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.     

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.     

Synthesis of Quinolinic Acid by 3-Hydroxyanthranilic Acid Oxygenase in Rat Brain Tissue In Vitro

In mammalian peripheral organs, 3-hydroxyanthranilic acid oxygenase (3HAO), catalyzing the conversion of 3-hydroxyanthranilic acid to quinolinic acid, constitutes a link in the catabolic pathway of tryptophan to NAD. Because of the possible involvement of quinolinic acid in the initiation of neurodegenerative phenomena, we examined the presence and characteristics of 3HAO in rat brain tissue. A simple and sensitive assay method, based on the use of [carboxy-14C]3-hydroxyanthranilic acid as a substrate, was developed and the enzymatic product, [14C]quinolinic acid, identified by chromatographic and biochemical means. Kinetic analysis of rat forebrain 3HAO revealed a Km of 3.6 +/- 0.5 microM for 3-hydroxyanthranilic acid and a Vmax of 73.7 +/- 9.5 pmol quinolinic acid/h/mg tissue. The enzyme showed pronounced selectivity for its substrate, since several substances structurally and metabolically related to 3-hydroxyanthranilic acid caused less than 25% inhibition of activity at 500 microM. Both the Fe2+ dependency and the distinct subcellular distribution (soluble fraction) of brain 3HAO indicated a close resemblance to 3HAO from peripheral tissues. Examination of the regional distribution in the brain demonstrated a 10-fold variation between the region of highest (olfactory bulb) and lowest (retina) 3HAO activity. The brain enzyme was present at the earliest age tested (7 days postnatum) and increased to 167% at 15 days before reaching adult levels. Enzyme activity was stable over extended periods of storage at -80 degrees C. Taken together, these data indicate that measurements of brain 3HAO may yield significant information concerning a possible role of quinolinic acid in brain function and/or dysfunction.     

Cloning of human 3-hydroxyanthranilic acid dioxygenase in Escherichia coli: characterisation of the purified enzyme and its in vitro inhibition by Zn2+

3-hydroxyanthranilic acid oxygenase (3-HAO) catalyses the conversion of 3-hydroxyanthranilic acid to quinolinic acid. Because of the involvement of quinolinic acid in the initiation of neurodegenerative phenomena, we have cloned human 3-HAO in Escherichia coli, overexpressed and purified it with the aim of studying its enzymatic activity and for future structural studies. The recombinant human protein, obtained in E. coli, retains its enzymatic activity which can occur only in the presence of Fe(II); several other metals have been tested but in no case the formation of the product has been observed. On the contrary, two of the ions tested inhibit the catalytic reaction and one of them, Zn2+, could be of physiological relevance. A circular dichroism analysis has also been performed, showing that the secondary structure is mainly of the beta type, with a minority of alpha.     

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.     

QSAR study on inhibition of brain 3-hydroxy-anthranilic acid dioxygenase (3-HAO): A molecular connectivity approach

The ability of 4,5-, 4,6-disubstituted and 4,5,6-trisubstituted 3-hydroxyanthranilic acid derivatives to reduce the production of the excitotoxin quinolinic acid (QUIN) by inhibition of brain 3-hydroxyanthranilic acid dioxygenase (3-HAO) has been investigated using molecular connectivity indices (⁰χ^v, ¹χ^v, ²χ^v). The in-vivo inhibition of 3-HAO in rat cortex (pIC₅₀, nM) is used for this purpose. The regression models obtained suggest that the degree of branching of the compounds under study have a dominant role in the observed inhibition potency. The data were used to generate quantitative structure–activity relationship (QSAR) models for estimating the potency of 3-HAO. The information obtained from the correlation should be useful in designing more potent analogues.     

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.     

High-performance liquid chromatographic assay of human lymphocyte kynureninase activity levels

Human lymphocyte kynureninase activity was assessed in homogenized cells by determination of 3-hydroxyanthranilic acid production as a function of time after addition of the substrate, 3-hydroxykynurenine. The product, 3-hydroxyanthranilic acid, was determined by isocratic high-performance liquid chromatography and fluorescence detection. Mean (± S.D.) lymphocyte kynureninase activity in a group (n = 12) of vitamin B₆-deficient men was 5.04 ± 0.81 pmol 3-hydroxyanthranilic acid formed per mg protein per min, which was significantly (p=0.005) lower than the 6.69 ± 1.70 pmol 3-hydroxyanthranilic acid formed per mg protein per min in men with a normal vitamin B₆ status. This indicates that lymphocyte kynureninase activity is depressed during a vitamin B₆ deficiency.     

