The presence of kynurenine aminotransferases in the human cornea: Evidence from bioinformatics analysis of gene expression and immunohistochemical staining.

PURPOSE: Kynurenine aminotransferases (KATs) catalyze the synthesis of kynurenic acid (KYNA), a compound of significant biological activity. The aim of this study is to investigate the presence and distribution of KAT immunoreactivity in the healthy human cornea. METHODS: Data on gene expression in human eye structures were extracted from public microarray experiments using Genevestigator software. Immunohistochemistry was conducted using polyclonal antibodies against KAT I, II, and III on sections of eight enucleated eyes from patients with choroidal melanoma. RESULTS: Bioinformatics analysis showed that all four KAT isoforms were actively transcribed in the cornea and the conjunctiva. Immunohistochemical analysis revealed the presence of KAT I, II, and III in all examined corneal sections. The corneal endothelium showed the strongest reactivity for all three KAT isoforms. There was a slight positive staining of the corneal stroma for KAT I and II. KAT III immunoreactivity was found only in the stroma of the limbal region. In the corneal epithelium, the expression of all three KAT isoforms showed a specific pattern of the stain with fine squatter granules throughout the cytoplasm. This reactivity was more pronounced in the basal cell layers. The intermediate cell layers showed only faint immunoreactivity, and occasionally, there was no staining. KAT I, II, and III were also present in the adjacent limbal conjunctiva. CONCLUSIONS: The results indicate that kynurenine can be metabolized to KYNA in the corneal epithelium, stroma, and endothelium.

Presence and distribution of L-kynurenine aminotransferases immunoreactivity in human cataractous lenses.

PURPOSE: To investigate the presence and distribution of l-kynurenine aminotransferases immunoreactivity in human and animal lenses during cataract formation. METHODS: Immunohistochemistry was conducted using polyclonal antibodies against KAT I, KAT II and KAT III on sections of 26 anterior capsules from patients undergoing surgical treatment of anterior subcapsular cataract (ASC) and 22 cataractous lenses from human eyes enucleated because of choroidal malignant melanoma. Additionally, the eyes of 11-month-old DBA/2J mice (6 eyes) were investigated (with KAT I and II). Ten clear human lenses and four BL6 mice lenses were used as controls. Spatial immunoreactivity patterns of enzymes were compared with Periodic Acid - Schiff (PAS)-stained sections. RESULTS: Immunohistochemical analysis revealed presence of KAT I, KAT II and KAT III in extracellular structures of all studied types of cataract in human eyes showing specific pattern of the stain. In cortical cataract, immunoreactivity was observed on cortical lens fibres. In nuclear cataract, KAT II revealed stronger and diffused staining than KAT I. Additionally, both KAT showed more pronounced staining at the edge of small clefts. In normal human lenses, KAT I, II and III, immunoreactivity was not observed. Presence of KAT I and KAT II in the intercellular substance of DBA/2J mice cataract was observed. In BL6 mice lenses without cataract, only weak KAT I and KAT II staining was observed. CONCLUSIONS: Presence of l-kynurenine aminotransferases in extracellular matrix (ECM) during human cataract formation suggests that products of l-kynurenine pathway might be involved in mechanisms of cataractogenesis.

Presence of L-kynurenine aminotransferase III in retinal ganglion cells and corpora amylacea in the human retina and optic nerve.

BACKGROUND: Corpora amylacea (CAm) are a hallmark of aging and neurodegeneration. The presence of kynurenine aminotransferases I and II (KAT I and II) in CAm in the human retina and optic nerve has been already shown. The present study aimed to examine kynurenine aminotransferase III (KAT III) immunoreactivity in CAm in the human retina and optic nerve. MATERIAL AND METHODS: Polyclonal antibody against KAT III was used on sections of human eyes enucleated due to malignant uveal melanoma. PAS-stained sections of CAm were compared with KAT III stained ones. RESULTS: KAT III immunoreactivity was observed in CAm in the retina, prelaminar, laminar and retrolaminar region of the optic nerve with similar location to PAS-stained sections. The most intense staining was observed in the retrolaminar part of the optic nerve. KAT III immunoreactivity was also present in the cytoplasm of retinal ganglion cells. CONCLUSIONS: Expression of KAT III in CAm in the human retina and optic nerve indicates that this enzyme may be relevant in mechanisms of neurodegeneration leading to CAm formation.

