Hypothesis kynurenic and quinolinic acids: The main players of the kynurenine pathway and opponents in inflammatory disease.

I hypothesize that the intermediates of the kynurenine (Kyn) pathway (KP) of tryptophan (Trp) degradation kynurenic acid (KA) and quinolinic acid (QA) play opposite roles in inflammatory diseases, with KA being antiinflammatory and QA being immunosuppressant. Darlington et al. have demonstrated a decrease in the ratio of plasma 3-hydroxyanthranilic acid to anthranilic acid ([3-HAA]/[AA]) in many inflammatory conditions and proposed that this decrease either reflects inflammatory disease or is an antiinflammatory response. I argue in favour of the latter possibility and provide evidence that KA is responsible for the decrease in this ratio by increasing AA formation from Kyn through activation of the kynureninase reaction. Immunosuppression has been attributed to some Kyn metabolites tested at concentrations far greater than could occur in microenvironments. So far, only QA has been shown using immunohistochemistry to reach immunosuppressive levels. Future immune studies of the KP should focus on QA as the potentially main microenvironmentally measurable immunosuppressant and should include KA as an antiinflammatory metabolite.

Ischemic Stroke and Kynurenines: Medicinal Chemistry Aspects.

Ischemic stroke is one of the leading causes of mortality and permanent disability in developed countries. Stroke induces massive glutamate release, which in turn causes N-Methyl-D-aspartate (NMDA) receptor over-excitation and thus, calcium overload in neurons leading to cell death via apoptotic cascades. The kynurenine pathway is a complex enzymatic cascade of tryptophan catabolism, generating various neuroactive metabolites. One metabolite, kynurenic acid (KYNA), is a potent endogenous NMDA glutamate receptor antagonist, making it a possible therapeutic tool to decrease excitotoxicity and neuroinflammation. Recently, clinical investigations have shown that during the acute phase of ischemic stroke, kynurenine pathway is activated and peripheral levels of metabolites correlated with worse outcome. In this review, we set out to summarize the current literature on the connection of the kynurenine pathway and ischemic stroke and set a course for future investigations and potential drug development.

Silencing of quinolinic acid phosphoribosyl transferase (QPT) gene for enhanced production of scopolamine in hairy root culture of Duboisia leichhardtii.

Scopolamine is a pharmaceutically important tropane alkaloid which is used therapeutically in the form of an anesthetic and antispasmodic drug. The present study demonstrates enhanced scopolamine production from transgenic hairy root clones of Duboisia leichhardtii wherein the expression of quinolinate phosphoribosyl transferase (QPT) gene was silenced using the QPT-RNAi construct under the control of CaMV 35 S promoter. The RNAi hairy roots clones viz. P4, P7, P8, and P12 showed the enhanced synthesis of scopolamine with significant inhibition of nicotine biosynthesis. Optimization of culture duration in combination with methyl jasmonate elicitor in different concentrations (50 µM-200 µM) was carried out. Maximum synthesis of scopolamine had obtained from HR clones P7 (8.84 ± 0.117 mg/gm) on the 30th day of cultivation. Conspicuously, elicitation with wound-associated hormone methyl jasmonate enhanced the yield of scopolamine 2.2 fold (19.344 ± 0.275 mg/gm) compared to the culture lacking the elicitor. The transgenic hairy roots cultures established with RNAi mediated silencing of quinolinate phosphoribosyl transferase gene provides an alternative approach to increase the yield of scopolamine in fulfilling the demand of this secondary metabolite.

Mechanism of partial agonism at NMDA receptors for a conformationally restricted glutamate analog.

The NMDA ionotropic glutamate receptor is ubiquitous in mammalian central neurons. Because partial agonists bind to the same site as glutamate but induce less channel activation, these compounds provide an opportunity to probe the mechanism of activation of NMDA-type glutamate receptors. Molecular dynamics simulations and site-directed mutagenesis demonstrate that the partial agonist homoquinolinate interacts differently with binding pocket residues than glutamate. Homoquinolinate and glutamate induce distinct changes in the binding pocket, and the binding pocket exhibits significantly more motion with homoquinolinate bound than with glutamate. Patch-clamp recording demonstrates that single-channel activity induced by glutamate or by homoquinolinate has identical single-channel current amplitude and mean open-channel duration but that homoquinolinate slows activation of channel opening relative to glutamate. We hypothesize that agonist-induced conformational changes in the binding pocket control the efficacy of a subunit-specific activation step that precedes the concerted global change in the receptor-channel complex associated with ion channel opening.

