Rat 3-Hydroxyanthranilic Acid Oxygenase: Purification from the Liver and Immunocytochemical Localization in the Brain

3-Hydroxyanthranilic acid oxygenase (3HAO; EC 1.13.11.6), the biosynthetic enzyme of the endogenous excitotoxin quinolinic acid, was purified to homogeneity from rat liver and partially purified from rat brain. The pure enzyme is a single subunit protein with a molecular weight of 37-38,000. Kinetic analyses of both pure liver and partially purified brain 3HAO revealed an identical Km of 3 microM for the substrate 3-hydroxyanthranilic acid. Evidence for the identity of liver and brain 3HAO was further provided by physicochemical (electrophoretic behavior, heat sensitivity) and biochemical (pH dependency, activation by Fe2+) means. Antibodies were produced against the pure liver enzyme and the identity of liver and brain 3HAO substantiated immunologically in immunotitration and Ouchterlony double-diffusion experiments. Immunohistochemical studies using purified anti-rat 3HAO antibodies were performed on tissue sections of perfused brains and demonstrated a preferential staining of astroglial cells. Notably, the cellular localization of 3HAO in the brain appears to be in part distinct from that of quinolinic acid phosphoribosyltransferase, the catabolic enzyme of quinolinic acid. Pure rat 3HAO and its antibodies can be expected to constitute useful tools for the further elucidation of the brain's quinolinic acid system.     

Quinolinic Acid Phosphoribosyltransferase in Rat Brain

Because of the possible participation of quinolinic acid in brain function and/or dysfunction, the characteristics of its catabolic enzyme, quinolinic acid phosphoribosyltransferase (QPRTase; EC 2.4.2.19), were examined in rat brain tissue. For this purpose, a sensitive radiochemical assay method, based on the conversion of quinolinic acid to nicotinic acid mononucleotide (NAMN), was developed. For brain QPRTase, the Mg2+ dependency, substrate specificity, and optimal assay conditions were virtually identical to those of the liver enzyme. Kinetic analyses of brain QPRTase revealed a Km of 3.17 +/- 0.30 microM for quinolinic acid and Km = 65.13 +/- 13.74 microM for the cosubstrate phosphoribosylpyrophosphate. The respective Vmax values were: 0.91 +/- 0.08 pmol NAMN/h/mg tissue for quinolinic acid and 11.65 +/- 1.55 fmol NAMN/h/mg tissue for phosphoribosylpyrophosphate. All kinetic parameters measured for the brain enzyme were significantly different from those determined for liver QPRTase, indicating structural differences or distinct regulatory processes for the brain and liver enzymes. Phthalic acid was a potent competitive inhibitor of brain QPRTase. Examination of the regional distribution of QPRTase in the rat CNS and retina indicated a greater than 20-fold difference between the area displaying the highest activity (olfactory bulb) and those of only moderate activity (frontal cortex, striatum, retina, hippo-campus). Enzyme activity was present at the earliest age tested, 2 days, and tended to increase in older animals. Brain QPRTase activity was preferentially located in the nerve-ending (synaptosomal) fraction. Enzyme activity was stable over extensive periods of storage at -80 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Presence of 3-Hydroxyanthranilic Acid in Rat Tissues and Evidence for Its Production from Anthranilic Acid in the Brain

As assessed by HPLC with electrochemical detection, 3-hydroxyanthranilic acid (3-HANA) was found to be present in the rat brain and peripheral organs. The highest concentrations were measured in the kidney (86 fmol/mg of tissue) and spleen (56 fmol/mg of tissue), whereas the adrenal gland, liver, heart, and several forebrain areas (hippocampus, striatum, parietal cortex, thalamus, amygdala/pyriform cortex, and frontal cortex) contained less 3-HANA (between 15 and 22 fmol/mg of tissue). Slightly lower concentrations of 3-HANA were found in the brainstem and the cerebellum. The metabolic disposition of 3-HANA was examined in tissue slices which were incubated in Krebs-Ringer buffer at 37 degrees C in vitro. Incubation for up to 2 h did not affect 3-HANA concentration in brain tissue. However, inhibition of 3-HANA degradation by the specific 3-hydroxyanthranilic acid oxygenase blocker 4-chloro-3-hydroxyanthranilic acid (4-Cl-3-HANA; 10 microM) resulted in a rapid (within 2.5 min) doubling of 3-HANA levels in slices from cerebral cortex. No further increases were observed after incubations of up to 120 min. Exposure of cortical slices to 3-HANA's putative bioprecursors, 3-hydroxykynurenine (3-HK) and anthranilic acid (ANA), in the absence of 4-Cl-3-HANA resulted in rapid, transient increases in 3-HANA production. Maximal 3-HANA synthesis from ANA exceeded the maximal effect of 3-HK by approximately 11-fold.2+ In the presence of 4-Cl-3-HANA, 1 mM ANA produced 9.0 +/- 0.3 and 89.0 +/- 9.3 (5 min) or 51.6 +/- 7.9 and 187.5 +/- 11.2 (120 min) fmol of newly synthesized 3-HANA/mg of brain tissue, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of mitochondria and o-methoxybenzoylalanine on 3-hydroxyanthranilic acid dioxygenase activity and quinolinic acid synthesis