Identification of radicals formed in the reaction mixtures of rat liver microsomes with ADP, Fe3+ and NADPH using HPLC EPR and HPLC EPR MS.

The reaction of rat liver microsomes with Fe(3+), ADP and NADPH was examined using EPR, HPLC-EPR and HPLC-EPR-MS combined use of spin trapping technique. A prominent EPR spectrum (alpha(N) = 1.58 mT and alpha(H)beta = 0.26 mT) was observed in the complete reaction mixture. The EPR spectrum was hardly observed for the complete reaction mixture without rat liver microsomes. The radicals appear to be derived from microsomal components. The EPR spectrum was also hardly observed in the absence of Fe(3+). Addition of some iron chelators such as EDTA, citrate and ADP resulted in the dramatic change in the EPR intensity. Iron ions seem to be essential for this reaction. For the complete reaction mixture with boiled microsomes, a weak EPR spectrum was observed, suggesting that enzymes participate in the reaction. Five peaks were separated on the HPLC-EPR elution profile of the complete reaction mixture of rat liver microsomes with ADP, Fe(3+) and NADPH. The retention times of the peaks 1 to 5 were 19.4, 22.5, 27.3, 29.8 and 31.4 min, respectively. To identify the radical adducts, HPLC-EPR-MS analyses were performed for the three prominent peaks. The HPLC-EPR-MS analyses showed that a new radical adduct, 4-POBN/1-hydroxypentyl radical, in addition to 4-POBN/ethyl radical adducts, forms in a reaction mixture of rat liver microsomes with ADP, Fe(3+) and NADPH.

Immunohistochemical identification of kynurenine aminotransferases in corpora amylacea in the human retina and optic nerve.

INTRODUCTION: Corpora amylacea (CAm) occur in the optic nerve and in retinal ageing and degeneration. Cellular expression of L-kynurenine aminotransferases (KAT I and II) in the avian and rodent retina and its changes in retinal development and neurodegeneration have been documented. This study examines KAT I and II immunoreactivity in CAm in the human retina and optic nerve. MATERIAL AND METHODS: Immunohistochemistry was performed using polyclonal antibodies against KAT I and KAT II on sections of 23 human eyes enucleated for malignant uveal melanoma. Occurrence and location of KAT I- or KAT II-stained CAm was compared with PAS-stained sections. RESULTS: KAT I and KAT II expression in CAm has been shown in the retina and optic nerve with similar location to PAS-stained sections. KAT I immunoreactivity was more intense than KAT II and its staining was more pronounced in the retrolaminar part of the optic nerve. Some of the CAm showed only faint KAT II expression and occasionally there was no staining. KAT II revealed no association of staining variability and localisation of CAm. Similarly to animal studies, in the human retina KAT I was observed on Müller cell endfeet while KAT II was expressed in retinal ganglion cells. CONCLUSIONS: Presence of kynurenine aminotransferases in CAm in the human retina and optic nerve suggest that both enzymes may be involved in mechanisms of retinal ageing and neurodegeneration leading to CAm formation.

Mitochondrial aspartate aminotransferase: a third kynurenate-producing enzyme in the mammalian brain.

The tryptophan metabolite kynurenic acid (KYNA), which is produced enzymatically by the irreversible transamination of l-kynurenine, is an antagonist of alpha7 nicotinic and NMDA receptors and may thus modulate cholinergic and glutamatergic neurotransmission. Two kynurenine aminotransferases (KAT I and II) are currently considered the major biosynthetic enzymes of KYNA in the brain. In this study, we report the existence of a third enzyme displaying KAT activity in the mammalian brain. The novel KAT had a pH optimum of 8.0 and a low capacity to transaminate glutamine or alpha-aminoadipate (the classic substrates of KAT I and KAT II, respectively). The enzyme was inhibited by aspartate, glutamate, and quisqualate but was insensitive to blockade by glutamine or anti-KAT II antibodies. After purification to homogeneity, the protein was sequenced and the enzyme was identified as mitochondrial aspartate aminotransferase (mitAAT). Finally, the relative contributions of KAT I, KAT II, and mitAAT to total KAT activity were determined in mouse, rat, and human brain at physiological pH using anti-mitAAT antibodies. KAT II was most abundant in rat and human brain, while mitAAT played the major role in mouse brain. It remains to be seen if mitAAT participates in cerebral KYNA synthesis under physiological and/or pathological conditions in vivo.