Enzyme activities along the tryptophan-nicotinic acid pathway in alloxan diabetic rabbits

Recent data from our laboratory have indicated that the rabbit is a suitable animal model for the study of enzyme activities of the tryptophan-nicotinic acid pathway. We report here the pattern of tryptophan metabolism in rabbits made diabetic with alloxan treatment, and hypercholesterolemic with a high-cholesterol diet. A group of rabbits with only hypercholesterolemia was also considered. The enzymes assayed were: liver tryptophan 2,3-dioxygenase (TDO), intestine indoleamine 2,3-dioxygenase (IDO), liver and kidney kynurenine 3-monooxygenase, kynurenine-oxoglutarate transaminase, kynureninase, 3-hydroxyanthranilate 3,4-dioxygenase and aminocarboxymuconate-semialdehyde decarboxylase.TDO showed a reduction of specific activity in liver of diabetic-hyperlipidemic and hyperlipidemic rabbits compared to controls. Intestine IDO activities and liver and kidney kynurenine monooxygenase were unchanged with respect to controls.Kynurenine-oxoglutarate transaminase and kynureninase activities were reduced in the kidneys, but not in the liver, of diabetic-hyperlipidemic rabbits.The main finding was the reduction of 3-hydroxyanthranilate 3,4-dioxygenase activity (expressed as activity per g of fresh tissue) in the liver and kidneys of diabetic-hypercholesterolemic and hyperlipidemic rabbits compared to controls. Conversely, aminocarboxymuconate-semialdehyde decarboxylase activity was significantly higher in diabetic hypercholesterolemic rabbits in comparison with control and hypercholesterolemic rabbits.These data demonstrate that also in diabetic rabbits there is an alteration of tryptophan metabolism at the level of 3-hydroxyanthranilic acid-->nicotinic acid step. Also dyslipidemia seems to be involved in enzyme activity variations of the tryptophan metabolism along the kynurenine pathway.     

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.     

The kinetic mechanism of inhibition of liver pyridoxal kinase with tryptophan metabolites

The activity of purified liver pyridoxal kinase (ATP:pyridoxal 5-phosphotransferase, EC 2.7.1.35) was determined in the presence of 13 different tryptophan metabolites. Only 3-hydroxykynurenine, 3-hydroxyanthranilic acid, xanthurenic acid and quinolinic acid were found to inhibit the enzyme with I50 values of 0.1, 0.12, 0.36 and 0.42 mM, respectively. The inhibition was not related to the presence of pyridine nucleus in the metabolites molecule as was proved from the patterns of inhibition.     

A radioenzymatic assay for quinolinic acid

A new and rapid method for the determination of the excitotoxic tryptophan metabolite quinolinic acid is based on its enzymatic conversion to nicotinic acid mononucleotide and, in a second step utilizing [3H]ATP, further to [3H] deamido-NAD. Specificity of the assay is assured by using a highly purified preparation of the specific quinolinic acid-catabolizing enzyme, quinolinic acid phosphoribosyltransferase, in the initial step. The limit of sensitivity was found to be 2.5 pmol of quinolinic acid, sufficient to conveniently determine quinolinic acid levels in small volumes of human urine and blood plasma.     

Mode of action of melinacidin, an inhibitor of nicotinic acid biosynthesis

Melinacidin, a new antibacterial agent, blocked the synthesis of nicotinic acid and its amide in Bacillus subtilis cells. The inhibitory activity of the agent was reversed by nicotinic acid, its amide, or nicotinamide adenine dinucleotides, but not by l-kynurenine, l-3-hydroxykynurenine, l-hydroxyanthranilic acid, or quinolinic acid. These properties indicated that the antibiotic interferes with the conversion of quinolinic acid to nicotinate ribonucleotide by the enzyme quinolinate phosphoribosyl-transferase. However, the activity of a purified preparation of this enzyme derived from a Pseudomonas strain was not impaired by the antibiotic. This suggested that, in B. subtilis, melinacidin interferes with a reaction which occurs before the formation of quinolinic acid in the biosynthetic pathway leading to nicotinic acid. Failure of quinolinic acid to reverse melinacidin inhibition in B. subtilis cultures might be due to insufficient penetration of the cell membranes by quinolinate.     