The use of o-methoxybenzoylalanine, a selective kynureninase inhibitor, has been proposed with the aim of reducing brain synthesis of quinolinic acid, an excitotoxic tryptophan metabolite. In liver homogenates, however, this compound caused unexpected accumulation of 3-hydroxyanthranilic acid, the product of kynureninase activity and the precursor of quinolinic acid. To explain this observation, we investigated the interaction(s) of o-methoxybenzoylalanine with 3-hydroxyanthranilic acid dioxygenase, the enzyme responsible for quinolinic acid formation. When the purified enzyme or partially purified cytosol preparations were used, o-methoxybenzoylalanine did not affect 3-hydroxyanthranilic acid dioxygenase activity. However, a significant reduction of this enzymatic activity did occur when o-methoxybenzoylalanine was tested in the presence of mitochondria. It is interesting that addition of purified mitochondria to 3-hydroxyanthranilic acid dioxygenase preparations reduced the enzymatic activity and the synthesis of quinolinic acid. In vivo, administration of o-methoxybenzoylalanine significantly reduced quinolinic acid synthesis and content in both blood and brain of mice. Our results suggest that mitochondrial protein(s) interact(s) with soluble 3-hydroxyanthranilic acid dioxygenase and cause(s) modifications in the enzyme resulting in a decrease in its activity. These modifications also allow the enzyme to interact with o-methoxybenzoylalanine, thus leading to a further reduction in quinolinic acid synthesis.     

Kynurenine Disposition in Blood and Brain of Mice: Effects of Selective Inhibitors of Kynurenine Hydroxylase and of Kynureninase

Abstract: To study the regulation of the synthesis of quinolinic and kynurenic acids in vivo, we evaluated (a) the metabolism of administered kynurenine by measuring the content of its main metabolites 3-hydroxykynurenine, anthranilic acid, and 3-hydroxyanthranilic acid in blood and brain of mice; (b) the effects of ( m -nitrobenzoyl)alanine, a selective inhibitor of kynurenine hydroxylase and of ( o -methoxybenzoyl)alanine, a selective inhibitor of kynureninase, on this metabolism; and (c) the effects of ( o -methoxybenzoyl)alanine on liver kynureninase and 3-hydroxykynureninase activity. The conclusions drawn from these experiments are (a) the disposition of administered kynurenine preferentially occurs through hydroxylation in brain and through hydrolysis in peripheral tissues; (b) ( m -nitrobenzoyl)alanine, the inhibitor of kynurenine hydroxylase, causes the expected changes in brain kynurenine metabolism, such as a decrease of 3-hydroxykynurenine, and an increase of kynurenic acid; and (c) ( o -methoxybenzoyl)alanine, the kynureninase inhibitor, increases brain concentration of the cytotoxic compound 3-hydroxykynurenine, and unexpectedly does not reduce brain concentration of 3-hydroxyanthranilic acid, the direct precursor of quinolinic acid. Taken together, the experiments suggest that the systemic administration of a kynurenine hydroxylase inhibitor is a rational approach to increase the brain content of kynurenate and to decrease that of cytotoxic kynurenine metabolites, such as 3-hydroxykynurenine and quinolinic acid.     

