in rats killed 2 h after injection of 1.5–1,500 µ M [5-³H]KYN. Labeled QUIN, KYNA, 3-hydroxykynurenine (3-HK), 3-hydroxyanthranilic acid, and xanthurenic acid (XA) were recovered from the striatum in every experiment. Following injection of 15 µ M [5-³H]KYN, a lesion-induced increase in KYN metabolism was noted. Thus, the proportional recoveries of [³H]KYNA (5.0 vs. 1.8%), [³H]3-HK (20.9 vs. 4.5%), [³H]XA (1.5 vs. 0.4%), and [³H]QUIN (3.6 vs. 0.6%) were markedly elevated in the lesioned striatum. Increases in KYN metabolism in lesioned tissue were evident at all time points and KYN concentrations used. Lesion-induced increases of the activities of kynurenine-3-hydroxylase (3.6-fold), kynureninase (7.6-fold), kynurenine aminotransferase (1.8-fold), and 3-hydroxyanthranilic acid oxygenase (4.2-fold) likely contributed to the enhanced flux through the pathway in the lesioned striatum. These data provide evidence for the existence of a functional KYN pathway in the normal rat brain and for a substantial increase in flux after neuronal ablation. This method should be of value for in vivo studies of cerebral KYN pathway function and dysfunction.     

On the relationship between the two branches of the kynurenine pathway in the rat brain in vivo

In the mammalian brain, kynurenine aminotransferase II (KAT II) and kynurenine 3-monooxygenase (KMO), key enzymes of the kynurenine pathway (KP) of tryptophan degradation, form the neuroactive metabolites kynurenic acid (KYNA) and 3-hydroxykynurenine (3-HK), respectively. Although physically segregated, both enzymes use the pivotal KP metabolite l -kynurenine as a substrate. We studied the functional consequences of this cellular compartmentalization in vivo using two specific tools, the KAT II inhibitor BFF 122 and the KMO inhibitor UPF 648. The acute effects of selective KAT II or KMO inhibition were studied using a radiotracing method in which the de novo synthesis of KYNA, and of 3-HK and its downstream metabolite quinolinic acid (QUIN), is monitored following an intrastriatal injection of ³H-kynurenine. In naïve rats, intrastriatal BFF 122 decreased newly formed KYNA by 66%, without influencing 3-HK or QUIN production. Conversely, UPF 648 reduced 3-HK synthesis (by 64%) without affecting KYNA formation. Similar, selective effects of KAT II and KMO inhibition were observed when the inhibitors were applied acutely together with the excitotoxin QUIN, which impairs local KP metabolism. Somewhat different effects of KMO (but not KAT II) inhibition were obtained in rats that had received an intrastriatal QUIN injection 7 days earlier. In these neuron-depleted striata, UPF 648 not only decreased both 3-HK and QUIN production (by 77% and 66%, respectively) but also moderately raised KYNA synthesis (by 27%). These results indicate a remarkable functional segregation of the two pathway branches in the brain, boding well for the development of selective KAT II or KMO inhibitors for cognitive enhancement and neuroprotection, respectively.     

Screening marine natural products for selective inhibitors of key kynurenine pathway enzymes

Abstract

Kynurenine, a metabolite of tryptophan along the ‘kynurenine pathway’, is at a branch point of the pathway which can lead to the synthesis of both quinolinic acid (QUIN) and kynurenic acid (KYNA). KYNA is an antagonist of glutamate receptors; however, QUIN is a selective agonist of NMDA receptors, and has been shown to act as an excitotoxic agent. A high QUIN/KYNA ratio has been implicated in a variety of neurological diseases in which excitotoxic neuronal cell death is found, e.g. AIDS-related dementia, stroke, etc. Inhibiting the key enzymes of this pathway (i.e. kynureninase and kynurenine 3-hydroxylase) would lower the QUIN/KYNA ratio, which may potentially have neuroprotective effects. We have developed high through-put assays for kynurenine pathway enzymes which allow us to screen extracts from marine organisms for selective enzyme inhibitors. Active metabolites are purified, isolated and identified by HPLC, high-field NMR and mass spectral techniques. Extracts from a sponge of the Aka species were found to contain a selective inhibitor of kynureninase. We have recently purified and identified the active principal as being serotonin sulfate. Related indoleamines, serotonin and 5-hydroxyindoleacetic acids are inactive. This finding may be suggestive of a novel interaction between the serotoninergic and excitatory amino acid pathways.     