A selective method for transfection of retinal ganglion cells by retrograde transfer of antisense oligonucleotides against kynurenine aminotransferase II.

PURPOSE: Intravitreal administration of specific antisense oligonucleotides (ODNs) effectively downregulates gene expression in the retina but does not modulate it exclusively in retinal ganglion cells (RGCs). Expression of kynurenine aminotransferase II (KAT II) in RGCs has been well described in the literature. We describe a new method for downregulating cellular KAT II expression via transfection of RGC by retrograde transfer of ODN. METHODS: Fluorescently labeled, specific ODNs against KAT II were injected into rats either intravitreally or into the superior colliculi. Fluorescence microscopy of retinal flat-mounts and radial sections was used to compare the location, duration, and degree of transfection for both methods of delivery. The effects of both methods on KAT II expression in RGCs were studied immunohistochemically with unlabeled ODN. Retinal kynurenic acid (KYNA) contents were measured using high pressure liquid chromatography (HPLC). RESULTS: After intravitreal injection, fluorescently labeled ODN reached all retinal layers, whereas injections into the superior colliculus resulted in transfection of the RGC layer alone. Immunohistochemistry showed that both methods of ODN application had a similar effect on downregulation of KAT II expression in RGC. Retinal KYNA content decreased significantly 4 days after both types of ODN administration. CONCLUSIONS: This study demonstrated that retrograde transfer of specific ODN into RGC is feasible and induces downregulation of KAT II cellular expression. This may become a useful tool for modulating gene expression in the retinal ganglion cell layer in vivo without direct transfer of ODN to other retinal cell layers.

Demonstration of kynurenine aminotransferases I and II and characterization of kynurenic acid synthesis in oligodendrocyte cell line (OLN-93).

In the present study we demonstrate for the first time that both kynurenine aminotransferase (KAT) isoforms I and II are present in the permanent immature rat oligodendrocytes cell line (OLN-93). Moreover, we provide evidence that OLN-93 cells are able to synthesize kynurenic acid (KYNA) from exogenously added L-kynurenine and we characterize its regulation by extrinsic factors. KYNA production in OLN-93 cells was depressed in the presence of aminotransferase inhibitor, aminooxyacetic acid and was not affected by depolarizing agents such as 50 mM K+ and 4-aminopyridine. Glutamate agonists, L-glutamate and D,L-homocysteine significantly decreased KYNA production. Selective agonist of ionotropic glutamate receptors Amino-2,3-dihydro-5-methyl-3-oxo-4-isoxazolepropionic acid (AMPA) lowered KYNA production in OLN-93 cell line, whereas N-methyl-D-aspartate (NMDA) had no influence on KYNA production. Furthermore, KYNA synthesis in OLN-93 cells was decreased in a concentration-dependent manner by amino acids transported by L-system, L-leucine, L-cysteine and L-tryptophan. The role of KYNA synthesis in oligodendrocytes needs further investigation.

Demonstration of kynurenine aminotransferases I and II and characterization of kynurenic acid synthesis in cultured cerebral cortical neurons.