Angiotensin II Regulation of Intracellular Calcium in Astroglia Cultured from Rat Hypothalamus and Brainstem

Abstract: Several pieces of evidence suggest a major role for brain macrophages in the overproduction of neuroactive kynurenines, including quinolinic acid, in brain inflammatory conditions. In the present work, the regulation of kynurenine pathway enzymes by interferon-γ (IFN-γ) was studied in immortalized murine macrophages (MT2) and microglial (N11) cells. In both cell lines, IFN-γ induced the expression of indoleamine 2,3-dioxygenase (IDO) activity. Whereas tumor necrosis factor-α did not affect enzyme induction by IFN-γ, lipopolysaccharide modulated IDO activity differently in the two IFN-γ-activated cell lines, causing a reduction of IDO expression in MT2 cells and an enhancement of IDO activity in N11 cells. Kynurenine aminotransferase, kynurenine 3-hydroxylase, and 3-hydroxyanthranilic acid dioxygenase appeared to be constitutively expressed in both cell lines. Kynurenine 3-hydroxylase activity was stimulated by IFN-γ. It was notable that basal kynureninase activity was much higher in MT2 macrophages than in N11 microglial cells. In addition, IFN-γ markedly stimulated the activity of this enzyme only in MT2 cells. IFN-γ-treated MT2 cells, but not N11 cells, were able to produce detectable amounts of radiolabeled 3-hydroxyanthranilic acid quinolinic acids from l -[5-³H]tryptophan. These results support the notion that activated invading macrophages may constitute one of the major sources of cerebral quinolinic acid during inflammation.     

On the Production and Disposition of Quinolinic Acid in Rat Brain and Liver Slices

Abstract: The de novo production and subsequent disposition of the endogenous excitotoxin quinolinic acid (QUIN) was investigated in vitro in tissue slices from rat brain and liver. Incubation of tissue with QUIN's immediate bioprecursor 3-hydroxyanthranilic acid (3-HANA) in oxygenated Krebs-Ringer buffer yielded measurable amounts of QUIN both in the tissue and in the incubation medium. Saturation was reached between 16 and 64 μM 3-HANA (166 pmol of QUIN formed per milligram of protein after a 60-min incubation with 64 μM 3-HANA). In the brain, more QUIN was recovered from the tissue than from the incubation medium at all time points examined (5 min to 4 h). In contrast, the tissue-to-medium ratio for QUIN in parallel experiments with hepatic slices was ? 1. The disposition of newly synthesized QUIN was further elaborated in tissue slices that had been preincubated for 60 min with 64 μM 3-HANA. Subsequent incubation of brain tissue in fresh buffer revealed a steady but relatively slow efflux of QUIN from the cellular compartment, with >30% remaining in the tissue after a 90-min incubation. Analogous experiments with liver slices showed that >93% of newly synthesized QUIN had entered the extracellular compartment within 30 min. Striatal and nigral slices obtained 7 days after an intrastriatal ibotenic acid injection showed severalfold increases in QUIN production compared with control tissues, in all likelihood due to astrogliosis and associated large increases in 3-hydroxyanthranilic acid oxygenase activity. In addition, the apparent tissue-to-medium ratio was markedly reduced in striatal slices from lesioned animals. Taken together, these data indicate that both brain and liver cells have a rather limited capacity to retain QUIN, and that 3-hydroxyanthranilic acid oxygenase activity is a critical determinant controlling extracellular QUIN concentrations in both organs. Changes in the activity of QUIN's biosynthetic enzyme in the brain can therefore be expected to influence the possible function of QUIN as an endogenous agonist at the N-methyl-D-aspartate receptor in health and disease.     

Effects of oxygen on 3-hydroxyanthranilate oxidase of the kynurenine pathway

Iron containing 3-Hydroxyanthranilate oxidase (3HAO) converts 3-hydroxyanthranilate (3HAA) and dioxygen into a precursor which spontaneously converts to quinolinic acid (QA). 3HAO participates in de novo biosynthesis of NAD in mammalian kidney and liver, and it is present in low concentrations in brain where its function is controversial. However, QA increases in spinal fluid and is associated with convulsions in AIDS dementia, Huntington’s disease, and CNS inflammation. QA is a known N-methyl, D-aspartate receptor agonist and excitotoxin that causes convulsions when injected into the brain. Hyperbaric oxygen (HBO) also causes convulsions and we investigated the interrelationships among the stimulating and toxic effects of oxygen and the role of iron in vitro using rat liver enzyme which is reported to be identical to brain enzyme and is more abundant. 3HAO requires dioxygen as a substrate but it was inactivated approximately 40% by 5.2 atm HBO in vitro in 15 min. The apparent K_m was 2.6 × 10⁻⁴ M for oxygen and 5 × 10⁻⁵ M for 3HAA, and these values did not change for enzyme that was half-inactivated by HBO oxygen. Thus, oxygen-inactivation appears to be all-or-none for individual enzyme molecules. Freshly prepared enzyme was activated about 3-fold by incubation with acidic iron. Iron-staining of 3HAO, separated by gel electrophoresis after partial purification by FPLC, showed that loss of iron and loss of enzyme activity during HBO exposure were correlated. The apparent oxygen K_m of 3HAO is far higher than the oxygen concentration in brain cells. Thus, 3HAO is capable of being stimulated initially in animals breathing HBO, and subsequently of being inactivated with potential significance for brain QA and convulsions.     

