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Human cytomegalovirus (HCMV) replication depends on the activities of several host regulators of metabolism. Hypoxia-inducible factor 1α (HIF1α) was previously proposed to support virus replication through its metabolic regulatory function. HIF1α protein levels rise in response to HCMV infection in nonhypoxic conditions, but its effect on HCMV replication was not investigated. We addressed the role of HIF1α in HCMV replication by generating primary human cells with HIF1α knocked out using CRISPR/Cas9. When HIF1α was absent, we found that HCMV replication was enhanced, showing that HIF1α suppresses viral replication. We used untargeted metabolomics to determine if HIF1α regulates metabolite concentrations in HCMV-infected cells. We discovered that in HCMV-infected cells, HIF1α suppresses intracellular and extracellular concentrations of kynurenine. HIF1α also suppressed the expression of indoleamine 2,3-dioxygenase 1 (IDO1), the rate-limiting enzyme in kynurenine synthesis. In addition to its role in tryptophan metabolism, kynurenine acts as a signaling messenger by activating aryl hydrocarbon receptor (AhR). Inhibiting AhR reduces HCMV replication, while activating AhR with an exogenous ligand increases virus replication. Moreover, we found that feeding kynurenine to cells promotes HCMV replication. Overall, our findings indicate that HIF1α reduces HCMV replication by regulating metabolism and metabolite signaling. IMPORTANCE Viruses, including human cytomegalovirus (HCMV), reprogram cellular metabolism using host metabolic regulators to support virus replication. Alternatively, in response to infection, the host can use metabolism to limit virus replication. Here, our findings show that the host uses hypoxia-inducible factor 1α (HIF1α) as a metabolic regulator to reduce HCMV replication. Further, we found that HIF1α suppresses kynurenine synthesis, a metabolite that can promote HCMV replication by signaling through the aryl hydrocarbon receptor (AhR). In infected cells, the rate-limiting enzyme in kynurenine synthesis, indoleamine 2,3-dioxygenase 1 (IDO1), is suppressed by a HIF1α-dependent mechanism. Our findings describe a functional connection between HIF1α, IDO1, and AhR that allows HIF1α to limit HCMV replication through metabolic regulation, advancing our understanding of virus-host interactions., (Copyright © 2021 Wise et al.)

Through structural modification of an oxalamide derived chemotype, a novel class of highly potent, orally bioavailable IDO1-specific inhibitors was identified. Representative compound 18 inhibited human IDO1 with IC 50 values of 3.9 nM and 52 nM in a cellular and human whole blood assay, respectively. In vitro assessment of the ADME properties of 18 demonstrated very high metabolic stability. Pharmacokinetic profiling in mice showed a significantly reduced clearance compared to the oxalamides. In a mouse pharmacodynamic model 18 nearly completely suppressed lipopolysaccharide-induced kynurenine production. Hepatocyte data of 18 suggest the human clearance to be in a similar range to linrodostat (1)., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Cisplatin resistance is a major barrier in the effective treatment of lung cancer. Cisplatin-resistant (CR) lung cancer cells do not primarily use glucose but rather consume amino acids such as glutamine and tryptophan (Trp) for survival. CR cells activate the kynurenine (KYN) pathway (KP) to cope with excessive reactive oxygen species (ROS) and maintain homeostasis for growth and proliferation. Consequently, indoleamine 2,3-dioxygenase-1 (IDO1) becomes an essential enzyme for CR cells' survival because it initiates and regulates the first step in the KP. Increased IDO1 activities and ROS levels are found in CR cells versus cisplatin-sensitive lung cancer. Importantly, significantly greater KYN/Trp ratio ( P = 0.005) is detected in serum of patients who fail cisplatin when compared with naïve treatment. Knocking down IDO1 using shRNA or IDO1 inhibitors heightens ROS levels and results in a significant growth inhibitory effect only on CR cells and not on cisplatin-sensitive cells. Exposing CR cells to antioxidant (TIRON) results in suppression of IDO1 activity and confers resistance to IDO1 inhibition, indicating an interrelationship between ROS and IDO1. Because KYN plays a critical role in reprogramming naïve T cells to the immune-suppressive regulatory T-cell (T-reg) phenotype, we observed higher expression of TGFβ, FoxP3, and CD4 + CD25 + in mice bearing CR tumors compared with tumors from cisplatin-sensitive counterparts. IMPLICATIONS: Findings suggest that the enzyme-inhibitory activity and antitumor efficacy of IDO1 inhibitors rely in part on ROS levels, arguing that IDO1 expression alone may be insufficient to determine the clinical benefits for this class of experimental cancer drugs. Importantly, IDO1 inhibitors may be more suitable to treat patients with lung cancer who failed cisplatin therapy than naïve treatment patients., (©2019 American Association for Cancer Research.)