The present study characterizes the synthesis of kynurenic acid (KYNA) from exogenously added kynurenine and its regulation by extrinsic factors, in cultured cerebral cortical neurons and, for comparison, in astrocytes incubated under identical conditions. The neuronal culture showed positive immunostaining for both kynurenic acid aminotransferase (KAT) isoforms I and II. Neurons synthesized KYNA at a rate about 2.3 times higher than astrocytes. Neuronal, but not astrocytic, KYNA synthesis was lowered approximately 30% by ionotropic glutamate receptor agonists [(R,S)-3-hydroxy-5-methoxyloxasole-4-propionic acid (AMPA; 100 microM) and N-methyl-D-aspartic acid (NMDA; 100 microM)] and depolarizing agents [KCl (50 mM) and 4-aminopyridine (4-AP; 10 microM)]. Neuronal and astrocytic synthesis alike were vulnerable to inhibition exerted by the aminotransferase inhibitor aminooxyacetic acid (AOAA), glutamate (IC50: 31 and 85 microM, respectively), substrates of the L-amino transport system [leucine (Leu); IC50: 19 and 42 microM, respectively] and 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH; IC50: 19 and 28 microM, respectively). Glutamine (Gln), which is a metabolic precursor of glutamate in astrocytes and L-system substrate in both cell types, inhibited KYNA synthesis both in neurons and in astrocytes (IC50: 268 and 318 microM, respectively). alpha-Ketoisocaproic acid (KIC), a Leu transamination product that is produced mainly in astrocytes and shuttled to neurons to modulate intraneuronal concentration of glutamate, stimulated KYNA synthesis in neurons but did not affect the synthesis in astrocytes. In conclusion, this study is the first to demonstrate active, regulation-prone KYNA synthesis in neurons.

Kynurenine aminotransferase in the supratentorial dura mater of the rat: effect of stimulation of the trigeminal ganglion.

Electrical stimulation of the trigeminal ganglion has been widely used as a model of nociception, characterizing migraine. This treatment is known to evoke release of neuropeptides and neurotransmitters from nerve fibers of the dura mater. On the basis of immunocytochemical investigations, we found that under normal conditions, surface membranes of Schwann cells surrounding nerve fibers in the supratentorial dura mater display kynurenine aminotransferase-immunoreaction (KAT-IR); also KAT-IR are the granules of mast cells and the cytoplasms of macrophages (histiocytes). In consequence of stimulation of the trigeminal ganglion, Schwann cells in the dura mater became conspicuously swollen while their KAT-IR decreased considerably; also KAT-IR of mast cells and macrophages decreased significantly. At the same time, nitric oxide synthase (NOS)-IR of nerve fibers in the dura mater increased, suggesting release of nitric oxide (NO), this is known to be involved in NMDA receptor activation leading to vasodilation followed by neurogenic inflammation. Because kynurenic acid (KYNA) is an antagonist of NMDA receptors, we hypothesize that KYNA and its synthesizing enzyme, KAT, may play a role in the prevention of migraine attacks.

Kynurenic acid production in cultured bovine aortic endothelial cells. Homocysteine is a potent inhibitor.

Kynurenic acid (KYNA) is a broad-spectrum antagonist at all subtypes of ionotropic glutamate receptors, but is preferentially active at the strychnine-insensitive glycine allosteric site of the N-methyl-D-aspartate (NMDA) receptor and is also a non-competitive antagonist at the alpha7 nicotinic receptor. KYNA occurs in the CNS, urine, serum and amniotic fluid. Whilst it possesses anticonvulsant and neuroprotective properties in the brain, its role in the periphery, however, is unknown. In this study we demonstrated the presence of kynurenine aminotransferase (KAT) I and II in the cytoplasm of bovine aortic endothelial cells (BAEC). BAEC incubated in the presence of the KYNA precursor L-kynurenine synthesized KYNA concentration- and time-dependently. KYNA production was inhibited by the aminotransferase inhibitor aminooxyacetic acid but was not affected by a depolarising concentration of K(+) or by 4-aminopyridine. The glutamate agonists L-aspartate and L-glutamate depressed KYNA production significantly. The selective ionotropic glutamate receptor agonists alpha-amino-2,3-dihydro-5-methyl-3-oxo-4-isoxazolepropionic acid (AMPA) and NMDA were ineffective in this respect. D,L-Homocysteine and L-homocysteine sulphinic acid lowered KYNA production in BAEC. Further investigations are needed to assess the role and importance of KYNA in vessels and peripheral tissues.

Age-dependent decrease of retinal kynurenate and kynurenine aminotransferases in DBA/2J mice, a model of ocular hypertension.