Effects of oxygen on kynurenine-3-monooxygenase activity

Kynurenine-3-monooxygenase (KM), the third enzyme in the kynurenine (KYN) pathway from tryptophan to quinolinic acid (QA), is a monooxygenase requiring oxygen, NADPH and FAD for the catalytic oxidation of L-kynurenine to 3-hydroxykynurenine and water. KM is innately low in the brain and similar in activity to indoleamine oxidase, the rate-limiting pathway enzyme. Accumulation in the CNS of QA, a known excitotoxin, is proposed to cause convulsions in several pathologies. Thus, we theorized that hyperbaric oxygen (HBO) induced convulsions arise from increased QA via oxygen K, effects on this pathway [Brown OR, Draczynska-Lusiak. Oxygen activation and inactivation of quinolinate-producing and iron-requiring 3-hydroxyanthranilic acid oxidase: a role in hyperbaric oxygen-induced convulsions? Redox Report 1995; 1: 383-385]. To complement prior studies on the effects of oxygen on pathway enzymes, in this paper we report the effects of oxygen on KM. Brain and liver KM enzyme are not known to be identical, and some systemically-produced KYN pathway intermediates can permeate the brain and might stimulate the brain pathway. Thus, KM from both brain and liver was assayed at various oxygen substrate concentrations to evaluate, in vitro, the potential effects of increases in oxygen, as would occur in mammals breathing therapeutic and convulsive HBO. In crude tissue extracts, KM was not activated during incubation in HBO up to 6 atm. The effects of oxygen as substrate on brain and liver KM activity was nearly identical: activity was nil at zero oxygen with an apparent oxygen Km of 20-22 microM. Maximum KM activity occurred at about 1000 microM oxygen and decreased slightly to plateau from 2000 to 8000 microM oxygen. This compares to approximately 30-40 microM oxygen typically reported for brain tissue of humans or rats breathing air, and an unknown but surely much lower value (perhaps below 1 microM) intracellularly at the site of KM. Thus HBO, as used therapeutically and at convulsive pressures, likely stimulates flux through the KM-catalyzed step of the KYN pathway in liver and in brain and could increase brain QA, by Km effects on brain KM, or via increased KM pathway intermediates produced systemically (in liver) and transported into the brain.     

3-Hydroxyanthranilic acid metabolism. IV. Spectrophotometric evidence for the formation of an intermediate

Evidence has been presented for the formation of an intermediate compound in the metabolism of 3-hydroxyanthranilic acid to quinolinic acid by 3-hydroxyanthranilase from rat liver preparations. The production of the intermediate was demonstrated by spectrophotometric analyses and quinolinic acid measurements of incubation mixtures in which small amounts of acetone powder extracts of rat liver were used as the enzyme source. The calculated extinction coefficient of the compound was more than double that of the substrate or of the final product, quinolinic acid.The intermediate was shown to be an oxidation product of 3-hydroxyanthranilate as indicated by Thunberg experiments. The data obtained indicate that the intermediate may be a quinone-type compound.     