Tryptophan 2,3-dioxygenase (TDO), an immunosuppressive enzyme, can involve in immune evasion and tumor tolerance. TDO inhibitors can boost the efficacy of chemotherapeutics by promoting immunity. Herein, a strategy to introduce a TDO inhibitor into Pt(IV) complexes for reversing tumor immune suppression was adopted. A mono-modified Pt(IV) complex, 3, displayed significant antitumor activity against human liver cancer cells. Flow cytometry study revealed that complex 3 could induce cell death via a mitochondrial-dependent apoptosis pathway and arrest the cell cycle at S phase. Furthermore, complex 3 was effective to enhance T-cell immune responses by inhibiting the TDO enzyme expression to block the kynurenine production and inactivating the downstream of aryl hydrocarbon receptor (AHR)., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Kynurenine is biosynthesised from tryptophan catalysed by indoleamine 2,3-dioxygenase (IDO). The abrogation of kynurenine production is considered a promising therapeutic target for immunological cancer treatment. In the course of our IDO inhibitor programme, formal cyclisation of the isothiourea moiety of the IDO inhibitor 1 afforded the 5-Cl-benzimidazole derivative 2b-6, which inhibited both recombinant human IDO (rhIDO) activity and cellular kynurenine production. Further derivatisation of 2b-6 provided the potent inhibitor of cellular kynurenine production 2i (IC 50  = 0.34 µM), which unexpectedly exerted little effect on the enzymatic activity of rhIDO. Elucidation of the mechanism of action revealed that compound 2i suppresses IDO expression at the protein level by inhibiting STAT1 expression in IFN-γ-treated A431 cells. The kynurenine-production inhibitor 2i is expected to be a promising starting point for a novel approach to immunological cancer treatment., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Our antiviral arsenal to fight influenza viruses is limited and we need novel anti-flu drugs. Recently, cellular drug targets came into focus and omics analysis were instrumental to suggest candidate factors. In this issue of The FEBS Journal, Kainov and colleagues used transcriptome data to investigate virus-induced changes in tryptophan metabolism that may serve as immunomodulatory approach against viruses., (© 2017 Federation of European Biochemical Societies.)

The kynurenine (KYN) pathway (KP) is a major degradative pathway of the amino acid, L-tryptophan (TRP), that ultimately leads to the anabolism of the essential pyridine nucleotide, nicotinamide adenine dinucleotide. TRP catabolism results in the production of several important metabolites, including the major immune tolerance-inducing metabolite KYN, and the neurotoxin and excitotoxin quinolinic acid. Dendritic cells (DCs) have been shown to mediate immunoregulatory roles that mediated by TRP catabolism. However, characterization of the KP in human DCs has so far only been partly delineated. It is critical to understand which KP enzymes are expressed and which KP metabolites are produced to be able to understand their regulatory effects on the immune response. In this study, we characterized the KP in human monocyte-derived DCs (MDDCs) in comparison with the human primary macrophages using RT-PCR, high-pressure gas chromatography, mass spectrometry, and immunocytochemistry. Our results show that the KP is entirely expressed in human MDDC. Following activation of the KP using interferon gamma, MDDCs can mediate apoptosis of T h cells in vitro. Understanding the molecular mechanisms regulating KP metabolism in MDDCs may provide renewed insight for the development of novel therapeutics aimed at modulating immunological effects and peripheral tolerance.