The study examines age-dependent changes of kynurenic acid (KYNA) content and kynurenine aminotransferases (KAT I and KAT II) celluar expression in the retinas of DBA/2J mice. Retinas were obtained from DBA/2J mice of different ages (3, 6 and 11 months). C57BL6 mice were used as controls. As measured with HPLC, KYNA content decreased (p < 0.01) in the retinas of 6-month-old DBA/2J mice and continued to decrease (p < 0.0074) in the retinas of 11-month-old animals compared to the controls. Immunohistochemistry showed that expression of both KAT I and KAT II decreased markedly in the retinas of 11-month-old DBA/2J mice compared to controls. The impairment in KYNA biosynthesis in the retinas of DBA/2J mice may be one of the mechanisms of retinal neurodegeneration related to ocular hypertension.

Expression of kynurenine aminotransferases in the rat retina during development.

The study investigates the cellular expression of kynurenine aminotransferases (KAT I and II) in the rat retina during development. At P1 (the day of birth) and P7 (the 7th day after birth), KAT I expression was observed in the inner plexiform layer (IPL), the fiber layer (FL), and in vertically running processes in the ganglion cell layer (GCL) (but not in the cell bodies). At P14 (the 14th day after birth) a strong KAT I immunoreactivity was observed in Müller cell endfeet. KAT II was expressed in the IPL, the FL, and in cells in the GCL at P1 and P7. From P14 on, KAT II expression in the IPL decreased. Double labeling revealed that KAT I was expressed in Müller cell endfeet, whilst KAT II both on retinal ganglion cells (RGC) and Müller cell endfeet. In conclusion, KAT I and II are present in the rat retina during development. The heterogeneity of the KAT developmental profiles possibly reflects a neuromodulatory role in the retinal differentiation.

L-alanine-glyoxylate aminotransferase II of rat kidney and liver mitochondria possesses cysteine S-conjugate beta-lyase activity: a contributing factor to the nephrotoxicity/hepatotoxicity of halogenated alkenes?

Several halogenated alkenes are metabolized in part to cysteine S-conjugates, which are mitochondrial toxicants of kidney and, to a lesser extent, other organs. Toxicity is due to cysteine S-conjugate beta-lyases, which convert the cysteine S-conjugate into pyruvate, ammonia and a reactive sulphur-containing fragment. A section of the human population is exposed to halogenated alkenes. To understand the health effects of such exposure, it is important to identify cysteine S-conjugate beta-lyases that contribute to mitochondrial damage. Mitochondrial aspartate aminotransferase [Cooper, Bruschi, Iriarte and Martinez-Carrion (2002) Biochem. J. 368, 253-261] and mitochondrial branched-chain aminotransferase [Cooper, Bruschi, Conway and Hutson (2003) Biochem. Pharmacol. 65, 181-192] exhibit beta-lyase activity toward S -(1,2-dichlorovinyl)-L-cysteine (the cysteine S-conjugate of trichloroethylene) and S -(1,1,2,2-tetrafluoroethyl)-L-cysteine (the cysteine S-conjugate of tetrafluoroethylene). Turnover leads to eventual inactivation of these enzymes. Here we report that mitochondrial L-alanine-glyoxylate aminotransferase II, which, in the rat, is most active in kidney, catalyses cysteine S-conjugate beta-lyase reactions with S -(1,1,2,2-tetrafluoroethyl)-L-cysteine, S -(1,2-dichlorovinyl)-L-cysteine and S -(benzothiazolyl-L-cysteine); turnover leads to inactivation. Previous workers showed that the reactive-sulphur-containing fragment released from S -(1,1,2,2-tetrafluoroethyl)-L-cysteine and S -(1,2-dichlorovinyl)-L-cysteine is toxic by acting as a thioacylating agent - particularly of lysine residues in nearby proteins. Toxicity, however, may also involve 'self-inactivation' of key enzymes. The present findings suggest that alanine-glyoxylate aminotransferase II may be an important factor in the well-established targeting of rat kidney mitochondria by toxic halogenated cysteine S-conjugates. Previous reports suggest that alanine-glyoxylate aminotransferase II is absent in some humans, but present in others. Alanine-glyoxylate aminotransferase II may contribute to the bioactivation (toxification) of halogenated cysteine S-conjugates in a subset of individuals exposed to halogenated alkenes.

Ontogenic changes of kynurenine aminotransferase I activity and its expression in the chicken retina.