Chemical modifications of the crystalline quinolinate phosphoribosyltransferase from hog liver.

Amino acid analysis and chemical modification of the crystalline quinolinate phosphoribosyltransferase (EC 2.4.2.19) from hog liver were performed. The enzyme contained 29 residues of half cystine per mol. The enzyme activity was strongly inhibited by sulfhydryl reagents. The number of reactive (exposed) sulfhydryl group was determined to be 10.2 and total sulfhydryl group was to be 25.2 per mol by using 5,5'-dithiobis(2-nitrobenzoic acid). The enzyme activity was also inhibited by lysine residue-, histidine residue-, and arginine residue-modifying reagents. These results and the effect of preincubation with the substrates on chemical modifications suggest that the lysine residue, histidine residue and sulfhydryl group may be closely related to the binding site of quinolinic acid.

Virus isolation and quinolinic acid in primary and chronic simian immunodeficiency virus infection.

OBJECTIVE: In this 2.5-year study of simian immunodeficiency virus (SIVsm) infection in rhesus monkeys, quinolinic acid (QUIN) levels and virus isolation determinations were made in serial cerebrospinal fluid (CSF) and blood samples to evaluate the relationship between these parameters over the course of infection. METHODS: Eight rhesus monkeys were inoculated in the saphenous vein with SIVsm. Four animals were maintained as uninoculated controls. CSF and blood samples were obtained every 1-4 weeks over the course of study. SIV isolation was determined in H9 cells for the CSF and in primary rhesus lymphocyte co-cultures for peripheral blood mononuclear cells (PBMC). QUIN was quantitated in CSF and serum by electron-capture negative chemical ionization gas chromatography mass spectrometry. RESULTS: All SIV-inoculated animals became CSF and PBMC isolation-positive by 1-3 weeks post-inoculation. Control animals remained SIV-negative. One SIV-positive animal was humanely euthanized at 2 weeks post-inoculation. The three SIV-inoculated animals that were CSF isolation-negative after the fifth week post-inoculation maintained CSF QUIN values < 100 nM, remained CSF and PBMC isolation-negative, and clinically healthy in the chronic course of disease. In contrast, the four SIV-inoculated animals that were CSF isolation-positive 6-8 weeks post-inoculation had CSF QUIN levels as high as 153-565 nM during the second month post-inoculation and remained CSF virus isolation-negative, persistently PBMC isolation-positive, and experienced clinical symptoms of SIV in the chronic course of disease. Three of these four animals have succumbed to SIV infection. DISCUSSION: Initial QUIN responses and viral isolation status in the first month post-inoculation were consistent among SIV-inoculated animals with CSF and serum QUIN values significantly higher than those of controls. A divergence within the SIV-inoculated group of animals became apparent within the second month of primary SIV infection and was maintained throughout the course of infection. Persistent PBMC viral isolation and marked elevations of QUIN were linked to symptomatic disease and a poor prognosis for survival. Predominantly negative PBMC viral isolation and slight, but significant, elevations of QUIN were linked to asymptomatic disease with a favorable prognosis for survival.

Delayed increases in regional brain quinolinic acid follow transient ischemia in the gerbil.

Excessive activity or release of excitatory amino acids has been implicated in the neuronal injury that follows transient cerebral ischemia. To investigate the metabolism of the endogenous excitotoxin, quinolinic acid, and its potential for mediating cell loss following ischemia, the concentrations of quinolinic acid, L-tryptophan, 5-hydroxytryptamine, and 5-hydroxyindoleacetic acid were quantified in gerbil brain regions at different times after 5 or 15 min of ischemia induced by bilateral carotid artery occlusion. Significant elevation of brain tryptophan levels, accompanied by increased 5-hydroxyindoleacetic acid concentrations, occurred during the first several hours of recirculation, but regional brain quinolinic acid concentrations were found either to decrease or remain unchanged during the first 24 h after the ischemic insult. However, significant increases in quinolinic acid concentrations occurred in striatum and hippocampus at 2 days of recirculation after 5 min of ischemia. After a further 4 and 7 days, strikingly large increases in quinolinic acid concentrations were observed in all regions examined, with the highest levels observed in the hippocampus and striatum, regions that also show the most severe ischemic injury. These delayed increases in brain quinolinic acid concentrations are suggested to reflect the presence of activated macrophages, reactive astrocytes, and/or microglia in vulnerable regions during and subsequent to ischemic injury. While the results do not support a role for increased quinolinic acid concentrations in early excitotoxic neuronal damage, the role of the delayed increases in brain quinolinic acid in the progression of postischemic injury and its relevance to postischemic brain function remain to be established.