The excitotoxin quinolinic acid, a by-product of the kynurenine pathway, is known to be involved in several neurological diseases including multiple sclerosis (MS). Quinolinic acid levels are elevated in experimental autoimmune encephalomyelitis rodents, the widely used animal model of MS. Our group has also found pathophysiological concentrations of quinolinic acid in MS patients. This led us to investigate the effect of quinolinic acid on oligodendrocytes; the main cell type targeted by the autoimmune response in MS. We have examined the kynurenine pathway (KP) profile of two oligodendrocyte cell lines and show that these cells have a limited threshold to catabolize exogenous quinolinic acid. We further propose and demonstrate two strategies to limit quinolinic acid gliotoxicity: 1) by neutralizing quinolinic acid's effects with anti-quinolinic acid monoclonal antibodies and 2) directly inhibiting quinolinic acid production from activated monocytic cells using specific KP enzyme inhibitors. The outcome of this study provides a new insight into therapeutic strategies for limiting quinolinic acid-induced neurodegeneration, especially in neurological disorders that target oligodendrocytes, such as MS.

During early brain development, N-methyl-d-aspartate (NMDA) receptors are involved in cell migration, neuritogenesis, axon guidance and synapse formation, but the mechanisms which regulate NMDA receptor density and function remain unclear. The kynurenine pathway of tryptophan metabolism includes an agonist (quinolinic acid) and an antagonist (kynurenic acid) at NMDA receptors and we have previously shown that inhibition of the pathway using the kynurenine-3-monoxygenase inhibitor Ro61-8048 in late gestation produces rapid changes in protein expression in the embryos and effects on synaptic transmission lasting until postnatal day 21 (P21). The present study sought to determine whether any of these effects are maintained into adulthood. After prenatal injections of Ro61-8048 the litter was allowed to develop to P60 when some offspring were euthanized and the brains removed for examination. Analysis of protein expression by Western blotting revealed significantly reduced expression of the GluN2A subunit (32%) and the morphogenetic protein sonic hedgehog (31%), with a 29% increase in the expression of doublecortin, a protein associated with neurogenesis. No changes were seen in mRNA abundance using quantitative real-time polymerase chain reaction. Neuronal excitability was normal in the CA1 region of hippocampal slices but paired-pulse stimulation revealed less inhibition at short interpulse intervals. The amount of long-term potentiation was decreased by 49% in treated pups and recovery after low-frequency stimulation was delayed. The results not only strengthen the view that basal, constitutive kynurenine metabolism is involved in normal brain development, but also show that changes induced prenatally can affect the brains of adult offspring and those changes are quite different from those seen previously at weaning (P21). Those changes may be mediated by altered expression of NMDAR subunits and sonic hedgehog., (Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.)

The oxidative pathway for the metabolism of tryptophan along the kynurenine pathway generates quinolinic acid, an agonist at N-methyl-D-aspartate receptors, as well as kynurenic acid which is an antagonist at glutamate and nicotinic receptors. The pathway has become recognized as a key player in the mechanisms of neuronal damage and neurodegenerative disorders. As a result, manipulation of the pathway, so that the balance between the levels of components of the pathway can be modified, has become an attractive target for the development of pharmacological agents with the potential to treat those disorders. This review summarizes some of the relevant background information on the pathway itself before identifying some of the chemical strategies for its modification, with examples of their successful application in animal models of infection, stroke, traumatic brain damage, cerebral malaria and cerebral trypanosomiasis., (© 2012 The Authors Journal compilation © 2012 FEBS.)