Kynurenine aminotransferases are key enzymes for the synthesis of kynurenic acid (KYNA), an endogenous glutamate receptor antagonist. The study described here examined ontogenic changes of kynurenine aminotransferase I (KAT I) activity and its expression in the chicken retina. KAT I activity measured on embryonic day 16 (E16) was significantly higher than at all other stages (E12, P0 and P7). Double labeling with antibodies against glutamine synthetase showed that on P7 KAT I was expressed in Müller cell endfeet and their processes in the inner retina. Since KAT I activity is high in the late embryonic stages, it is conceivable that it plays a neuromodulatory role in the retina during the late phase of embryogenesis.

Effect of 3-nitropropionic acid on kynurenine aminotransferase in the rat brain.

Activation of excitatory amino acid receptors by endogenous excitotoxins results in degenerative changes characteristic of neurodegenerative brain diseases such as Huntington's disease. Excitatory amino acid receptors are present in the highest concentration in the striatum, the hippocampal region, and the temporal lobe. The most potent, naturally occurring excitatory amino acid receptor antagonist is kynurenic acid (KYNA) which acts preferentially on N-methyl-D-aspartate (NMDA) receptors. KYNA is produced from L-kynurenine, by the action of the enzymes kynurenine aminotransferases (KAT I and KAT II). Several inhibitors of mitochondrial energy metabolism result in an indirect excitotoxic neuronal degeneration. We examined whether systemic administration of the mitochondrial toxin 3-nitroproprionic acid (3-NP), an irreversible inhibitor of succinate dehydrogenase, which also acts by an indirect excitotoxic mechanism, would produce alterations in the immunohistochemical pattern of KAT I. Our present investigations demonstrate that after 15 days of administration of 3-NP, an inhibitor of mitochondrial Complex II, the most severe depletion of KAT I occurred in the striatum; less severe depletion occurred in other brain areas investigated, following a striatum > hippocampus > temporal cortex gradient. The alterations induced by 15 days of 3-NP treatment were less conspicuous in 6-week-old (young) animals than in 3-month-old adults. In these adult animals, 3-NP induced necrotic cores in the striatum, characterized by destruction of neuronal and glial elements, similar to that seen in the histologic and neurochemical features of Huntington's disease. It appears that immunohistochemical depletion of KAT after administration of 3-NP to adult animals may contribute to the pathological processes that characterize Huntington's disease.

Expression of kynurenine aminotransferase in the subplate of the rat and its possible role in the regulation of programmed cell death.

The neurons of the transient subplate zone, considered important for the prenatal development of the cerebral cortex, were shown here to express kynurenine aminotransferase (KAT)-I from embryonic day (E) 16 until postnatal day (P) 7 in the rat. No other cells of brain tissue exerted KAT-I immunoreactivity during this period. From P3 on, the neurons of the subplate gave rise to KAT-I immunoreactive, varicose axons, which entered the thalamus and terminated around thalamic nerve cells that are devoid of KAT-I immunoreactivity. Other subplate markers displayed a different expression pattern during development. Thus, subplate neurons displayed parvalbumin (PV) immuno-reactivity from E16 to P10 and an intense NPY immunoreaction from P7 to P1. They also exhibited nitric oxide synthase immunoreactivity from E16 to P10, whereas on the surface of the subplate neurons, the alpha7 subunit of the nicotinic acetylcholine receptor (nAChR) was present from P1 to P10. The cells of Cajal-Retzius were nAChR-immunoreactive during this period. Between P1 and P7, the perikarya of subplate neurons also showed an intense immuno-reaction with the N-methyl-D-aspartate (NMDA) receptor subtype R2A. After the first postnatal week, many of the KAT-I positive subplate neurons display a gradual decrease of immunoreactivity and undergo programmed cell death. Since KAT-I persists in the subplate through the period E16-P7, we conclude that KAT-I is a useful and reliable subplate marker in the rat. Since it is assumed that migration of nerve cells is regulated by NMDA receptors, and since kynurenic acid--the only naturally occurring NMDA receptor antagonist--is synthesized by KAT, we suggest that a temporary breakdown of the delicate equilibrium between NMDA and KAT might induce abnormal neuronal migration, giving rise to developmental abnormalities.