Measurement of Rat Brain Kynurenine Aminotransferase at Physiological Kynurenine Concentrations

The production of the neuroinhibitory and neuroprotective metabolite kynurenic acid (KYNA) was investigated in rat brain by examining its biosynthetic enzyme, kynurenine aminotransferase (KAT). By using physiological (low micromolar) concentrations of the substrate L-kynurenine (KYN) and by determining the irreversible conversion of [3H]KYN to [3H]KYNA as a measure of KAT activity, a novel, simple, and sensitive assay was developed which permitted the detailed characterization of the enzyme. Only a single protein, which under routine assay conditions showed approximately equal activity with 2-oxoglutarate and pyruvate as the aminoacceptor, was found in rat brain. The enzyme was distributed heterogeneously between the nine brain regions studied, with the KAT-rich olfactory bulb displaying approximately five times higher activity than the cerebellum, the area with lowest KAT activity. In subcellular fractionation studies, the majority of KAT was recovered in mitochondria. In contrast to many known aminotransferases, partially purified KAT was shown to be highly substrate-specific. Thus, of the amino acids tested, only alpha-aminoadipate and tryptophan displayed moderate competition with KYN. Notably, 3-hydroxykynurenine, reportedly a very good substrate of KAT, competed rather poorly with KYN as well. Aminooxyacetic acid, a nonspecific transaminase inhibitor, blocked KAT activity with an apparent Ki of 5 microM. Kinetic analyses with partially purified rat brain KAT revealed a Km of 17 microM for KYN with 1 mM 2-oxoglutarate, but a much higher Km (910 microM) with 1 mM pyruvate. Km values for 2-oxoglutarate and pyruvate were 150 and 160 microM, respectively. The cellular localization of KAT was examined in striatal homogenates obtained from rats 7 days after an intrastriatal injection of quinolinate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence that quinolinic acid severely impairs energy metabolism through activation of NMDA receptors in striatum from developing rats

In the present study we investigated the effect of intrastriatal administration of 150 nmol quinolinic acid to young rats on critical enzyme activities of energy production and transfer, as well as on 14CO2 production from [1-14C]acetate at distinct periods after quinolinic acid injection. We observed that quinolinic acid injection significantly inhibited complexes II (50%), III (46%) and II-III (35%), as well as creatine kinase (27%), but not the activities of complexes I and IV and citrate synthase in striatum prepared 12 h after treatment. In contrast, no alterations of these enzyme activities were observed 3 or 6 h after quinolinic acid administration. 14CO2 production from [1-14C]acetate was also significantly inhibited (27%) by quinolinic acid in rat striatum prepared 12 h after injection. However, no alterations of these activities were observed in striatum homogenates incubated in the presence of 100 microm quinolinic acid . Pretreatment with the NMDA receptor antagonist MK-801 and with creatine totally prevented all inhibitory effects elicited by quinolinic acid administration. In addition, alpha-tocopherol plus ascorbate and the nitric oxide synthase inhibitor l-NAME completely abolished the inhibitions provoked by quinolinic acid on creatine kinase and complex III. Furthermore, pyruvate pretreatment totally blocked the inhibitory effects of quinolinic acid injection on complex II activity and partially prevented quinolinic acid-induced creatine kinase inhibition. These observations strongly indicate that oxidative phosphorylation, the citric acid cycle and cellular energy transfer are compromised by high concentrations of quinolinic acid in the striatum of young rats and that these inhibitory effects were probably mediated by NMDA stimulation.     

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.     

Delta 5-3 beta-hydroxysteroid acyl transferase activity in the rat brain.

Pregnenolone and dehydroepiandrosterone accumulate in brain as sulfate and fatty acid esters and unconjugated steroids. The steroid fatty acid ester-synthesizing activity was investigated in rat brain microsomes. Endogenous fatty acids in the microsomal fraction were used for the esterification of steroids. The enzyme system had a pH optimum of 4.5 in acetate buffer with [3H]dehydroepiandrosterone as substrate. The apparent Km was 9.2 +/- 3.1 x 10(-5) M and Vmax was 18.6 +/- 3.4 nmol/h/mg protein (mean +/- SEM). The inhibition constants of pregnenolone and testosterone were 123 and 64 microM, respectively. Results were compatible with a competitive type of inhibition. A high level of synthetic activity was found in the brain of 1- to 3-week-old male rats, which rapidly decreased with aging. Saponification of purified [3H]pregnenolone esters yielded pregnenolone and a mixture of palmitate, oleate, linoleate, stearate, and myristate as the predominant fatty acids. Contrasting with the high rates of esterification of several radioactive delta 5-3 beta-hydroxysteroids or 17 beta-hydroxysteroids, no fatty acid esters of either cholesterol, epitestosterone (with a hydroxyl group at position C-17 alpha), or corticosterone (with hydroxyl groups at C-21 and C-11 beta) were formed in the same incubation conditions.     