Ommochrome deficiency in an albino strain of a terrestrial isopod, Armadillidium vulgare.

In order to clarify the cause of ommochrome deficiency in an albino strain of the terrestrial isopod, Armadillidium vulgare, levels of xanthommatin, 3-hydroxykynurenine, 3-hydroxyanthranilic acid and tryptophan in whole body extracts of the albino and the wild type individuals were determined together with enzyme activities of kynurenine-3-hydroxylase, kynureninase and tryptophan-2,3-dioxygenase. Xanthommatin could not be detected in the albinos. The levels of 3-hydroxykynurenine and 3-hydroxyanthranilic acid were determined by high-performance liquid chromatography (HPLC) with electrochemical detection and were markedly low in the albinos compared with the wild type individuals. In contrast to those, the tryptophan levels determined by HPLC with fluorescence detection did not differ significantly between the two phenotypes. In the albino A. vulgare, kynurenine-3-hydroxylase activity was lower and kynureninase activity was higher than in the wild type, although the differences were not statistically significant. Tryptophan-2,3-dioxygenase activity in the albinos was less than 10% that in the wild type. Thus, ommochrome deficiency in the albino A. vulgare is considered to be caused by the extremely low activity of tryptophan-2,3-dioxygenase.     

Ommochrome Deficiency in an Albino Strain of a Terrestrial Isopod,Armadillidium vulgare

In order to clarify the cause of ommochrome deficiency in an albino strain of the terrestrial isopod, Armadillidium vulgare, levels of xanthom-matin, 3-hydroxykynurenine, 3-hydroxyanthranilic acid and tryptophan in whole body extracts of the albino and the wild type individuals were determined together with enzyme activities of kynurenine-3-hydroxylase, kynureninase and tryptophan-2,3-dioxygenase. Xanthommatin could not be detected in the albinos. The levels of 3-hydroxykynurenine and 3-hydroxyanthranilic acid were determined by high-performance liquid chro-matography (HPLC) with electrochemical detection and were markedly low in the albinos compared with the wild type individuals. In contrast to those, the tryptophan levels determined by HPLC with fluorescence detection did not differ significantly between the two phenotypes. In the albino A. vulgare, kynurenine-3-hydroxylase activity was lower and kynureninase activity was higher than in the wild type, although the differences were not statistically significant. Tryptophan-2,3-dioxygenase activity in the albinos was less than 10% that in the wild type. Thus, ommochrome deficiency in the albino A. vulgare is considered to be caused by the extremely low activity of tryptophan-2,3-dioxygenase.     

4-Methyl-3-hydroxyanthranilic acid activating enzyme from actinomycin-producing Streptomyces chrysomallus

A 4-methyl-3-hydroxyanthranilic acid (4-MHA) activating enzyme was purified 24-fold from a crude protein extract of Streptomyces chrysomallus . The enzyme catalyzes both 4-MHA-dependent ATP/PPi exchange and the formation of the corresponding adenylate. No AMP was formed during the reaction, indicating that no covalent binding of 4-MHA takes place. Besides 4-MHA, the enzyme also catalyzes the formation of adenylates from 3-hydroxyanthranilic acid (3-HA), anthranilic acid (AA), benzoic acid (BA), 3-hydroxybenzoic acid (3-HB), 4-methyl-3-hydroxybenzoic acid (4-MHB), 4-methyl-3-methoxybenzoic acid (4- MMB ), and 4-aminobenzoic acid (4-AB). No such adenylates were formed from 2-aminophenol (2-AP), 2-hydroxybenzoic acid (2-HB), 3-hydroxykynurenine (3-HK), and tryptophan (Trp). 3-HA, 4-MHB, and 4-AB were among the structural analogues of 4-MHA that were the most effective for adenylate synthesis. In the case of 3-HA, considerable AMP release was observed, most probably due to nonenzymatic hydrolysis of the corresponding adenylate. A molecular weight between 53 000 and 57 000 was estimated. The specific activity of the enzyme was correlated with the titer of antibiotic in the cultures, and feeding experiments with whole mycelium of S. chrysomallus showed that 4-MHB was a strong inhibitor of actinomycin synthesis in vivo. The data strongly suggest that the enzyme is involved in the biosynthesis of actinomycin.     