Kynurenine (Kyn), a metabolite of tryptophan (Trp), is known to be a key regulator of human immune responses including cancer immune tolerance. Therefore, abrogation of Kyn production from cancer cells by small molecules may be a promising approach to anticancer therapy. Indeed, several small molecule inhibitors of indoleamine 2,3-dioxygenase (IDO), a rate-limiting enzyme in the catabolism of Trp to Kyn, exert antitumor effects in animal models. We screened our chemical libraries using a cell-based Kyn production assay to identify a new type of small molecules that regulate Kyn production, and for the first time identified a benzenesulfonamide derivative (compound 1) as a hit with the ability to inhibit Kyn production in interferon-γ (IFN-γ)-stimulated A431 and HeLa cells. Unlike the previously identified S-benzylisothiourea derivative, compound 2, compound 1 had little effect on the enzymatic activity of recombinant human IDO in vitro but suppressed the expression of IDO at the mRNA level in cells. Furthermore, compound 1 suppressed STAT1-dependent transcriptional activity and DNA binding, whereas no decrement in either the expression or phosphorylation level of STAT1 was observed. The inhibition of IDO expression by several benzenesulfonamide derivatives is associated with the suppression of STAT1. Thus, compound 1 and its analogs might be useful for analyzing the regulation of IDO activation, and STAT1-targeting could be an alternative to the IDO-directed approach for the regulation of Kyn levels by small molecules in the tumor microenvironment., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Amyotrophic lateral sclerosis (ALS) is an adult onset, progressive and fatal motor neuron degenerative disease [1]. The aetiology of ALS is currently unknown, though strongly suggested to be multifactorial. Recently, the kynurenine pathway (KP) has emerged as a potential contributing factor [2]. The KP is a major route for the metabolism of tryptophan, generating neuroactive intermediates in the process. These catabolites include the excitotoxic N-methyl-D-aspartate (NMDA) receptor agonist, quinolinic acid (QUIN) [3] and the neuroprotective NMDA receptor antagonist, kynurenic acid (KYNA) [4,5]. These catabolites appear to play a key role in the communication between the nervous and immune systems, and also in modulating cell proliferation and tissue function [6]. As the cause of ALS is still unknown, there is presently no efficient treatment for it. Currently, Riluzole is the drug of choice but its effect is relatively modest [7]. Targeting the KP, hence, could offer a new therapeutic option to improve ALS treatment [8]. Several drugs that block the KP are already under investigation by our laboratory and others, some of which are in or about to enter clinical trials for other diseases. For example, the KP inhibitors, Teriflunomide (Sanofi-Aventis) and Laquinimod (Teva Neuroscience). Recently, a KP inhibitor has also reached the Japan market as an immunomodulative drug [9]: Tranilast/Rizaben (Angiogen Ltd.) is an anthranilic acid derivative [8]. Finally, the 8-hydroxyquinolinine metal attenuating compounds, Clioquinol and PBT2, interestingly have close structural similarity with KYNA and QUIN. Such drugs would open a new and important therapeutic door for ALS.

Human plasmacytoid dendritic cells (PDCs) can drive naive, allogeneic CD4(+)CD25(-) T cells to differentiate into CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs). However, the intracellular mechanism or mechanisms underlying PDC-induced Treg generation are unknown. In this study, we show that human PDCs express high levels of IDO, an intracellular enzyme that catabolizes tryptophan degradation. Triggering of TLR 9 with CpG oligodeoxynucleotides activates PDCs to up-regulate surface expression of B7 ligands and HLA-DR Ag, but also significantly increases the expression of IDO and results in the generation of inducible Tregs from CD4(+)CD25(-) T cells with potent suppressor cell function. Blocking IDO activity with the pharmacologic inhibitor 1-methyl-D-tryptophan significantly abrogates PDC-driven inducible Treg generation and suppressor cell function. Adding kynurenine, the immediate downstream metabolite of tryptophan, bypasses the 1-methyl-D-tryptophan effect and restores PDC-driven Treg generation. Our results demonstrate that the IDO pathway is essential for PDC-driven Treg generation from CD4(+)CD25(-) T cells and implicate the generation of kynurenine pathway metabolites as the critical mediator of this process.

The amino acid tryptophan is a precursor for the neurotransmitter serotonin as well as for kynurenic and quinolinic acids. These latter molecules are antagonists and agonists, respectively, of the excitatory amino acid glutamate and arise through the kynurenine pathway of tryptophan metabolism. Significant differences exist in the sites and physiological control of serotonin versus kynurenine. While serotonin is formed within serotonin neurons (in the brain and intestine) and neuroendocrine cells of the intestine, kynurenine is formed by liver cells (as a precursor to nicotinic acid) and in macrophages, activated by inflammatory cytokines. Our studies are based on the hypothesis that inhibition of kynurenine metabolism (at the kynurenine hydroxylase [KH] step) allows the amino acid to be converted to kynurenic acid, a neuroprotective antagonist of excitatory amino acid receptors. Inhibition of KH also prevents formation of the neurotoxic species 3-hydroxykynurenine and quinolinic acid. To accomplish this end, inhibitors were identified and are described.