Screening marine natural products for selective inhibitors of key kynurenine pathway enzymes

Kynurenine, a metabolite of tryptophan along the 'kynurenine pathway', is at a branch point of the pathway which can lead to the synthesis of both quinolinic acid (QUIN) and kynurenic acid (KYNA). KYNA is an antagonist of glutamate receptors; however, QUIN is a selective agonist of NMDA receptors, and has been shown to act as an excitotoxic agent. A high QUIN/KYNA ratio has been implicated in a variety of neurological diseases in which excitotoxic neuronal cell death is found, e.g. AIDS-related dementia, stroke, etc. Inhibiting the key enzymes of this pathway (i.e. kynureninase and kynurenine 3-hydroxylase) would lower the QUIN/KYNA ratio, which may potentially have neuroprotective effects. We have developed high through-put assays for kynurenine pathway enzymes which allow us to screen extracts from marine organisms for selective enzyme inhibitors. Active metabolites are purified, isolated and identified by HPLC, high-field NMR and mass spectral techniques. Extracts from a sponge of the Aka species were found to contain a selective inhibitor of kynureninase. We have recently purified and identified the active principal as being serotonin sulfate. Related indoleamines, serotonin and 5-hydroxyindoleacetic acids are inactive. This finding may be suggestive of a novel interaction between the serotoninergic and excitatory amino acid pathways.     

Kynurenine Pathway Enzymes in Brain: Responses to Ischemic Brain Injury Versus Systemic Immune Activation

Accumulation of l -kynurenine and quinolinic acid (QUIN) in the brain occurs after either ischemic brain injury or after systemic administration of pokeweed mitogen. Although conversion of l -[¹³C₆]tryptophan to [¹³C₆]-QUIN has not been demonstrated in brain either from normal gerbils or from gerbils given pokeweed mitogen, direct conversion in brain tissue does occur 4 days after transient cerebral ischemia. Increased activities of enzymes distal to indoleamine-2,3-dioxygenase may determine whether l -kynurenine is converted to QUIN. One day after 10 min of cerebral ischemia, the activities of kynureninase and 3-hydroxy-3,4-dioxygenase were increased in the hippocampus, but local QUIN levels and the activities of the indoleamine-2,3-dioxygenase and kynurenine-3-hydroxylase were unchanged. By days 2 and 4 after ischemia, however, the activities of all of these enzymes in the hippocampus as well as QUIN levels were significantly increased. Kynurenine aminotransferase activity in the hippocampus was unchanged on days 1 and 2 after ischemia but was decreased on day 4, at a time when local kynurenic acid levels were unchanged. A putative precursor of QUIN, [¹³C₆]anthranilic acid, was not converted to [¹³C₆]-QUIN in the hippocampus of either normal or 4-day postischemic gerbils. Gerbil macrophages stimulated by endo-toxin in vitro converted l -[¹³C₆]tryptophan to [¹³C_e]QUIN. Kinetic analysis of kynurenine-3-hydroxylase activity in the cerebral cortex of postischemic gerbils showed that V_max increased, without changes in K_m. Systemic administration of pokeweed mitogen increased indoleamine-2,3-dioxygenase and kynureninase activities in the brain without significant changes in kynurenine-3-hydroxylase or 3-hydroxyanthranilate-3,4-dioxygenase activities. Increases in kynurenine-3-hydroxylase activity, in conjunction with induction of indoleamine-2,3-dioxygenase, kynureninase, and 3-hydroxyanthranilate-3,4-dioxygenase in macro-phage infiltrates at the site of brain injury, may explain the ability of postischemic hippocampus to convert l -[¹³C₆]tryptophan to [¹³C₆]QUIN.     