Quinolinate phosphoribosyl transferase is not the oxygen-sensitive site of nicotinamide adenine dinucleotide biosynthesis.

Quinolinate phosphoribosyl transferase (QPT) activity was not affected when Escherichia coli were treated with hyperbaric oxygen. This result is not in accord with a previous report (Biochem. Biophys. Res. Comm. 91:982-990; 1979) in which the enzyme was shown to be rapidly inactivated in E. Coli exposed to 4.2 atmospheres of oxygen. Our data rule out QPT as a site of oxygen toxicity and suggest other mechanisms for the inhibitory effects of the hyperbaric oxygen on pyridine nucleotide biosynthesis.

Neuroactive metabolites of L-tryptophan, serotonin and quinolinic acid, in striatal extracellular fluid. Effect of tryptophan loading.

Extracellular fluid levels of the neurotoxin quinolinic acid in the corpus striatum of rats, measured by in vivo microdialysis, were increased in a dose-dependent manner following the intraperitoneal administration of tryptophan. The lowest dose of tryptophan (12.5 mg/kg), equivalent to about 5% of the normal daily intake, increased peak quinolinic acid levels nearly 3-fold. At higher doses of tryptophan (up to 250 mg/kg), concentrations of quinolinic acid increased over 200-fold and exceeded potentially neurotoxic levels (10 microM). In contrast, the increase in extracellular serotonin following even the highest tryptophan dose was small (less than 2-fold). These data indicate that quinolinic acid is present in the extracellular fluid where it may function as a neuromodulator and that it is very responsive to physiological changes in precursor availability.

Differential complementary localization of metabolic enzymes for quinolinic acid in olfactory bulb astrocytes.

The cellular localizations of the synthetic [3-hydroxyanthranilic acid oxygenase (3HAO)] and degradative [quinolinic acid phosphoribosyltransferase (QPRT)] enzymes of the endogenous excitotoxin quinolinic acid were studied in the adult rat main olfactory bulb by immunohistochemical techniques. 3HAO and QPRT were expressed only in astrocytes. The two enzymes were differentially expressed by astrocytes in a complementary pattern: 3HAO staining was strongest at the glomerular-external plexiform layer junction; QPRT staining was strongest at the glomerular-olfactory nerve layer junction. The complementary distributions of these metabolic enzymes suggests that there could be a gradient of quinolinic acid across the glomerular layer of the main olfactory bulb. Such a gradient could function to restrict the ingrowth of new olfactory axons to the glomeruli and/or to stabilize the formation of new synapses.

Neuroactive kynurenines in Lyme borreliosis.

Although neurologic dysfunction occurs frequently in patients with Lyme borreliosis, it is rarely possible to demonstrate the causative organism within the neuraxis. This discordance could arise if neurologic symptoms were actually due to soluble neuromodulators produced in response to infection. Since immune stimulation is associated with the production of quinolinic acid (QUIN), an excitotoxin and N-methyl-D-aspartate (NMDA) agonist, we measured levels of CSF and serum QUIN, and lymphokines. Samples were obtained from 16 patients with CNS Borrelia burgdorferi infection, eight patients with Lyme encephalopathy (confusion without intra-CNS inflammation), and 45 controls. CSF QUIN was substantially elevated in patients with CNS Lyme and correlated strongly with CSF leukocytosis. In patients with encephalopathy, serum QUIN was elevated with corresponding increments in CSF QUIN. Lymphokine concentrations were not consistently elevated. We conclude that CSF QUIN is significantly elevated in B burgdorferi infection--dramatically in patients with CNS inflammation, less in encephalopathy. The presence of this known agonist of NMDA synaptic function--a receptor involved in learning, memory, and synaptic plasticity--may contribute to the neurologic and cognitive deficits seen in many Lyme disease patients.