3-Hydroxykynurenine (3-HK), an endogenous tryptophan metabolite, is known to have toxic effects in brain. However, the molecular mechanism of the toxicity has not been well identified. In this study, we investigated the involvement of MAPK/extracellular signal-regulated kinase (ERK) in the 3-HK-induced neuronal cell damage. Our results showed that 3-HK induced apoptotic neuronal cell death and ERK phosphorylation occurred during cell death. Inhibition of ERK activation using PD98059 considerably increased cell death. Furthermore, cell death was preceded by mitochondrial malfunction including collapse of mitochondrial membrane potential (DeltaPsi(m)) and cytochrome c release from mitochondria to the cytosol. Interestingly, inhibition of ERK dramatically increased mitochondrial malfunction, and enhanced caspase activation, resulting in enhanced neuronal cell death. Thus, our results show that ERK plays a protective role by maintaining mitochondrial function and regulating caspase activity under conditions of cellular stress.

Age-dependent human lens colouration and fluorescence may stem primarily from the covalent binding of UV filters to crystallins. The tendency of the kynurenine (Kyn) UV filters to deaminate at neutral pH, with the generation of reactive alpha,beta-ketoalkenes, underlies this phenomenon. In this study the authors examined the ability of small molecular weight antioxidants, which are known to be present in the lens, to inhibit this process. Crystallins were incubated with Kyn at pH 7 in the presence of glutathione (GSH), ascorbate or NADH. Ascorbate, even at high (15 m M) levels, was not found to significantly retard the time-dependent covalent binding of Kyn to the proteins. GSH, and to a lesser extent NADH, however, had a major impact in preventing this modification. The increase in protein UV absorbance and fluorescence was inhibited by GSH intercepting the reactive ketone intermediate, to form a GSH-Kyn adduct. NADH seemed to protect by both reduction of the reactive ketone intermediate and by competing with Kyn for presumably hydrophobic sites on the crystallins. This may indicate that the covalent attachment of aromatic Kyn molecules could be facilitated by initial hydrophobic interactions. Since GSH is present at far greater concentrations than NADH, these results show that in primate lenses, GSH is the key agent responsible for protecting the crystallins from covalent modification., (Copyright 2002 Elsevier Science Ltd.)

3-hydroxykynurenine (3HK), an endogenous metabolite of tryptophan in the kynurenine pathway, is a potential neurotoxin in several neurodegenerative disorders. Stabilizing protein structure, heat shock proteins (HSPs) have diverse roles as molecular chaperones to mediate stress tolerance. In the present study, we investigated the possible protective role of HSPs against 3HK induced neuronal cell death. Here we report that 3HK induced in a dose- and time-dependent manner neuronal cell death in neuroblastoma SK-N-SH cells. The cell death showed characteristic apoptotic features such as cell shrinkage, plasma membrane blebbing, chromatin condensation, and nuclear condensation and fragmentation. Furthermore, SK-N-SN cells were protected from 3HK induced cytotoxicity by prior elevation of HSPs expression. Our results show that the protective effect was abolished by HSP90 anti-sense oligonucleotides while not by HSP27 and HSP70 anti-sense oligonucleotides. Also, our result shows that HSP90 effectively inhibits caspases activities leading to the apoptosis. These results suggest that 3HK induces apoptosis in neuroblastoma SK-N-SN cells and that HSP90 is major contributing protein component of protection against 3HK induced apoptosis., (Copyright 2001 Academic Press.)

The kynurenine pathway accounts for the metabolism of around 80% of non-protein tryptophan metabolism. It includes both an agonist (quinolinic acid) at NMDA receptors and an antagonist (kynurenic acid). Since their discovery, there has been a major development of kynurenic acid analogues as neuroprotectants for the treatment of stroke and neurodegenerative disease. Several prodrugs of kynurenic acid or its analogues that can be hydrolysed within the CNS are also available. More recently, the pathway itself has proved to be a valuable drug target, affected by agents which reduce the synthesis of quinolinic acid and increase the formation of kynurenic acid. The change in the balance of these, away from the excitotoxin and towards the neuroprotectant, has anticonvulsant and neuroprotective properties.