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     

Changes in kynurenine pathway metabolism in the brain, liver and kidney of aged female Wistar rats

The kynurenine pathway of tryptophan catabolism plays an important role in several biological systems affected by aging. We quantified tryptophan and its metabolites kynurenine (KYN), kynurenine acid (KYNA), picolinic acid (PIC) and quinolinic acid (QUIN), and activity of the kynurenine pathway enzymes indoleamine 2,3-dioxygenase (IDO), tryptophan 2,3-dioxygenase (TDO) and quinolinic acid phosphoribosyltransferase (QPRTase), in the brain, liver and kidney of young, middle-aged and old female Wistar rats. Tryptophan levels and TDO activity decreased in all tissues with age. In contrast, brain IDO activity increased with age, while liver and kidney IDO activity decreased with age. The levels of KYN, KYNA, QUIN and PIC in brain all increased with age, while the levels of KYN in the liver and kidney showed a tendency to decrease. The levels of KYNA in the liver did not change, but the levels of KYNA in the kidney increased. The levels of PIC and QUIN increased significantly in the liver but showed a tendency to decrease in the kidney. QPRTase activity in both brain and liver decreased with age but was elevated in the kidney in middle-aged (12-month-old) rats. These age-associated changes in tryptophan metabolism have the potential to impact upon major biological processes, including lymphocyte function, pyridine (NAD(P)(H)) synthesis and N-methyl-d-aspartate (NMDA)-mediated synaptic transmission, and may therefore contribute to several degenerative changes of the elderly.     

Kynurenic Acid Inhibits the Release of the Neurotrophic Fibroblast Growth Factor (FGF)-1 and Enhances Proliferation of Glia Cells, <Emphasis Type="Italic">in vitro</Emphasis>

1. Kynurenic (KYNA) and quinolinic (QUIN) acids are neuroactive tryptophan metabolites formed along the kynurenine pathway: the first is considered a non-competitive antagonist and the second an agonist of glutamate receptors of NMDA type. The affinity of these compounds for glutamate receptors is, however, relatively low and does not explain KYNA neuroprotective actions in models of post-ischemic brain damage. 2. We evaluated KYNA effects on the release of fibroblast growth factor (FGF)-1, a potent neurotrophic cytokine. Because KYNA exhibits a neuroprotective profile in vitro and in vivo, we anticipated that it could function as an autocrine/paracrine inducer of FGF-1 release. Studies were performed in several models of FGF-1 secretion (FGF-1 transfected NIH 3T3 cells exposed to heat shock, A375 melanoma cells exposed to serum starvation, growth factor deprived human endothelial cells). To our surprise, KYNA, at low concentration, inhibited FGF-1 release in all cellular models. QUIN, a compound having opposite effects on glutamate receptors, also reduced this release, but its potency was significantly lower than that of KYNA. 3. KYNA and QUIN also displayed a major stimulatory effect on the proliferation rate of mouse microglia and human glioblastoma cells, in vitro. 4. Our data suggest that minor changes of local KYNA concentration may modulate FGF-1 release, cell proliferation, and ultimately tissue damage in different pathological conditions.     

Nicotinylalanine Increases the Formation of Kynurenic Acid in the Brain and Antagonizes Convulsions