Localization of quinolinic acid metabolizing enzymes in the rat brain. Immunohistochemical studies using antibodies to 3-hydroxyanthranilic acid oxygenase and quinolinic acid phosphoribosyltransferase.

Specific antibodies raised in rabbits against 3-hydroxyanthranilic acid oxygenase (EC 1.13.11.6) and quinolinic acid phosphoribosyltransferase (EC 1.13.11.6) and quinolinic acid phosphoribosyltransferase (EC 2.4.2.19) were used in immunohistochemical studies to map the cellular localization of the quinolinic acid metabolizing enzymes in the adult male rat brain. 3-Hydroxyanthranilic acid oxygenase immunoreactivity was found to be present in glial cells of presumed astroglial identity, as judged by co-localization with glial fibrillary acidic protein. 3-Hydroxyanthranilic acid oxygenase-immunoreactive glial cells were present in all brain regions and within major fiber tracts. The density of 3-hydroxyanthranilic acid oxygenase-immunoreactive glial cells as well as the intensity of staining of these cells differed among brain regions. In general, telencephalic acid diencephalic areas harbored a larger number of 3-hydroxyanthranilic acid oxygenase-positive cells than did mesencephalic regions. In the former regions the caudate nucleus, septum, nucleus accumbens, neocortex and hippocampus were particularly enriched in 3-hydroxyanthranilic acid oxygenase-immunoreactive cells. In the thalamus, regional differences were noted with regard to the intensity of staining among glial cells with high densities of 3-hydroxyanthranilic acid oxygenase cells in the anteroventral, reticular and ventromedial nuclei. In the inferior and superior colliculi, numerous 3-hydroxyanthranilic acid oxygenase-positive glial cells were found in all layers. In the hypothalamus, 3-hydroxyanthranilic acid oxygenase-immunoreactive glial cells were encountered in the zona incerta, the lateral hypothalamic area, the caudal preoptic region and in the dorsomedial nucleus. In the mesencephalon, the substantia nigra contained numerous, moderately stained cells. At caudal levels of the brain-stem, a relatively large number of cells was detected in the nucleus of the solitary tract, the pontine nucleus and in the fascial nerve nucleus, while other nuclei, such as the reticular formation and the area postrema were relatively poor in 3-hydroxyanthranilic acid oxygenase-immunoreactive cells. In addition to staining of glial cells, neuronal cell bodies containing 3-hydroxyanthranilic acid oxygenase immunoreactivity were detected in the main and in the accessory olfactory bulb, as well as in the ventromedial nucleus of the hypothalamus. Quinolinic acid phosphoribosyltransferase immunoreactivity was observed within glial cells and in association with neuronal cell bodies. Some, but not all, quinolinic acid phosphoribosyltransferase positive glial cells contained glial fibrillary acidic protein (Köhl

4-halo-3-hydroxyanthranilic acids: potent competitive inhibitors of 3-hydroxy-anthranilic acid oxygenase in vitro.

The mechanism of action of three potent inhibitors of 3-hydroxyanthranilic acid oxygenase (3HAO), the enzyme responsible for the production of the endogenous excitotoxin quinolinic acid, was examined in vitro. Using either liver homogenate or purified 3HAO, and following the rapid synthesis of the immediate enzymatic product alpha-amino-beta-carboxymuconic acid omega-semialdehyde spectrophotometrically, 4-halogenated (F, Cl, Br) 3-hydroxyanthranilic acids were found to inhibit enzymatic activity in a reversible fashion. Because of the very tight binding of the drugs to 3HAO, reversibility was detected only after warming the protein-inhibitor complexes at 37 degrees. Further studies showed that enzyme inhibition was competitive in nature (apparent Ki values: 190, 6 and 4 nM for the F-, Cl- and Br-compounds, respectively), and suggested that the drugs are metabolized by the enzyme. Specific, reversible, and tightly binding 3HAO inhibitors can be expected to become valuable tools for the study of quinolinate neurobiology. The drugs could also be of interest for the diagnostics and therapeutics of brain diseases which have been speculatively linked to a pathological overabundance of quinolinic acid.