Strokes (intracranial thomboses or haemorrhaging) cause death and disability, but effective treatments are lacking. The metabolism of tryptophan leads to the generation of quinolinic acid, an agonist potentially neurotoxic at glutamate receptors, and kynurenic acid, an antagonist at the same population of receptors. The commercial development of the kynurenine pathway has included the use of analogues of kynurenic acid as antagonists at glutamate receptors. A second has been to use prodrugs of kynurenic acid or its analogues. Alternatively, it is proving possible to interfere directly with the kynurenine pathway to block the synthesis of quinolinic acid and promote the formation of kynurenic acid. This change yields neuroprotectant and anticonvulsant compounds.

The AIDS dementia complex (ADC) is a consequence of excessive immune activation driven at least in part by systemic HIV infection and probably brain infection. Quinolinic acid (QUIN) is a neurotoxic tryptophan metabolite produced by macrophages in response to stimulation with cytokines or infection with HIV-1. Consequently it has been implicated in ADC pathogenesis. However, macrophages infected with HIV-1 synthesize numerous neurotoxic substances. Therefore we conducted experiments using human fetal brain tissue to determine the relative importance of QUIN as a neurotoxin in ADC. Human macrophages were infected with HIV-1 in vitro using a viral isolate from a demented patient. 6-Chloro-D-tryptophan, an inhibitor of QUIN biosynthesis, was added to half the macrophage cultures to block formation of QUIN. Supernatants containing QUIN (SQpos) or in which QUIN biosynthesis had been inhibited (SQneg) were then added to human fetal brain aggregate cultures. Toxicity was evaluated using lactate dehydrogenase efflux, trypan blue exclusion, immunohistochemistry, image analysis, and electron microscopy. Each technique showed a reduction of toxicity in SQneg-treated cultures. These studies confirm the significance of QUIN as a neurotoxin in ADC and suggest that neuroprotective strategies may have a place in the treatment of this disease.

The self-injurious movements of Lesch-Nyhan patients are considered extremely severe and currently intractable. Lesch-Nyhan syndrome is a genetic disorder of purine metabolism resulting in several abnormalities, one of which is elevated levels of xanthine. The author reasons that elevated plasma xanthine sets off a chain of events that produce excessive endogenous convulsants and diminished endogenous anticonvulsants. Treatment is proposed for self-injury in Lesch-Nyhan that entails reducing production of two endogenous convulsants (kynurenine and quinolinic acid) and enhancing two endogenous anticonvulsants (serotonin and gamma aminobutyric acid).

Previous studies have indicated that the generation of H2O2 may be a key step in the mechanism mediating the in vitro cytotoxicity of 3-hydroxykynurenine (3HK). An exposure protocol resulting in a delayed toxicity was utilized in order to further examine the role of H2O2 in the in vitro toxicity of 3HK in a neural hybrid cell line. 3HK-induced cell lysis was significantly attenuated by administration of catalase after termination of 3HK exposure and was abolished when intracellular peroxidase activity was elevated by pretreatment of cultures with horseradish peroxidase. In addition, a dose-dependent attenuation of 3HK toxicity was observed when cultures were exposed to 3HK in the presence of the iron chelator, desferrioxamine (DFO). Pretreatment with DFO also resulted in a significant attenuation of 3HK toxicity. These data suggest a direct role for H2O2 and metal ions in the cytotoxic action of 3HK and indicate that cell lysis results from the intracellular accumulation of toxic levels of H2O2.

Benzobarbital, primidone, phenobarbital, phynytoin and trimethadionum (in order of decreasing activity) were moderately effective against DL-kynurenine (50 micrograms, i. c. v.)-induced seizures in SHR albino male mice. Diazepam was ineffective even in doses of 15 and 25 mg/kg. Against seizures induced by quinolinic acid (5 micrograms, i. c. v.) anticonvulsants, particularly primidone and phenytoin, were more effective than against kynurenine-induced seizures. Diazepam was slightly effective in high doses of 7-15 mg/kg. Primidone and benzobarbital in doses used (10-50 mg/kg) were ineffective against strychnine and pentylenetetrazol. Selective efficacy of primidone and benzobarbital in this model of seizures suggests that kynurenine and quinolinic acid could be involved in the grand mal fits in epileptic patients.