Crystal structure of 3-hydroxyanthranilic acid 3,4-dioxygenase from Saccharomyces cerevisiae: a special subgroup of the type III extradiol dioxygenases

3-Hydroxyanthranilic acid 3,4-dioxygenase (3HAO) is a non-heme ferrous extradiol dioxygenase in the kynurenine pathway from tryptophan. It catalyzes the conversion of 3-hydroxyanthranilate (HAA) to quinolinic acid (QUIN), an endogenous neurotoxin, via the activation of N-methyl-D-aspartate (NMDA) receptors and the precursor of NAD(+) biosynthesis. The crystal structure of 3HAO from S. cerevisiae at 2.4 A resolution shows it to be a member of the functionally diverse cupin superfamily. The structure represents the first eukaryotic 3HAO to be resolved. The enzyme forms homodimers, with two nickel binding sites per molecule. One of the bound nickel atoms occupies the proposed ferrous-coordinated active site, which is located in a conserved double-strand beta-helix domain. Examination of the structure reveals the participation of a series of residues in catalysis different from other extradiol dioxygenases. Together with two iron-binding residues (His49 and Glu55), Asp120, Asn51, Glu111, and Arg114 form a hydrogen-bonding network; this hydrogen-bond network is key to the catalysis of 3HAO. Residues Arg101, Gln59, and the substrate-binding hydrophobic pocket are crucial for substrate specificity. Structure comparison with 3HAO from Ralstonia metallidurans reveals similarities at the active site and suggests the same catalytic mechanism in prokaryotic and eukaryotic 3HAO. Based on sequence comparison, we suggest that bicupin of human 3HAO is the first example of evolution from a monocupin dimer to bicupin monomer in the diverse cupin superfamilies. Based on the model of the substrate HAA at the active site of Y3HAO, we propose a mechanism of catalysis for 3HAO.     

4-chloro-3-hydroxyanthranilate inhibits brain 3-hydroxyanthranate oxidase

Quinolinic acid is synthesized from 3-hydroxyanthranilic acid via 3-hydroxyanthranilic acid oxidase. In liver, 4-chloro-3-hydroxyanthranilic acid inhibits 3-hydroxyanthranilic acid oxidase. To determine whether 4-chloro-3-hydroxyanthranilic acid also inhibits 3-hydroxyanthranilic acid oxidase in brain, 3-hydroxyanthranilic acid was injected into the cisterna magna of rats either with or without 4-chloro-3 hydroxyanthranilic acid. 3-Hydroxyanthranilic acid increased quinolinic acid concentrations throughout the brain. 4-Chloro-3-hydroxyanthranilic acid attenuated increases in brain quinolinic acid. These observations indicate that 4-chloro-3-hydroxyanthranilic acid inhibits 3-hydroxyanthranilic acid oxidase in brain.Quinolinic acid is a well established systemic metabolite of l-tryptophan which has been shown to be present in brain (Wolfensberger et al., 1983; Heyes and Markey, 1988a). QUIN has proved to be a convulsant (Lapin, 1982), neurotoxin (Schwarcz et al., 1983) and agonist of N-methyl-D-aspartate receptors (Perkins and Stone, 1983) when injected directly into the central nervous system of experimental animals. Therefore increased concentrations of QUIN in brain may have neoropathologic consequences. l-Tryptophan is converted to QUIN via the kynurenine pathway. The precursor of QUIN, 2-amino-3-carboxymuconic semialdehyde is synthesized from 3-hydroxyanthranilic acid (3-HAA) by the action of 3-hydroxyanthranilic acid oxidase (3-HAA/OX) in liver and brain (Foster et al., 1986; Okuno et al., 1987). QUIN is then formed from 2-amino-3-carboxymuconic semialdehyde by a spontaneous, non-enzymatic reaction. In liver, 3-HAA/OX is inhibited by 4-chloro-3-hydroxyantranilic acid (CL-HAA; Parli et al., 1980). In the present study, rats were given an intracisternal injection of 3-HAA and the resultant increases in regional brain QUIN concentrations quantified by gas chromatography/mass spectrometry (Heyes and Markey, 1988a,b). To determine whether CL-HAA inhibit 3-hydroxyanthranilic acid oxidase in brain, CL-HAA was co-administered with 3-HAA to see whether increases in QUIN were attenuated.     