The effect of tryptophan administration on fatty acid synthesis in the livers of rats under various nutritional conditions.

1. Tryptophan was administered to rats under various nutritional conditions: fasted for 24 hr, fasted and refed with glucose or corn-oil, fasted and administered glycerol intramuscularly, and nonfasted. 2. The changes in the contents of glycolytic intermediates in the livers indicated that the phosphoenolpyruvate carboxykinase [EC 4.1.1.32] reaction is inhibited by tryptophan administration in all groups of rats. The inversely related changes in the contents of malate and phosphoenolpyruvate were associated with the accumulation of quinolinate in the livers. The content of quinolinate which exhibited the half-maximal effect on the contents of both metabolites was 0.1-0.2 mumole per g liver. 3. The rate of incorporation of 3H from 3H2O into the total hepatic fatty acids was increased about 2-fold by the administration of this amino acid to the fasted rats. The enhancement of the rate was closely related to the increase in the citrate content. The hyperlipogenesis was also related to the decrease of acetyl-CoA and the increase of malonyl-CoA. The content of long-chain acyl-CoA was not affected. These effects of tryptophan administration on the hepatic fatty acid metabolism were found in all groups of rats. The liver content of glycerol 3-phosphate was decreased by tryptophan administration was markedly increased by glycerol injection. The injection of glycerol into the control and the tryptophan-treated rats produced a marked increase of glycerol 3-phosphate but did not affect the rate of fatty acid synthesis in the livers of either group. 4. It may be concluded that, in the livers of rats under various nutritional conditions, the short-term control of fatty acid synthesis by tryptophan administration is most likely due to the activation of acetyl-coenzyme A carboxylase [EC 6.4.1.2] by citrate.

Quinolinic acid concentrations in striatal extracellular fluid reach potentially neurotoxic levels following systemic L-tryptophan loading.

Following a systemic tryptophan load, striatal extracellular fluid levels of quinolinic acid in the rat were quantified using intracerebral microdialysis. After an intraperitoneal dose of L-tryptophan (250 mg/kg), quinolinic acid levels in striatal perfusates increased by 230 fold. Peak concentrations of quinolinic acid exceeded 10(-5)M, a concentration previously shown to be neurotoxic in vitro. These results indicate that quinolinic acid is markedly precursor responsive and that its concentration in striatal extracellular fluid may reach neurotoxic levels following an acute tryptophan load.

Increased quinolinic acid metabolism following neuronal degeneration in the rat hippocampus.

The excitotoxic brain metabolite quinolinic acid (QUIN) has been hypothetically linked to the pathogenesis of seizure disorders and other neurodegenerative events affecting the hippocampal formation. Its biosynthetic enzyme, 3-hydroxyanthranilic acid oxygenase (3-HAO) and its catabolic enzyme, quinolinic acid phosphoribosyltransferase (QPRT), can be used as markers for the cellular localization of the brain's QUIN system. Measured between 2 days and 2 months following intrahippocampal ibotenic acid injections, the activities of both enzymes increased at the lesion site due to the synthesis of new enzyme protein. The time course of the increase in 3-HAO activity coincided with that of the known astrocytic proliferation following excitotoxic insults. It is less obvious if the elevation in QPRT activity, too, is related to an increase in the number of reactive glial cells. No changes in the activity of hippocampal 3-HAO or QPRT were noted 7 or 60 days after cholinergic deafferentation by fornix-fimbria transection nor were any changes observed in the contralateral hippocampus at any time-point following the ibotenate lesion. These data raise the possibility that a feed-forward mechanism, resulting in ever increasing amounts of QUIN in the brain, may be operant in situations of progressive hippocampal nerve cell loss.

Systemic endotoxin increases L-tryptophan, 5-hydroxyindoleacetic acid, 3-hydroxykynurenine and quinolinic acid content of mouse cerebral cortex.