L-Kynurenine (KYN), an intermediary product in the kynurenine pathway of tryptophan metabolism, is the common precursor from which are formed both quinolinic acid, a potent endogenous "excitotoxin," and kynurenic acid, a nonselective antagonist of excitotoxins. The present work examines 3H-KYN transport in primary astrocyte cultures derived from the cerebra of newborn mice. Influx and efflux of 3H-KYN were attributable almost entirely to carrier-mediated transport. The tritium recovered in uptake experiments was identifiable as 3H-KYN, indicating a low rate of KYN metabolism during incubations up to 30 min. KYN uptake decreased in the presence of extracellular Na+, at least in part because KYN efflux was accelerated. Marked trans stimulation of KYN efflux by extracellular KYN provided evidence of the exchanging nature of the carrier. Saturation curves for the initial velocity of KYN uptake conformed to a 1-component saturable system with Km of 32 microM and Vmax of 2.1 nmol mg-1 protein min-1. KYN was notably concentrated by the astrocytes, with an estimated steady-state distribution ratio of 180-fold for 1 microM KYN. Analog inhibition studies showed that the KYN transporter exhibited a clear preference for large neutral amino acids; leucine, tryptophan, and phenylalanine were recognized with relatively higher affinity than KYN. In summary, KYN is concentratively transported into astrocytes by a Na+-independent exchanger with high affinity for branched-chain and aromatic neutral amino acids. The substrate specificity and high affinity of this transport system resemble the properties of neutral amino acid transport across the blood-brain barrier in the rat and human.

Nicotinamide (NAM, 1000 mg/kg), inosine (INS, 1000 mg/kg), hypoxanthine (HXT, 500 mg/kg), putative endogenous ligands of the benzodiazepine receptor, and nicotinic acid (NA, 500 mg/kg) diminished DL-kynurenine-(DL-K, 50 micrograms ICV) induced seizures in C57BL/6 adult male mice and only prolonged the latency of pentylenetetrazol (PTZ, 500 micrograms iCV) seizures. The same effect was previously observed when PTZ was administered IP. In albino male BALB/c and SHR (bred from Swiss) mice only NA was effective against DL-K. Diazepam in a dose of 0.5 mg/kg prevented PTZ-induced seizures in half of the animals but even in dose of 10 and 20 mg/kg it was ineffective against DL-K. When injected ICV NAM (1 and 10 micrograms), INS (10 micrograms) and HXT (10 micrograms) prevented seizures induced by DL-K and were ineffective against seizures induced by PTZ. It is suggested that if NAM, INS and HXT are of functional importance in the central nervous system, they can act as antagonists of endogenous brain kynurenine. NA and NAM are suggested to be functional feedback inhibitory regulators of the kynurenine pathway of metabolism of tryptophan.

Picolinic, kynurenic, xanthurenic and anthranilic acids are metabolites of L-kynurenine which, when administered intraperitoneally (i.p.) antagonized (in descending order of potency) the seizures induced by intracerebroventricular (i.c.v.) injections of l-kynurenine sulfate in SHR and C57BL/6 mice. Picolinic and anthranilic acids were also effective after oral administration. Picolinic acid completely prevented seizures. Kynurenic acid, when injected i.c.v. prior to l-kynurenine sulfate, appeared to be more effective than after i.p. administration while picolinic and anthranilic acids were less effective. This suggest that the antikynurenine effect of metabolites of kynurenine (kynurenines) is related to different brain structures, i.e. kynurenic acid predominantly affects structures adjacent to ventricles (e.g. hippocampus, caudate nucleus) while picolinic and anthranilic acids act on other brain structures or the periphery. Xanthurenic, kynurenic and picolinic acids merely prolonged the latency of seizures induced by i.c.v. quinolinic acid (another metabolite of kynurenine) or by subcutaneous strychnine sulfate and i.p. pentylenetetrazole, and did not modify seizures induced by i.p. caffeine and thiosemicarbazide. This selective antagonism of the tested kynurenines against kynurenine might be an important anticonvulsant factor in kynurenine-dependent seizures. It is suggested that increased excretion of xanthurenic, kynurenic and picolinic acids in patients with convulsive states may be manifestations of compensatory processes.