Kynurenic acid (KYNA) was quantified in the extracellular spaces of the rat hippocampus using microdialysis and HPLC (fluorimetric detection) to study the possible role of this tryptophan metabolite in the modulation of the function of the N-methyl-D-aspartate (NMDA) receptor. Addition of probenecid (1 mM), which is an inhibitor of the organic acid transport system, to the Ringer's solution perfusing the dialysis probe increased the KYNA concentration in the dialysate from 10.4 +/- 0.9 to 48 +/- 6 nM. Addition of 2 mM aminooxyacetic acid, a nonspecific inhibitor of KYNA synthesis, reduced this concentration by 50%. These data suggest that KYNA is continuously synthesized in the rat hippocampus. Nicotinylalanine (NAL), 200-400 mg/kg i.p., an analogue of kynurenine that is able to direct the flow of tryptophan metabolites toward the synthesis of KYNA, significantly increased the KYNA concentration in the hippocampal dialysate and significantly potentiated the effect of tryptophan on the accumulation of KYNA in the brain and other organs. This increase resulted in pharmacological actions compatible with an antagonism of the NMDA receptors. In fact, NAL antagonized sound-induced seizures and prevented death in DBA/2 mice. Pretreatment of the mice with D-serine (100 micrograms intracerebroventricularly), a glycine agonist and a competitive antagonist of KYNA, completely prevented the anticonvulsive action of NAL. These data suggest that changes in the extracellular concentration of KYNA in the brain are associated with a modulation of NMDA receptor function.     

Drosophila eye color mutants as therapeutic tools for Huntington disease

Huntington disease (HD) is a fatal inherited neurodegenerative disorder caused by a polyglutamine expansion in the huntingtin protein (htt). A pathological hallmark of the disease is the loss of a specific population of striatal neurons, and considerable attention has been paid to the role of the kynurenine pathway (KP) of tryptophan (TRP) degradation in this process. The KP contains three neuroactive metabolites: 3-hydroxykynurenine (3-HK), quinolinic acid (QUIN), and kynurenic acid (KYNA). 3-HK and QUIN are neurotoxic, and are increased in the brains of early stage HD patients, as well as in yeast and mouse models of HD. Conversely, KYNA is neuroprotective and has been shown to be decreased in HD patient brains. We recently used a Drosophila model of HD to measure the neuroprotective effect of genetic and pharmacological inhibition of kynurenine monoxygenase (KMO)-the enzyme catalyzing the formation of 3-HK at a pivotal branch point in the KP. We found that KMO inhibition in Drosophila robustly attenuated neurodegeneration, and that this neuroprotection was correlated with reduced levels of 3-HK relative to KYNA. Importantly, we showed that KP metabolites are causative in this process, as 3-HK and KYNA feeding experiments modulated neurodegeneration. We also found that genetic inhibition of the upstream KP enzyme tryptophan-2,3-dioxygenase (TDO) was neuroprotective in flies. Here, we extend these results by reporting that genetic impairment of KMO or TDO is protective against the eclosion defect in HD model fruit flies. Our results provide further support for the possibility of therapeutic KP interventions in HD.     

Drosophila eye color mutants as therapeutic tools for Huntington disease

Huntington disease (HD) is a fatal inherited neurodegenerative disorder caused by a polyglutamine expansion in the huntingtin protein (htt). A pathological hallmark of the disease is the loss of a specific population of striatal neurons, and considerable attention has been paid to the role of the kynurenine pathway (KP) of tryptophan (TRP) degradation in this process. The KP contains three neuroactive metabolites: 3-hydroxykynurenine (3-HK), quinolinic acid (QUIN), and kynurenic acid (KYNA). 3-HK and QUIN are neurotoxic, and are increased in the brains of early stage HD patients, as well as in yeast and mouse models of HD. Conversely, KYNA is neuroprotective and has been shown to be decreased in HD patient brains. We recently used a Drosophila model of HD to measure the neuroprotective effect of genetic and pharmacological inhibition of kynurenine monoxygenase (KMO)—the enzyme catalyzing the formation of 3-HK at a pivotal branch point in the KP. We found that KMO inhibition in Drosophila robustly attenuated neurodegeneration, and that this neuroprotection was correlated with reduced levels of 3-HK relative to KYNA. Importantly, we showed that KP metabolites are causative in this process, as 3-HK and KYNA feeding experiments modulated neurodegeneration. We also found that genetic inhibition of the upstream KP enzyme tryptophan-2,3-dioxygenase (TDO) was neuroprotective in flies. Here, we extend these results by reporting that genetic impairment of KMO or TDO is protective against the eclosion defect in HD model fruit flies. Our results provide further support for the possibility of therapeutic KP interventions in HD.     