The metabolism of [carboxyl-14C]anthranilic acid. I. The incorporation of radioactivity into NAD+ and NADP+.

A new pathway of NAD+ synthesis from anthranilic acid was found in the livers of rats. Starting from [carboxyl-14C]anthranilic acid, radioactive NAD+ and NADP+ were produced as judged by Dowex-1 X 8-formate column chromatography followed by radiochromatography. Several intermediate compounds, such as quinolinic acid, nicotinic acid mononucleotide, and nicotinic acid adenine dinucleotide were also identified with the aid of various chromatographic techniques. In the experiments with liver microsomal hydroxylation systems, anthranilic acid was converted into not only 5-hydroxyanthranilic acid but also 3-hydroxyanthranilic acid.     

Purification of quinolinic acid phosphoribosyltransferase from rat liver and brain

Quinolinic acid phosphoribosyltransferase (EC 2.4.2.19) was purified 3600-fold from rat liver and 280-fold from rat brain. Kinetic analyses (K_m = 12 μM for the substrate quinolinic acid and K_m 23 μM for the cosubstrate phosphoribosylpyrophosphate), physicochemical properties of the purified enzymes, inhibition by phthalic acid (K_i = 1.4 μM) and molecular weight determination (M_r 160 000 for the holoenzyme, consisting of five identical 32 kDa subunits) indicated the structural identity of quinolinic acid phosphoribosyltransferase from the two rat tissues. This was further confirmed immunologically, using antibodies raised against purified rat liver quinolinic acid phosphoribosyltransferase. Rat quinolinic acid phosphoribosyltransferase differs in several aspects from quinolinic acid phosphoribosyltransferase isolated from other organisms. The purified enzyme will prove a useful tool in the examination of a possible role of quinolinic acid in cellular function and/or dysfunction.     

Quinolinic acid phosphoribosyltransferase: purification and partial characterization from human liver and brain

Quinolinic acid phosphoribosyltransferase (QPRT) [EC 2.4.2.19] from human liver and brain was purified to homogeneity. Identity of the pure enzymes isolated from the two organs was proven by biochemical, physiocochemical and, following the production and partial purification of anti-liver QPRT antibodies, immunological techniques. Human QPRT has a molecular weight of 170,000 and consists of five identical subunits. Kinetic analyses revealed a Km of 5.6 microM for the substrate (quinolinic acid) and 23 microM for the co-substrate (phosphoribosylpyrophosphate). Enzyme activity was dependent on Mg2+ (optimal concentration: 1 mM) and was inhibited by the enzymatic by-product, inorganic pyrophosphate. Pure QPRT and its antibodies will constitute useful tools in the examination of the possible role of quinolinic acid in the pathogenesis of human neurodegenerative disorders.     

Cinnabarinic acid formation in Malpighian tubules of the silkworm, Bombyx mori. Participation of catalase in cinnabarinic acid formation in the presence of manganese ion

Cinnabarinic acid was formed from 3-hydroxyanthranilic acid during incubation with a soluble fraction from Malpighian tubules of the silkworm, Bombyx mori, in the presence of manganese ion. The enzyme having this activity was purified to homogeneity by ammonium sulfate fractionation, gel filtration and ion exchange chromatography. Enzyme activity was accompanied by parallel catalase activity at all steps of purification; the two activities could not be separated from each other. The purified protein was concluded to be catalase. Manganese was shown to be present in 0.1 mM concentration in Malpighian tubules of Bombyx mori. These findings suggest that in Malpighian tubules catalase participates in the formation of cinnabarinic acid. A possible mechanism for the formation of cinnabarinic acid from 3-hydroxyanthranilic acid by catalase in the presence of manganese ion is proposed.