Chloramphenicol a potent inhibitor of bacterial and some mammalian cell protein synthesis, was administered i.p. to a group of mice for 6 consecutive days. Another group of animals was treated similarly with thiamphenicol and a third group served as control. The effects of the two antibiotics on the activity of some liver enzymes; the two pyridoxal 5-phosphate dependent enzymes, kynurenine hydrolase and kynurenine amino-transferase; pyridoxal phosphokinase; beta-glucuronidase and acid ribonuclease were determined. Chloramphenicol and thiamphenicol decreased significantly the activities of kynurenine hydrolase, beta-glucuronidase and acid ribonuclease and both drugs increased the activity of pyridoxal phosphokinase significantly. Their effect on kynurenine amino-transferase was different, chloramphenicol decreased while thiamphenicol increased the enzyme activity. Results are discussed and possible explanations suggested.

Derivatives of GABA which are ineffective against seizures produced by typical convulsants (strychnine, pentylenetetrazole, etc.), phenibut and sodium hydroxybutyrate in doses of 200 mg/kg and higher possess marked anticonvulsant action against seizures induced by intraventricular injection of L-kynurenine and quinolinic acid in mice. Phepiron, a cyclic form of phenibut, had dissimilar action on the effects of L-kynurenine and a quinolinic acid, enhancing that produced by the former agent and diminishing tonic extension and lethality after the latter one. GABA and piracetam appeared to be ineffective. Efficacy of GABA derivatives against seizures produced by L-kynurenine and quinolinic acid suggests that the system of GABA plays a significant role in the genesis of seizures produced by kynurenines.

Serotonin and 5-hydroxykynurenamine caused dose-related contractions in the spiral strips of dog basilar arteries. The potency of 5-hydroxykynurenamine was approximately 1/100 that of serotonin, the former frequently causing a transient relaxation preceding the contraction. The contractile responses to 5-hydroxykynurenamine and serotonin were attenuated by methysergide. Treatment with 5-hydroxykynurenamine inhibited the response to serotonin, and this inhibitory effect was not completely reversed by removal of 5-hydroxykynurenamine from the bathing medium. The contractile response to K(+) was only slightly attenuated by high concentrations of 5-hydroxykynurenamine. It appears that 5-hydroxykynurenamine and serotonin share receptors in dog basilar arteries and that the effect of serotonin is specifically antagonized by 5-hydroxykynurenamine.

L-glycine (1-12.5 micrograms, intracerebroventricularly, i.c.v.) completely prevented seizures induced by i.c.v. administration of L-kynurenine, and practically did not modify those induced by another convulsant quinolinic acid, a metabolite of tryptophan, and by strychnine. L-Glycine administered intraperitoneally (i.p.) (1000 mg/kg) decreased lethality after K-kynurenine and quinolinic acid; at doses of 3000 and 4000 mg/kg which are sedative and hypothermic it prolonged the latency of strychnine and L-kynurenine seizures. The convulsant action of pentylenetetrazol was not modified. Kynurenine seizures are suggested to be related to the action of kynurenine on glycine receptors in the central nervous system.

In experiments on mice the existence of common mechanisms of action was revealed in picrotoxin and kynurenine and quinolinic acid. Picrotoxin selectively potentiated convulsions induced by kynurenine and quinolinic acid and the antagonists of these endogenous convulsants--picolinic, kynurenic and xanthurenic acids--selectively antagonized picrotoxin convulsions. GABA-ergic preparations, effective in kynurenine convulsions and the anticonvulsants diazepam and phenobarbital, were highly effective against picrotoxin-induced convulsions also. Puphemid and trimethin indicated for treating petit mal epilepsy, proved ineffective. The obtained data suggest the possibility of action of kynurenine and quinolinic acid via picrotoxin-sensitive subunits of the benzodiazepine-GABA-receptor-ionophore complex.