Blood–Brain Barrier Transport of Kynurenines: Implications for Brain Synthesis and Metabolism

To evaluate the potential contribution of circulating kynurenines to brain kynurenine pools, the rates of cerebral uptake and mechanisms of blood-brain barrier transport were determined for several kynurenine metabolites of tryptophan, including L-kynurenine (L-KYN), 3-hydroxykynurenine (3-HKYN), 3-hydroxyanthranilic acid (3-HANA), anthranilic acid (ANA), kynurenic acid (KYNA), and quinolinic acid (QUIN), in pentobarbital-anesthetized rats using an in situ brain perfusion technique. L-KYN was found to be taken up into brain at a significant rate [permeability-surface area product (PA) = 2-3 x 10(-3) ml/s/g] by the large neutral amino acid carrier (L-system) of the blood-brain barrier. Best-fit estimates of the Vmax and Km of saturable L-KYN transfer equalled 4.5 x 10(-4) mumol/s/g and 0.16 mumol/ml, respectively. The same carrier may also mediate the brain uptake of 3-HKYN as D,L-3-HKYN competitively inhibited the brain transfer of the large neutral amino acid L-leucine. For the other metabolites, uptake appeared mediated by passive diffusion. This occurred at a significant rate for ANA (PA, 0.7-1.6 x 10(-3) ml/s/g), and at far lower rates (PA, 2-7 x 10(-5) ml/s/g) for 3-HANA, KYNA, and QUIN. Transfer for KYNA, 3-HANA, and ANA also appeared to be limited by plasma protein binding. The results demonstrate the saturable transfer of L-KYN across the blood-brain barrier and suggest that circulating L-KYN, 3-HKYN, and ANA may each contribute significantly to respective cerebral pools. In contrast, QUIN, KYNA, and 3-HANA cross the blood-brain barrier poorly, and therefore are not expected to contribute significantly to brain pools under normal conditions.     

Gas chromatography/tandem mass spectrometry detection of extracellular kynurenine and related metabolites in normal and lesioned rat brain

We describe here a gas chromatography-tandem mass spectrometry (GC/MS/MS) method for the sensitive and concurrent determination of extracellular tryptophan and the kynurenine pathway metabolites kynurenine, 3-hydroxykynurenine (3-HK), and quinolinic acid (QUIN) in rat brain. This metabolic cascade is increasingly linked to the pathophysiology of several neurological and psychiatric diseases. Methodological refinements, including optimization of MS conditions and the addition of deuterated standards, resulted in assay linearity to the low nanomolar range. Measured in samples obtained by striatal microdialysis in vivo, basal levels of tryptophan, kynurenine, and QUIN were 415, 89, and 8 nM, respectively, but 3-HK levels were below the limit of detection (<2 nM). Systemic injection of kynurenine (100 mg/kg, i.p.) did not affect extracellular tryptophan but produced detectable levels of extracellular 3-HK (peak after 2-3 h: ~50 nM) and raised extracellular QUIN levels (peak after 2h: ~105 nM). The effect of this treatment on QUIN, but not on 3-HK, was potentiated in the N-methyl-D-aspartate (NMDA)-lesioned striatum. Our results indicate that the novel methodology, which allowed the measurement of extracellular kynurenine and 3-HK in the brain in vivo, will facilitate studies of brain kynurenines and of the interplay between peripheral and central kynurenine pathway functions under physiological and pathological conditions.     

Characterisation of kynurenine pathway metabolism in human astrocytes and implications in neuropathogenesis

The role of astrocytes in the production of the neurotoxin quinolinic acid (QUIN) and other products of the kynurenine pathway (KP) is controversial. Using cytokine-stimulated human astrocytes, we assayed key enzymes and products of the KP. We found that astrocytes lack kynurenine-hydroxylase so that large amounts of kynurenine (KYN) and kynurenic acid (KYNA) were produced, while minor amounts of QUIN were synthesised that were completely degraded. We then showed that kynurenine added to macrophages led to significant production of QUIN. These results suggest that astrocytes alone are neuroprotective by minimising QUIN production and maximising synthesis of KYNA. However, it is likely that, in the presence of macrophages and/or microglia, astrocytes are neurotoxic by producing large concentrations of KYN that can be metabolised by neighbouring monocytic cells to QUIN.     