[3H]Norepinephrine Release from Hippocampal Slices Is an In Vitro Biochemical Tool for Investigating the Pharmacological Properties of Excitatory Amino Acid Receptors

[3H]Norepinephrine ([3H]NE) efflux from preloaded rat hippocampal slices was increased in a dose-dependent manner by excitatory amino acids, with the following order of potencies: N-methyl-D-aspartate (NMDA) greater than kainic acid (KA) greater than L-glutamate greater than or equal to D,L-homocysteate greater than L-aspartate greater than quinolinic acid greater than quisqualic acid. The effect of the excitatory amino acids was blocked by physiological concentrations of Mg2+, with the exception of KA. D,L-2-Amino-7-phosphonoheptanoic acid dose-dependently inhibited the NMDA effect (ID50 = 69 microM), whereas at 1 mM it was ineffective versus KA. The release of [3H]-NE induced by quinolinic acid was blocked by 0.1 mM D,L-2-amino-7-phosphonohepatanoic acid. gamma-D-Glutamylglycine dose-dependently inhibited the KA effect with an ID50 of 1.15 mM. Tetrodotoxin (2 microM) reduced by 40 and 20% the NMDA and KA effects, respectively. The data indicate that [3H]NE release from hippocampal slices can be used as a biochemical marker for pharmacological investigations of excitatory amino acid receptors and their putative agonists and antagonists.     

Changes in kynurenic, anthranilic, and quinolinic acid concentrations in rat brain tissue during development

Kynurenic, anthranilic, and quinolinic acid, brain tissue concentrations and indoleamine 2,3-dioxygenase [EC 1 13.11.17] activity were determined in rat brain, during pre- and postnatal development. Quinolinic acid brain tissue concentration was significantly increased at birth as compared with the prenatal level, then it declined rapidly in the postnatal period. By the contrary, kynurenic and anthranilic acids brain tissue concentrations in rat brain were significantly lower at birth as compared with those found prenatally; then kynurenic acid concentration decreased in the first postnatal week and increased thereafter, while anthranilic acid concentration increased in the first postnatal week and decreased thereafter. Indoleamine 2,3-dioxygenase [EC 1 13.11.17] activity were found unchanged in pre and post natal rat brain. The described opposite changes in quinolinic and kynurenic acids concentrations, occurring in pre- and postnatal period, despite the lack of knowledge on the precise role played by these compounds on the different neurotransmitter systems in the brain, could be involved in brain ontogenetic development.     

The effect of pyrazines on the metabolism of tryptophan and nicotinamide adenine dinucleotide in the rat. Evidence of the formation of a potent inhibitor of aminocarboxy-muconate-semialdehyde decarboxylase from pyrazinamide

The intraperitoneal or oral administration of pyrazinamide and pyrazinoic acid (pyrazine 2-carboxylic acid) resulted in a marked increase of the NAD content in rat liver. The injections of pyrazine and pyrazine 2,3-dicarboxylic acid exhibited no significant effect on the hepatic NAD content. The boiled extract obtained from liver and kidney of rat injected with either pyrazinamide or pyrazinoic acid exhibited a potent inhibitory effect on the aminocarboxymuconate-semialdehyde decarboxylase (EC 4.1.1.45) activity in either lier or kidney, although pyrazinamide or pyrazinoic acid per se did not inhibit the enzyme activity. The unknown inhibitor of aminocarboxymuconate-semialdehyde decarboxylase was dialysable and heat-stable, and mostly excreted in urine by 6 and 12 h after injected of pyrazinoic acid and pyrazinamide, respectively. Pyrazine 2,3-dicarboxylic acid, pyrazine, nicotinamide, nicotinic acid, tryptophan, anthranilic acid, 5-hydroxyanthranilic acid and quinolinic acid exhibited no significant effect on the aminocarboxymuconate-semialdehyde decarboxylase activity in liver and kidney at the concentration of 1 mM in the reaction mixture. The expired 14CO2 from L-[benzen ring-U-14C]tryptophan was markedly decreased by the pyrazinamide injection, while the urinary excretion of 14C-labeled metabolites from L-tryptophan, mainly quinolinic acid, was markedly increased. These results suggest that the glutarate pathway of L-tryptophan was strongly inhibited by the inhibitor produced after the administration of pyrazinoic acid and pyrazinamide. Pyrazinamide but not pyrazinoic acid also exhibited a significant inhibition of the nuclear enzyme poly(ADP-ribose) synthetase in rat liver.