Systemic infections and injection of endotoxin are known to increase L-tryptophan release from skeletal muscle and increase systemic L-tryptophan catabolism through the kynurenine pathway. To investigate the effects of systemically administered endotoxin on brain L-tryptophan metabolites. C57BL6/6NCR mice were given an intraperitoneal injection of 10 micrograms of lipopolysaccharide from Salmonella abortus equii and samples of serum and cerebral cortex collected. After 9 h, serum L-tryptophan concentration was decreased by 51%. At 9 h and 24 h, increases in L-tryptophan metabolites in cerebral cortex were: L-tryptophan, 42% and 39%; 5-hydroxyindoleacetic acid, 38% and 67%; 3-hydroxykynurenine, 235% and 381%; and quinolinic acid, 76% and 306%. Cortical quinolinic acid concentration was still elevated at 48 h (88%) and 72 h (79%) after lipopolysaccharide. No significant changes in cortical serotonin concentrations were found at the time points examined. When L-tryptophan (0.37 mmol/kg) was administered systemically to either normal or lipopolysaccharide-treated mice, increases in cortical L-tryptophan, serotonin, 5-hydroxyindoleacetic acid and 3-hydroxykynurenine concentrations were largest in mice treated with both lipopolysaccharide and L-tryptophan. These results suggest that disturbances in L-tryptophan metabolism that follow systemic endotoxin administration extend to the central nervous system. The consequences of these changes in L-tryptophan metabolites remain to be determined.

Chronic effects of gamma-interferon on quinolinic acid and indoleamine-2,3-dioxygenase in brain of C57BL6 mice.

Chronic infections are associated with increased concentrations of the neuroactive kynurenine pathway metabolite, quinolinic acid (QUIN), in blood and cerebrospinal fluid. In the present study, repeated injections of gamma-interferon (5000 IU, every 3 days for 39 days) to C57BL6 mice were associated with persistent activation of indoleamine-2,3-dioxygenase (IDO), the first enzyme of the kynurenine pathway, in lung and brain, sustained increases in brain QUIN concentration and increases in plasma L-kynurenine and QUIN levels. Mice chronically treated with gamma-interferon offer an animal model to investigate the effects of sustained immune stimulation on kynurenine pathway metabolism.

Basal ganglia lesions in the rat: effects on quinolinic acid metabolism.

Experimental basal ganglia lesions were produced in order to examine the effect of neuronal loss on quinolinic acid (QUIN) metabolism. The latter was investigated by measuring the activities of QUIN's biosynthetic enzyme, 3-hydroxyanthranilic acid oxygenase (3-HAO) and its degradative enzyme, quinolinic acid phosphoribosyltransferase (QPRT). Striatal ibotenic acid lesions caused a steady increase in striatal QPRT activity, reaching 280% of control levels 21 days after the lesion. In the same tissue, 3-HAO activity, too, was elevated. It rose to 436% of control after 7 days and to a lesser degree (+309%) after 3 weeks. Immunotitration experiments using anti-rat 3-HAO antibodies and kinetic analysis of lesioned and control striata showed that the increase in 3-HAO was due to de novo production of enzyme protein. The large increases in striatal enzyme activities after 7 days were accompanied by smaller increases in both 3-HAO and QPRT activities in the ipsilateral substantia nigra. Physical destruction of corticostriatal glutamatergic fibers resulted in increases in striatal 3-HAO (+216%) and QPRT (+243%) activities after one week. No changes in nigral or striatal QUIN metabolism were recorded 7 days after an intranigral injection of 6-hydroxydopamine. These data confirm the notion of a largely glial localization of the QUIN system in the basal ganglia, and correlate well with recent observations in brain tissue from Huntington's disease victims.

No changes in central quinolinic acid levels in Alzheimer's disease.

Levels of the endogenous excitotoxin quinolinic acid were measured in brain and lumbar spinal fluid from Alzheimer patients and age-matched controls. Values in post mortem brain tissue, unlike those in spinal fluid, showed considerable variability among subjects. In the control group, frontal cortex and caudate nucleus had higher concentrations of quinolinic acid compared to other regions studied. No significant differences were found between Alzheimer brains and controls in any of the regions analyzed. Studies in lumbar spinal fluid showed no gradient for quinolinic acid along the neuraxis, a trend for increasing levels with normal aging, and no difference between Alzheimer patients and age-matched control subjects. The lack of increased central quinolinic acid levels in Alzheimer's disease does not necessarily negate the possibility of excitotoxins contributing to cell death in this disorder.