Characterisation of kynurenine pathway metabolism in human astrocytes and implications in neuropathogenesis

Abstract

The role of astrocytes in the production of the neurotoxin quinolinic acid (QUIN) and other products of the kynurenine pathway (KP) is controversial. Using cytokine-stimulated human astrocytes, we assayed key enzymes and products of the KP. We found that astrocytes lack kynurenine-hydroxylase so that large amounts of kynurenine (KYN) and kynurenic acid (KYNA) were produced, while minor amounts of QUIN were synthesised that were completely degraded. We then showed that kynurenine added to macrophages led to significant production of QUIN. These results suggest that astrocytes alone are neuroprotective by minimising QUIN production and maximising synthesis of KYNA. However, it is likely that, in the presence of macrophages and/or microglia, astrocytes are neurotoxic by producing large concentrations of KYN that can be metabolised by neighbouring monocytic cells to QUIN.     

Purinergic signalling at the plasma membrane: a multipurpose and multidirectional mode to deal with amyotrophic lateral sclerosis and multiple sclerosis

At endogenous brain concentrations, the neuroinhibitory tryptophan metabolite kynurenic acid (KYNA) is a preferential antagonist of the α7 nicotinic acetylcholine receptor (α7nAChR). In the present study, male Wistar rats were fed a high tryptophan diet (adding 0.1-1.5% tryptophan) for 24 h to examine (i) the effect of increased tryptophan on extracellular dopamine (DA) and KYNA levels and (ii) to determine any possible interactions between DA and KYNA. Brain KYNA levels were dose-dependently increased by tryptophan intake, and these increase were consistent with kynurenine (KYN), the precursor to KYNA, levels in the brain, plasma and liver. Administration of the 1.5% tryptophan added diet reduced the extracellular DA level to 60%, and increased the extracellular KYNA to 320% in the striatum. The DA reduction was attenuated through inhibiting KYNA synthesis with 2-aminoadipic acid. These results indicate that a high tryptophan diet can induce KYNA production and suppress DA release. One possible mechanism is that as more KYN is metabolized from the high doses of tryptophan in the liver and released into the blood stream, KYNA production in astrocytes is enhanced and the increased extracellular KYNA inhibits DA release by blocking α7nAChRs. Dietary manipulation of KYNA formation in astrocytes may offer a unique strategy to modulate DA.     

Kynurenine 3-mono-oxygenase inhibitors attenuate post-ischemic neuronal death in organotypic hippocampal slice cultures

Kynurenine 3-mono-oxygenase (KMO) inhibitors reduce 3-hydroxykynurenine (3-HK) and quinolinic acid (QUIN) neosynthesis and facilitate kynurenine metabolism towards kynurenic acid (KYNA) formation. They also reduce tissue damage in models of focal or transient global cerebral ischemia in vivo. We used organotypic hippocampal slice cultures exposed to oxygen and glucose deprivation (OGD) to investigate KMO mechanism(s) of neuroprotective activity. Exposure of the slices to 30 min of OGD caused CA1 pyramidal cell death and significantly decreased the amount of KYNA released in the incubation medium. The KMO inhibitors (m-nitrobenzoyl)-alanine (30-100 micro m) or 3,4-dimethoxy-[-N-4-(nitrophenyl)thiazol-2yl]-benzenesulfonamide (1-10 micro m) reduced post-ischemic neuronal death and increased KYNA concentrations in slice incubation media. The maximal concentration of KYNA detected in the incubation media of slices treated with KMO inhibitors was approximately 50 nm and was too low to efficiently interact with alpha7 nicotinic acetylcholine receptors or with the glycineb site of N-methyl-d-aspartate (NMDA) receptors. On the other hand, the addition of either 3-HK or QUIN (1-10 micro m) to OGD-exposed hippocampal slices prevented the neuroprotective activity of KMO inhibitors. Our results suggest that KMO inhibitors reduce the neuronal death found in the CA1 region of organotypic hippocampal slices exposed to 30 min of OGD by decreasing the local synthesis of 3-HK and QUIN.     

Effect of indoleamine dioxygenase-1 deficiency and kynurenine pathway inhibition on murine cerebral malaria

Cerebral malaria (CM) can be a fatal manifestation of Plasmodium falciparum infection. In this study, two different approaches were used to examine the role of indoleamine 2,3-dioxygenase-1 (IDO-1) and its metabolites in the development of murine CM. Mice genetically deficient in IDO-1 were not protected against CM, but partial protection was observed in C57BL/6 mice treated with Ro 61-8048, an inhibitor of kynurenine-3-hydroxylase. This protection was associated with suppressed levels of picolinic acid (PA) within the brain, but not with changes in the levels of kynurenic acid (KA) or quinolinic acid (QA). These data suggest that although IDO-1 is not directly involved in the pathogenesis of CM in C57BL/6 mice, the production of the kynurenine pathway metabolite PA may contribute to the development of murine CM.     

Mechanism of Delayed Increases in Kynurenine Pathway Metabolism in Damaged Brain Regions Following Transient Cerebral Ischemia

Abstract: Delayed increases in the levels of an endogenous N-methyl-D-aspartate receptor agonist, quinolinic acid (QUIN), have been demonstrated following transient ischemia in the gerbil and were postulated to be secondary to induction of indoleamine-2,3-dioxygenase (IDO) and other enzymes of the L-tryptophan-kynurenine pathway. In the present study, proportional increases in IDO activity and QUIN concentrations were found 4 days after 10 min of cerebral ischemia, with both responses in hippocampus > striatum > cerebral cortex > thalamus. These increases paralleled the severity of local brain injury and inflammation. IDO activity and QUIN concentrations were unchanged in the cerebellum of postischemic gerbils, which is consistent with the preservation of blood flow and resultant absence of pathology in this region. Blood QUIN and L-kynurenine concentrations were not affected by ischemia. Brain tissue QUIN levels at 4 days postischemia exceeded blood concentrations, minimizing a role for breakdown of the blood–brain barrier. Marked increases in the activity of kynureninase, kynurenine 3-hydroxylase, and 3-hydroxyanthranilate-3,4-dioxygenase were also detected in hippocampus but not in cerebellum on day 4 of recirculation. In vivo synthesis of [¹³C₆]QUIN was demonstrated, using mass spectrometry, in hippocampus but not in cerebellum of 4-day postischemic animals 1 h after intracisternal administration of L-[¹³C₆]tryptophan. However, accumulation of QUIN was demonstrated in both cerebellum and hippocampus of control gerbils following an intracisternal injection of 3-hydroxyanthranilic acid, which verifies the availability of precursor to both regions when administered intracisternally. Notably, although IDO activity and QUIN concentrations were unchanged in the cerebellum of ischemic gerbils, both IDO activity and QUIN content were increased in cerebellum to approximately the same degree as in hippocampus, striatum, cerebral cortex, and thalamus 24 h after immune stimulation by systemic pokeweed mitogen administration, demonstrating that the cerebellum can increase IDO activity and QUIN content in response to immune activation. No changes in kynurenic acid concentrations in either hippocampus, cerebellum, or cerebrospinal fluid were observed in the postischemic gerbils compared with controls, in accordance with the unaffected activity of kynurenine aminotransferase activity. Collectively, these results support roles for IDO, kynureninase, kynurenine 3-hydroxylase, and 3-hydroxyanthranilate-3,4-dioxygenase in accelerating the conversion of L-tryptophan and other substrates to QUIN in damaged brain regions following transient cerebral ischemia. Immunocytochemical results demonstrated the presence of macrophage infiltrates in hippocampus and other brain regions that parallel the extent of these biochemical changes. We hypothesize that increased kynurenine pathway metabolism after ischemia reflects the presence of macrophages and other reactive cell populations at sites of brain injury.